MLS 400 Quiz Questions (Exam 1)

Herpesviruses (DNA Virus)

***All herpesviruses have the following features in common: morphology, basic mode of replication and capacity to establish latent and recurrent infections General Properties of Herpesviruses: 1.) Family: Herpesviridae 2.) Size: 120 to 200 nm 3.) Icosahedral 4.) Enveloped 5.) Double-stranded linear DNA genome

Macromolecule Synthesis

- Macromolecule synthesis involves the production of protein polymers and nucleic acids: - Viral transcription results in the synthesis of viral messenger RNA (mRNA) - mRNA codes for the early proteins (nonstructural elements such as enzymes) and late proteins (structural components) - Replication of viral nucleic acid is necessary to synthesize viral genomes that are incorporated into the progeny virus particles. - The mechanics of macromolecular synthesis varies depending on the characteristics of the viral genome (DNA or RNA, positive or negative sense strand, single- or double-stranded).

Overview of Diseases:

- Many adenovirus infections are self-limiting, asymptomatic diseases - Respiratory diseases and gastroenteritis most common symptomatic infections 1.) Respiratory diseases: - Accounts for up to 10% of respiratory infections in children 2.) Gastroenteritis (gastrointestinal disease) due to types 40 and 41: - Causes up to 15% of gastroenteritis in children that requires hospitalization 3.) Type 14: cause severe, acute respiratory disease - Outbreaks seen in military recruits - Potentially fatal - Incidence of respiratory infection in military recruits also caused by serotypes 4 & 7

Regarding rotavirus, which of the following statements is NOT correct?

- The viral genome is double-stranded segmented RNA. - Rotavirus is a common cause of viral gastroenteritis with diarrhea. - It is a member of the Hepadnaviridae family. - Use of available vaccines has reduced the incidence of disease, especially in young children. answer: It is a member of the Hepadnaviridae family. explanation: Rotavirus is a member of the Reoviridae family; Hepatitis B is a member of the Hepadnaviridae family.

Hemadsorption / Hemagglutination Assay (Detection of Virus-Infected Cells)

1.) Cells infected with certain viruses (influenza, mumps, etc.) express a viral glycoprotein (hemagglutinin) that binds erythrocytes to the infected cell surface (hemadsorption). 2.) Such viruses can be detected in cell culture medium due to the agglutination of erythrocytes (hemagglutination). 3.) The reaction may positive before cytopathic changes are visible 4.) Hemagglutination can be inhibited by antibodies to the hemagglutinin (hemagglutination inhibition)

Adenoviruses (DNA Viruses)

1.) Family: Adenoviridae 2.) Genus: Mastadenovirus 3.) 7 groups: HAdV-A to HAdV-G (HAdV = Human Adenovirus) - At least 57 human adenovirus serotypes 4.) Size: 80 to 100 nm 5.) Icosahedral 6.) Nonenveloped 7.) Double-stranded linear DNA 8.) Fiber protein protruding from capsid a. Viral attachment protein (VAP) b. Main type-specific antigen - Stimulates neutralizing antibody only to serotype causing infection

Caliciviruses Replicative Process

1.) Genome functions as mRNA and is translated into one very large polypeptide. 2.) The polypeptide is cleaved by virus-encoded protease 3.) The RNA polymerase synthesizes negative strand RNA that serves as the template for the positive strand genomic RNA. 4.) Viral progeny are released by cell lysis.

In a double-stranded DNA polymer, which DNA sequence below would be complementary to the sequence 5'GATTCTCAAAGGACT3'?

3' CTAAGAGTTTCCTGA 5'

Flaviviruses (RNA Viruses)

General Properties: 1.) Family: Flaviviridae 2.) Genus: Hepacivirus 3.) Species: Hepatitis C Virus (HCV) 4.) Enveloped 5.) Icosahedral 6.) Single-stranded positive sense RNA genome 7.) 6 genotypes (1 to 6) and more than 50 subtypes (lower case letter designations) - Due to differences in envelope glycoprotein caused by high mutation rate Overview of HCV Disease: 1.) 20% are self-limited acute infections and are able to be cleared without treatment 2.) 80% of acute cases develop into chronic hepatitis 3.) Chronic infection can predispose patient to cirrhosis and liver cancer (hepatocellular carcinoma) - One of the leading causes of liver transplants

Which of the following classes of enzyme are responsible for digesting nucleic acids?

Nucleases

Caliciviruses Pathogenesis & Immunity

Pathogenesis: 1.) Infection is usually limited to mucosal cells of intestinal tract. 2.) Many asymptomatic infections occur Immunity: 1.) Immunity following infection appears to be brief, and reinfection can occur.

VZV Laboratory Diagnosis

a. Most diagnoses are made clinically b. Laboratory Testing: 1.) Observation of CPEs in VZV-infected cells - Multinucleated giant cells seen in vesicles - Identification of intranuclear inclusions using direct fluorescent antibody procedure 2.) Can perform PCR on blister fluid - PCR may also be used on CSF to aid in diagnosis of VZV encephalitis 3.) Rise in antibody titer can be used in diagnosis of varicella - Less useful in diagnosis of zoster

Most genes contain large amounts of non-coding regions, called introns, that are interspersed among the coding regions, called __________, that provide the blueprint for the final protein.

exons

In the diagnosis of infectious mononucleosis caused by EBV, which of the following antibodies would NOT typically be expected to be present in the patient's serum during the acute stage of the disease?

- Heterophile antibodies - IgM VCA antibodies - IgG VCA antibodies - EBNA antibodies answer: EBNA antibodies

Cytopathogenesis

- Replication of the virus can initiate changes in cells that lead to cell death or to alterations in the cell's appearance, functional properties or antigenicity. - The effects on the cell may result from viral takeover of macromolecular synthesis, accumulation of viral proteins, modification of cellular structures, etc. - The outcomes of a viral infection of a cell include: A. Failed infection (no viral replication) - Do not multiply and therefore disappear - May be associated with a host cell that lacks the appropriate receptor, important enzyme pathway, etc. B. Cell death (lytic infection): 1.) A lytic infection results when virus replication ultimately kills the host cell 2.) May see characteristic changes in the appearance of the target cells - These changes in the host cells due to the viral infection are referred to as cytopathic effect (CPE) and can include: - Inclusion bodies in nucleus or cytoplasm (may be due to the accumulation of viral proteins, etc.) - Presence of inclusion bodies are used in laboratory diagnosis as the nature and location of inclusion bodies may be characteristic of particular viral infections - Vacuolization (a small cavity or vesicle in the cytoplasm containing air or fluid) - Rounding of cells - Fusion of neighboring cells into multinucleated giant cells called syncytia - Allows viral infection to spread cell-to-cell and escape antibody detection C. Persistent infection involving replication of virus without cell death: a. Viruses continue to replicate despite the host's defense mechanisms - Cell might not be destroyed if the virus is released through budding b. Individuals chronically infected can be referred to as carriers 1.) Can serve as reservoirs of infectious virions 2.) Low levels of virus may be detectable with little or no damage (shedding) 3.) In a population with a high proportion of carriers, the disease is endemic D. Presence of virus without viral replication, but with the potential for reactivation of the virus (latent - recurrent infections) - May include: a. Latent infection: limited viral macromolecular synthesis 1.) No clinical symptoms 2.) Virus present in resting or inactive state, with absence of viral replication - Nonproductive response - Viral genetic material persists indefinitely 3.) Cell often functions normally 4.) Virus may be dormant for months or years - Stress and other stimuli can activate cells to viral replication, resulting in: b. Recurrent infection: periods of latency followed by viral production 1.) Reactivation can occur with immune suppression 2.) Results in full viral replication 3.) Recurrence of clinically apparent disease E. Transforming (immortalizing) infections caused by oncogenic viruses: - Viruses are an established cause of cancer in humans - Persistent infections can stimulate uncontrolled cell growth, causing transformation of the cell - Causes alteration in cell morphology and metabolism - Oncogenic viruses have mechanisms for immortalizing cells - Viral transformation is first step, but not sufficient to cause tumor formation - Over time, immortalized cells accumulate other mutations that promote development of tumor

Prevention

1.) Live attenuated vaccines against serotypes 4 and 7 a. Each is monovalent b. Must be administered separately c. Only available for military personnel 2.) Conjunctivitis is preventable by asepsis and hand washing 3.) Environmental surfaces disinfected with bleach Adequate chlorination of swimming pools

Picornaviruses Replicative Process

1.) RNA genome functions as mRNA and is translated into one very large polypeptide. 2.) Polypeptide is cleaved by a protease to form capsid and noncapsid proteins. 3.) Replication of genome occurs by synthesis of a complementary negative strand, which serves as template for synthesis of positive strands. 4.) Positive strands function as mRNA and also serve as progeny virion genomic RNA. 5.) Virions are released by cell lysis.

All of the following statements about immunofluorescent assays are true except:

A. Fluorescent dyes called fluorochromes absorb short wavelength light and emit energy at a longer wavelength. B. These fluorescent dyes can be attached to antibody molecules. C. The reaction is observed using a brightfield microscope. D. A positive reaction will appear brightly illuminated against a dark background. answer: The reaction is observed using a brightfield microscope.

Immunologic Status of Patient

A. The ultimate goals of functional host antiviral immune responses are to prevent entry, reduce spread and eliminate the virus and the cells harboring the virus. 1.) The immune response is the best and in many cases the only means of controlling a viral infection. Innate, humoral and cellular immune response are all important for antiviral immunity. 2.) The hypersensitivity and inflammatory reactions initiated by antiviral immunity can sometimes also be a major cause of the pathologic manifestations and symptoms of viral diseases (immunopathogenesis) 3.) Immune status a. Naïve: not previously exposed and therefore susceptible to viral infection b. Immunized to virus: previously exposed and possibly protected - Prior immunity activates the specific immune response much sooner and more effectively - Immune responses can be produced by vaccines c. Immunocompromised: the host's immune system is impaired - These patients are more likely to get a viral infection and more likely to have a severe illness - Ultimate goal of immune response: elimination of virus and infected host cells - Failure to resolve both aspects may lead to chronic infection 4.) Intact natural barriers (skin, mucous membranes, gastric acids, fever etc.) are the first opportunity to prevent entry of the virus - Skin and mucous membranes are excellent barriers to infection - The immune system will be activated if the natural barriers are breached 5.) First line of defense: innate (nonspecific) immune system a. Involves cells and mechanisms such as inflammation b. Nonspecific process which attempts to limit and control viral replication and spread 6.) Next line of defense: adaptive or specific immune system - Attempts to resolve the infection by eliminating all infectious virus and virus-infected cells - The two arms of the specific immune response include: a. Humoral immunity: production of specific antibodies - Antibodies are effective against the extracellular virus and are essential to control virus spread to target organs 1.) Neutralizing antibodies: generated toward viral attachment proteins that interact with host cell surface receptors 2.) IgM antibodies: first produced but usually transient (temporary) - Typically drops below detectable limits within 3 to 6 months - Primary and Secondary Immune Responses - Usually present during the acute stage of infection - IgM is an indicator of recent or current infection 3.) IgG antibodies: appear later in infection but are typically produced indefinitely - Indicates an infection sometime in the past b. Cell-mediated immunity: involves T cells that can recognize and destroy viral-infected cells through the activation of cytotoxic T cells c. Disease resolution may occur when specific antibody and cell-mediated immune mechanisms halt continued replication of virus - Resolution requires elimination of free virus and virus-producing cells d. The immune response may be source of pathogenesis for many viral diseases 1.) Antibody interacting with large amounts of viral antigen can lead to immune complex disease 2.) Tissue damage may result from cell-mediated and inflammatory responses 3.) Aggressive NK-cell and T-cell responses in adults may exacerbate diseases benign in children 4.) Viral infections may provide initial activation trigger that allows immune system to respond to self-antigens e. Viruses have developed mechanisms to evade the protective immune response

Papillomaviruses (DNA Virus)

General Properties 1.) Family: Papillomaviridae 2.) Icosahedral 3.) Nonenveloped 4.) Double-stranded circular DNA 5.) The HPV genome has 7 early genes (E1 to E7) a. E2 is an important regulator of gene expression b. E6 and E7 encode proteins that inactivate proteins from tumor suppressor genes - p53 - Retinoblastoma (RB) gene **Important step in process by which normal cell becomes cancer cell 6.) Genome has 2 late genes (L1 and L2) a. Encode for structural proteins 7.) Can cause lytic, chronic, latent and transforming infections

Caliciviruses (RNA Virus)

General Properties: 1.) Family: Caliciviridae 2.) Genus: Norovirus (NoV) a.Species: Norwalk virus 3.) Nonenveloped virus 4.) Icosahedral structure 5.) Positive sense single-stranded RNA genome 6.) Distinctive spikes and cup-like depressions on the virus surface Overview of Diseases: 1.) Norovirus is one of the most common causes of viral gastroenteritis in adults. - Responsible for more than 80% of nonbacterial acute gastroenteritis - Cause of more than 50% of food borne outbreaks 2.) Also now the most common cause of viral gastroenteritis in children, since the vaccine has reduced the incidence of gastroenteritis due to rotavirus infection

Hepeviruses Transmission & Epidemiology

Transmission 1.) Fecal-oral route through contaminated water 2.) May occur following consumption of uncooked shellfish (mollusks) Epidemiology: 1.) Believed to be uncommon in US 2.) Usually the result of travel to developing country where HEV is endemic 3.) HEV common in developing countries with inadequate water supply and sanitation - Also reported with overcrowded temporary housing after natural disasters - Epidemics reported in Asia, the Middle East, Africa and Central America

EBV Transmission & Epidemiology

Transmission a. Primarily through exchange of saliva b. Saliva from patients with reactivation as well as active infection may be source of virus - Shedding can occur from oropharynx for several years after clinical recovery from acute infection Epidemiology a. One of the most common infections worldwide - Up to 90% of adults in US have antibody b. Infections in childhood usually asymptomatic - Generally seen in children under the age of 6 - In developing countries over 90% of infection occurs by the age of 2 c. Clinically apparent infectious mononucleosis: exposed later in life (young adults ages 15 to 24)

VZV Treatment and Prevention

Treatment: a. No antiviral therapy needed for chickenpox in immunocompetent children b. Immunocompromised children and adults should be treated with acyclovir Prevention: - Live, attenuated VZV vaccines: 1.) Varivax vaccine: designed to prevent varicella 2.) Shingrix vaccine: designed to prevent zoster a. Recommended for individuals over 50 who have had varicella b. More than 90% effective at preventing shingles c. Does not eradicate latent state of VZV

CMV Laboratory Diagnosis

a. Isolation of CMV from saliva or urine confirms congenital infection b. Preferred culture technique: shell vials with an immunofluorescent antibody, can make diagnosis in 24 hours c. Histological staining of giant (cytomegalic) cells in urine and tissue: - Cells demonstrate large intranuclear inclusion bodies with characteristic owl's eye appearance d. PCR-based assays for CMV DNA in blood, tissue, body fluids e. CMV antigenemia: detecting the pp65 antigen within white blood cells f. IgM antibody is useful acute CMV infection, as well as congenital infection g. A four-fold or greater rise in CMV IgG antibody titer can be used diagnostically

Indirect ELISA

a. Patient antibody (primary antibody) attaches to viral antigen in the well of a microtiter plate. b. Enzyme-labeled antibody (secondary antibody) detects the patient's antibody attached in the first step. c. Enzyme attached to the detecting antibody converts the colorless substrate to a colored product, which can be detected with a spectrophotometer. d. Color development indicates viral-specific antibodies in patient's specimen.

Your summer research project is to study viruses that cause upper respiratory tract infections. You have isolated a virus from a patient's throat and find that its genome is single-stranded RNA. You also find that the genome is the complement of the viral mRNA within the infected cell. Of the following, which is the most appropriate conclusion you could draw?

a. The RNA genome is segmented. b. The virion has a lipoprotein envelope. c. The RNA genome is circular. d. The virion contains an RNA polymerase. answer: The virion contains an RNA polymerase.

Hemagglutination Inhibition

a. Viruses such as influenza have the envelope protein hemagglutinin (HA) which binds to the surface of red blood cells causing them to agglutinate (hemagglutination). b. Antibodies to the virus neutralize the virus, preventing the attachment of the virus to the red blood cells. c. Therefore, hemagglutination is inhibited when antibodies are present.

HSV Transmission

***Both types of HSV can cause oral or genital lesions, but classic associations are: a. HSV-1 is usually spread by oral contact in saliva - HSV-1 infections occur mainly on the face b. HSV-2 primarily by sexual contact - HSV-2 infections occur in genital area - Also spread from infected mother to infant at birth due to genital lesions c. Alternate transmission in at least 20% of cases - HSV-1 infection in genital area and HSV-2 lesions in oral cavity d. Transmission most likely when active lesions are present e. Infected person is lifelong source of contagion - Asymptomatic shedding role in transmission

CMV Immunity

- Host defenses: includes both antibodies and cell-mediated immunity 1.) Cell-mediated immunity is essential for controlling CMV infection and for maintaining latency 2.) The role of antibodies in infection is limited

EBV Clinical Findings

- Many primary EBV infections are asymptomatic. - However, infections in the second decade of life often lead to clinically apparent Infectious Mononucleosis A. Infectious Mononucleosis: 1.) Characterized by: a. Fever b. Pharyngitis c. Lymphadenopathy - Major complaint: fatigue 2.) May also see: a. Hepatomegaly b. Splenomegaly 3.) Spontaneous recovery usually in 2 to 3 weeks B. Epstein-Barr Virus-Induced Lymphoproliferative Disorders: - If the initial EBV-induced proliferation of B cells is not contained, an EBV lymphoproliferative disease ensues. - Greatest risk in patients with primary EBV infection rather than reactivation - Seen with lacking or deficient T-cell immunity: B cell proliferation not contained 1.) Congenital deficiencies of T-cell function a. X-linked lymphoproliferative syndrome (Duncan's syndrome) - Severe often fatal EBV infection - Occurs in young boys - Inherited immunodeficiency (defective T cell responses) - Displacement of normal bone marrow precursor cells with EBV infected cells - Hemorrhage or secondary opportunistic infections - Mortality rate up to 75% by age 10 2.) EBV associated with several malignancies: - EBV initiating event that causes cells to divide abnormally - Requires additional steps for malignant transformation to occur a. Endemic Burkitt's Lymphoma - Initial evidence of association was production of EBV by lymphoma cells in culture - Seen primarily in children (3 to 14 years) in central Africa 3.) Malignancy of jaw 4.) Reciprocal translocation of c-myc oncogene from chromosome 8 to a site adjacent to a gene promoter on chromosome 14 - Enhances synthesis of c-myc protein, a potent oncoprotein 5.) Found in regions where malaria is also present - May be cofactor as it may reduce cell-mediated immunity b. Nasopharyngeal carcinoma 1.) Malignancy of squamous epithelial cells in nasopharynx 2.) Primarily affects males in southern China 3.) EBV DNA found in malignant cells c. Some forms of Hodgkin's lymphoma d. Lymphomas may occur following bone marrow and organ transplants 1.) Post-transplant lymphoproliferative disorder (PTLD) 2.) Main predisposing factor is immunosuppression 3.) Lymphoma regresses if immunosuppression is reduced 3.) May be seen in patients with acquired immunodeficiency syndrome (AIDS) - 10% AIDS patients develop B cell lymphomas (CNS) 4.) Individuals with lymphoproliferative disorders are at risk for productive EBV infections such as hairy leukoplakia - Whitish, nonmalignant lesion with "hairy" surface on side of tongue - Occurs in immunocompromised individuals, especially AIDS patients

Summary of Replicative Process

- Replicates best in epithelial cells 1.) Attachment to cell surface through fiber protein 2.) Genome moves to nucleus 3.) Transcription/translation of early (nonstructural) genes 4.) Viral DNA replication 5.) Transcription/translation of late (structural) genes 6.) Released by lysis

Nucleic Acid Sequencing

- Sequencing provides information about the precise sequence of nucleotides in the viral nucleic acid. - The sequence of viral nucleic acid can be used to characterize the particular strain infecting a patient. - Sequencing can be used to predict drug resistance for certain viruses.

Indirect Examination: Serologic Procedures

- The humoral immune response, which results in the production of antibodies, can provide a history of a patient's infections. - Serologic testing for specific antibodies in a patient's serum can be used to identify a virus responsible for an infection (including its strain or serotype), whether the infection is acute or chronic and determine whether it is a primary infection or a reinfection. a. Diagnosis of infection using serologic methods is based on detection of specific antibodies to viral antigens produced due to the host's immune response to the virus i. The first antibodies to be detected are directed against antigens expressed on the surface of the virus. ii. Later in the infection, antibodies directed against the intracellular viral proteins and enzymes may be detected. b. A significant rise in titer of an IgG class antibody to the virus can be used to diagnose current or recent infection. i. May observe seroconversion, in which antibody is detected when the patient previously had no antibody. ii. A change in (or the appearance of) antibodies can be demonstrated when testing acute-phase serum specimen collected when symptoms are present (acute specimen) and a convalescent-phase specimen collected 10 to 14 days later. - A fourfold increase in the antibody titer between acute and convalescent sera is required to confirm recent infection. c. IgM antibody can diagnose an acute primary infection, as it is the first antibody produced after exposure to the virus. - Indirect immunoassays use viral antigens in the test system to detect the presence of antibodies in the patient's specimen that were produced on exposure to the virus.

CMV Transmission & Epidemiology

- Transmitted by direct contact with blood, body fluids and tissues a. Transmitted across placenta, in the birth canal and through breast milk to infants b. In young children, most common mode of transmission is through saliva - Tends to be transmitted between children in daycare facilities, etc. c. Later in life it is transmitted sexually Epidemiology: a. CMV infection occurs worldwide b. Up to 80% of adults have antibodies to this virus

Coronaviruses Epidemiology

1.) A CoV infection causing cold symptoms occurs worldwide. - Infection typically occurs in childhood, as evidenced by finding antibodies - Outbreaks occur primarily in the winter on a 2 to 3 year cycle. 2.) SARS-CoV originated in Guangdong province in China in November, 2002 a. 8,098 cases with 774 deaths b.Human-to-human transmission c. Respiratory disease of zoonotic origin - The virus apparently jumped to man from animals (masked-palm civets present in live animal markets in China - more than 80% showed evidence of coronavirus infection) d. Horseshoe bat natural reservoir for SARS-CoV, with masked palm civet intermediate host 3.) MERS-CoV originated in Saudi Arabia and neighboring countries in 2012 and 2013 a. Over 1,600 cases with fatality rate up to 50% of those identified as infected with MERS b. Causative agent MERS-CoV (HCoV-EMC) c. Respiratory disease of zoonotic origin d. Virus appears to have originated in bats e. Dromedary camels appear to be reservoir, and may be associated with transmission to humans f. The risk of person-to-person transmission was thought to be low 4.) SARS-CoV-2: causative agent of coronavirus disease 2019 (COVID-19) - Closely related to the original SARS-CoV - It is 96% identical at the whole genome level to other bat coronavirus samples a. As of 2/14/21: - The disease has spread worldwide and is classified as a pandemic - Global Statistics: 108,630,991 confirmed cases 2,395,619 deaths - US Statistics: 27,837,755 confirmed cases 488,364 deaths - Michigan Statistics: 628,956 confirmed cases 16,119 deaths b. It is thought to have an animal (zoonotic) origin - it is likely a single, recent emergence of this virus from an animal reservoir - Early on, many patients had some link to a large seafood and live animal markets in China, suggesting animal-to-person spread. c. Later, patients did not have exposure to animal markets, indicating person-to-person spread. d. The United States reported the first confirmed instance of person-to-person spread with this virus on January 30, 2020. - Most often, spread from person-to-person happens among close contacts (within 6 feet) - thought to occur mainly via respiratory droplets - During human-to-human transmission, an average of 1,000 infectious SARS-CoV-2 virions are thought to initiate a new infection e. Three known variants of COVID-19 are currently spreading among global populations: i. A UK Variant (referred to as B.1.1.7) - Modeling suggests variant transmits more easily than other strains - CDC Director stated that UK variant could be dominant strain in US by March ii. A variant discovered in South Africa (referred to as 1.351) iii. A variant discovered in Brazil (referred to as P.1)

Asymptomatic Infections (Clinical Presentation)

1.) A lack of symptoms may be associated with a viral infection if the extent of damage is below a functional threshold 2.) Despite the lack of symptoms, virus-specific antibodies are produced 3.) Asymptomatic infections can be a major source of contagion

Hepadnaviridae Laboratory Diagnosis

1.) Acute hepatitis B infection: a. Hepatitis B surface antigen (HBsAg): - Present in most patients during acute disease - Drops below detectable levels if acute HBV infection successfully resolved ***Anti-HBs will appear when acute HBV infection successfully resolves - IgM antibody to Hepatitis B core antigen (IgM anti-HBc) ***Appears early in HBV infection Note: HBcAg does NOT appear in the blood in any stage of the infection b. HBeAg is present during acute infection - Not as useful in the diagnosis of acute HBV as HBsAg and IgM anti-HBc. - Window phase (core window or serologic window): - Period in recovery of acute HBV infection where HBsAg has disappeared but anti-HBs is not yet detectable - Anti-HBc will be detectable during this period and can confirm diagnosis - May be only marker detected in core window 2.) Chronic hepatitis B infection: a. Prolonged presence of HBsAg for at least 6 months b. Antibody to HBV surface antigen (anti-HBs) is NOT detectable in the chronic HBV infection as the HBsAg continues to be detectable. c. Total antibody to HBV core antigen (anti-HBc) is present in chronic HBV. - The IgM form of anti-HBc (present during acute infection) will disappear approximately 6 months after infection. d. The presence of HBeAg in chronic active HBV indicates a high likelihood of transmissibility. - Antibody to HBeAg (anti-HBe) is not detectable when HBeAg is present. e. The absence of HBeAg in chronic HBV carriers with chronic persistent hepatitis indicates a low likelihood of transmissibility of HBV - Antibody to HBeAg (anti-HBe) is detectable when HBeAg disappears. 3.) The detection of viral DNA (viral load) by real-time PCR a. Monitor response to therapy, particularly in chronic HBV infections.

Transmission

1.) Aerosol droplet 2.) Fecal-oral transmission 3.) Direct inoculation (including fomites such as door knobs, bedding, utensils, etc.) 4.) Swimming pools with insufficient chlorine ***Epidemiology: 1.) Endemic worldwide

Hepadnaviridae Pathogenesis

1.) After entering the blood, the hepatitis B virus infects hepatocytes (liver cells) a. Viral antigens are displayed on the surface of the infected cells b. Cytotoxic T cells attack viral antigens on the infected hepatocytes. c. The pathogenesis of HBV infection is probably due to this cell-mediated immune response. d. Infants and children have immature cell-mediated immune response and are less able to resolve infection - Have milder symptoms and less tissue damage e. Antibodies to HBsAg can protect against infection of hepatocytes, indicating immunity. f. Immune (antigen-antibody) complexes cause some of the symptoms of acute and chronic hepatitis. 2.) Chronic HBV due to continuing infection of the hepatocytes. a. Patients with chronic HBV demonstrate the presence of HBsAg in the blood for 6 months or longer. * - About 5 % of adult patients with HBV infection have chronic HBV. - 90% of infected newborns are chronically infected with HBV. b. The determining factor of whether infection clears or becomes chronic infection is the adequacy of the cytotoxic T-cell response. - Newborns more likely to become chronically infected because immune system is less competent. - Chronic infection takes one of two forms based on the presence of the e antigen: c. Some patients with chronic HBV produce the e antigen. - High probability of producing infectious viral particles. - Indicator of transmissibility. - Classified as chronic active hepatitis d. Some chronic carriers do not produce the e antigen. - Low probability of producing infectious particles. - Less likely to transmit disease. - Classified as chronic persistent hepatitis. - These patients tend to be asymptomatic - Individuals with chronic persistent hepatitis are referred to as carriers 3.) High rate of hepatocellular carcinoma (HCC) occurs with chronic HBV. a. Almost all HCC cells have HBV DNA integrated into the cell DNA - Could be the result of insertional mutagenesis - Could activate cellular oncogene, leading to loss of growth control. - the HBx protein inactivates the p53 tumor suppressor protein.

Human Herpes Viruses by Subfamily Include:

1.) Alpha Herpesvirinae: - Infect epithelial cells primarily and cause latent infection in neurons a. Genus Simplexvirus - Herpes simplex virus type 1 (HSV-1) (Human Herpesvirus 1, or HHV-1) - Herpes simplex virus type 2 (HSV-2) (Human Herpesvirus 2, or HHV-2) b. Genus Varicellovirus - Varicella zoster virus (VZV or HHV-3)) - Varicella is primary infection and zoster is recurrent disease 2.) Beta Herpesvirinae: - Infect and become latent in a variety of tissues a. Genus Cytomegalovirus (CMV or HHV-5) 3.) Gamma Herpesvirinae a. Lymphocryptovirus (Epstein-Barr virus (EBV) or HHV- b. Rhadinovirus (Kaposi's sarcoma-associated herpesvirus (KSHV) or HHV-8): tumor of connective tissue.

Coronaviruses Pathogenesis

1.) Approximately 45% of infections are asymptomatic 2.) Viruses appear to attach to host cells using a viral surface glycoprotein spike that interacts with the angiotensin-converting enzyme 2 (ACE2) found on host cell membranes i. Enzyme is abundant in alveolar cells in lungs, causing respiratory infections - May result in acute respiratory distress syndrome (ARDS) ii. ACE2 is expressed on gastrointestinal cells, resulting in infections of gastrointestinal organs iii. Virus can cause acute myocardial injury related to ACE2 receptors in the heart - Blood vessel dysfunction and clot formation appear to play a significant role in mortality 3.) Patients with severe COVID-19 also have symptoms of hyperinflammation, indicative of cytokine release syndrome (CRS) which may be an underlying immunopathology

Coronaviruses Prevention

1.) COVID-19 Vaccines intended to provide immunity against SARS-CoV-2 include: a. mRNA Vaccines: - Uses viral mRNA to instruct host cells to produce part of the "spike" protein (S protein) unique to SARS-CoV-2 - Once displayed on the cell surface, the spike protein causes the immune system to produce antibodies - The Pfizer-BioNTech and the Moderna vaccines are mRNA vaccines b. Viral Vector Vaccines: - Uses an inactivated or attenuated live virus (for example non-replicating adenovirus) as the viral vector - Genetic material from SARS-CoV-2 is inserted into viral vector shell - Viral vector enters host cell and uses host cell macromolecule machinery to make SARS-CoV-2 proteins to stimulate the immune response c. Protein Subunit (Peptide) Vaccines: - Uses viral proteins which immune system recognizes as foreign to stimulate immune system to produce antibodies

Papillomaviruses Laboratory Diagnosis

1.) Digene Hybrid Capture 2 hybridization assay performed in a 96-well plate: 1. Release Nucleic Acids Clinical specimens are combined with an alkaline solution which releases the target DNA. 2. Hybridize the Target DNA with an RNA Probe Target DNA combines with specific RNA probes creating RNA:DNA hybrids. 3. Capture Hybrids RNA:DNA hybrids are captured in wells coated with monoclonal capture antibodies specific for RNA:DNA hybrids. 4. Signal Amplification Captured RNA:DNA hybrids are detected with a second monoclonal antibody conjugated to alkaline phosphatase. 5. Detection and Result Interpretation The bound alkaline phosphatase is detected with a chemiluminescent dioxetane substrate. Upon cleavage by alkaline phosphatase, the substrate produces light signal. 2.) Cervista Assay (Hologic): a. Cervista HPV assay uses Invader technology, a signal amplification method that utilizes isothermal reactions. b. The primary reaction demonstrates a fluorescent signal if the HPV target DNA sequence is present. c. A secondary fluorescent signal functions as an internal control to indicate that sufficient cellularity was present to correctly interpret negative results. d. The screening assay uses probes to cover all 14 HR HPV types, and uses a second assay with probes specific for HPV 16 and 18 for genotyping assay. 3.) Real-time polymerase chain reaction: a. Amplifies target DNA, generating millions of copies using repeated cycles of heating and cooling. Step One: Denaturation by Heat Step Two: Annealing - Binding to Primer Step Three:Extension b. Each cycle results in a doubling of the target DNA molecules and leads to the production of billions of copies of the DNA sequence. c. A DNA probe is a strand of DNA that can be labeled with fluorescent detector molecules. d. The probe hybridizes or binds to the target DNA molecule, and detection of the target nucleic acid can be recognized through the bound detector molecules. - Channel detection to measure different targets: • Channel 1 = hrHPV panel (12 of the 14 HR Types) • Channel 2 = HPV 16 genotyping • Channel 3 = HPV 18 genotyping • Channel 4 = beta-globin target e. Approved by FDA for first-line primary screening for cervical cancer in US 4.) The APTIMA HPV Assay - A nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 HR HPVs - HPV mRNA is amplified using Transcription Mediated Ampification or TMA. - First, a DNA copy of the target RNA is created using the enzyme reverse transcriptase. - Next, a primer binds to the DNA copy and a new strand DNA is synthesized, creating a double-stranded DNA molecule. - RNA polymerase produces multiple copies of RNA amplicons from the DNA template. These RNA amplicons reenter the TMA process, serving as templates for a new round of replication. - Detection of the RNA amplicons: single-stranded DNA probe with a chemiluminescent label that hybridizes specifically to the RNA amplicon. - Light is emitted from the labeled RNA-DNA hybrids.

Retroviruses Replicative Process

1.) Entry into the cell involves gp120 binding to CD4 protein on the surface of cells such as CD4+ T lymphocytes, dendritic cells and macrophages 2.) The gp120 glycoprotein then interacts with a chemokine receptor, forming a secondary attachment 3.) The gp41 glycoprotein mediates fusion of viral envelope with cell membrane 4). Virion core containing nucleocapsid, RNA genome, and reverse transcriptase enters the host cell cytoplasm 5.) Reverse transcriptase synthesizes dsDNA from genomic RNA template, which migrates to nucleus and integrates into host cell DNA, becoming a "provirus". - Integration is mediated by integrase 6.) Viral mRNA is transcribed from proviral DNA and translated into several large polyproteins 7.) The immature virion containing precursor polyproteins forms in the cytoplasm. 8.) Cleavage by the protease enzymes occur as the immature virion buds from the cell membrane. a. The Gag polyprotein is cleaved to form the main core protein (p24), the matrix protein (p17), and several smaller proteins. b. The Pol polyprotein is cleaved to form the reverse transcriptase, integrase, and protease. c. The gp160 polyprotein is cleaved to form the envelope glycoproteins gp120 and gp41. 9.) It is this cleavage process that results in the mature, infectious virion.

Flaviviruses Replicative Process

1.) Flaviviruses replicate in the cytoplasm. 2.) Positive-sense RNA genome can be directly translated into large polyproteins 3.) Polyproteins cleaved into functional proteins by virus-encoded proteases 4.) Flavivirus envelope (E) glycoprotein binds to host cell to initiate endocytosis - Antibodies to E glycoprotein may provide immunity 5.) Viral genome replication involves a double-stranded RNA intermediate synthesized from the single-stranded positive sense RNA genome

Varicella-Zoster Virus (VZV): Human herpesvirus 3 (HHV-3) (DNA Virus)

1.) General Properties: a. Structurally and morphologically similar to other herpesviruses b. Antigenically different from other herpesviruses c. Has a single serotype d. The same virus causes both varicella and zoster e. Humans are the natural host 2.) Overview of Diseases a. Varicella (chickenpox) is primary disease b. Zoster (shingles) is recurrent form - Occurs later in lie from reactivation of VZV

Retroviruses Epidemiology

1.) Global statistics: a. As of 2018, approximately 37.9 million people worldwide are living with HIV. b. Worldwide, there were about 1.7 million new cases of HIV in 2018. c. An estimated 35 million people have died from AIDS-related illnesses since the start of the epidemic. 2.) US statistics: a. At the end of 2018, there were approximately 1.2 million adults and adolescents with diagnosed HIV in the US b. Approximately 38,000 people in the US were diagnosed with an HIV infection in 2018. c. In 2018, there were 15,800 deaths among adults and adolescents with diagnosed HIV in the United States d. The risk of being infected after percutaneous exposure to HIV-infected blood is estimated to be about 0.3%.

Hepadnaviridae Replication Process

1.) HBsAg serves as viral attachment protein. 2.) Nucleocapsid delivers genome to the nucleus. 3.) DNA-dependent DNA polymerase synthesizes the missing portion of DNA, forming circular double-stranded DNA. 4.) Double-stranded DNA serves as template for mRNA synthesis, transcribed using cellular RNA polymerase. 5.) The positive sense RNA strand serves as the template for synthesis of a negative sense (antisense) strand of DNA. 6.) Viral RNA-dependent DNA polymerase reverse transcribes RNA into DNA. 7.) The negative sense DNA strand serves as the template for the synthesis of the positive sense DNA strand, creating the DNA genome for the viral progeny. 8.) Synthesis of the positive sense DNA strand stops when the genome and core are enveloped, resulting in a partially double-stranded circular DNA genome. 9.) Progeny HBV with HBsAg-containing envelope are released by exocytosis. 10.) Hepadnavirus: only virus that produces genomic DNA by reverse transcription using positive sense viral RNA as template.

Papillomaviruses Pathogenesis

1.) HPV initially infects the basal layer of the skin. 2.) In the basal layer, only E1 and E2 proteins are expressed. 3.) Basal stem cells maintain HPV genome indefinitely in a latent state - Can produce symptoms or disease if the immune system weakens 4.) As basal cells differentiate and progress to the surface, HPV DNA replicates and is transcribed. 5.) Active viral replication occurs in the superficial layers of the skin. - Late genes (L1 and L2) are expressed only in upper layers. 6.) Viral stimulation of cell growth changes the epidermal layers, causing them to thicken, resulting in wart or condyloma. 7.) Most warts are benign and don't progress to malignancy. 8. Process of malignant transformation: a. 14 specific high-risk HPV (HR HPV) types in genital tract have been associated with the development of cervical cancer - High Risk: 16,18,31,33,35,39,45,51,52,56,58,59,66,68 b. Infection with HR HPV types which persist for years is the primary risk factor for cervical cancer. c. Cervical cancer is a continuous disease process that involves 4 major steps: 1. HR HPV infection 2. Viral persistence 3. Progression from mild dysplasia to more severe dysplasia in cervical cells 4. Invasive cancer - Progression from HPV infection to invasive cancer may take 15 to 20 years d. Proteins encoded by viral genes E6 and E7 interfere with the p53 and RB tumor suppressor proteins. - With the circular viral genome, expression of the E2 gene normally downregulates E6 and E7 through stimulation of a control region (LCR) e. However, in invasive cancers associated with HR HPVs, the viral genome is integrated into the cellular DNA - Can be inserted anywhere in the chromosome f. Integration of linear viral genome usually occurs with a break in the E2 region. g. The E6 and E7 genes are released from the inhibitory effects of the E2 protein. h. E6 and E7 genes are expressed at high levels in invasive cancers. i. The two tumor suppressor proteins (p53 and retinoblastoma) normally control cell growth in an HPV-infected cell. j. If the two tumor suppressor genes are inactivated, cells demonstrate uncontrolled proliferation and may lead to malignancy and tumor development. k. The E6 HPV protein complexes with the p53 and targets it for destruction. - E7 binds to and inactivates retinoblastoma tumor suppressor protein l. The functional inactivation of the tumor suppressor proteins results in cell proliferation without allowing time for repair of DNA damage. - The cell is more susceptible to mutations, which results in an uncontrolled malignant transformation m. E6 and E7 also protect the infected cell from apoptosis (programmed cell death), which confers immortality. - the cells demonstrate increased genetic instability and more damaged DNA which cannot be repaired. n. Thus, infections with high-risk HPVs allow genetic alterations that underlie cervical cancer by causing transformation to malignant cells.

Hepadnaviridae Epidemiology

1.) Hepatitis B is found worldwide (endemic) 2.) Globally, estimated 400 million people are chronically infected with HBV. - 50 million new cases annually 3.) In the US, there are more than 1 million chronically infected with HBV - Each year, 200,000 people will become infected with HBV in the US. 4.) HBV is associated with the development of hepatocellular carcinoma (HCC). - Hepatitis B is a leading cause of liver cancer 5.) The HBV vaccine is first vaccine able to prevent the development of cancer.

Overview of Diseases from Herpesvirus

1.) Herpesviruses generally produce an initial productive infection - Viral replication with release of virions resulting in acute disease (symptoms) 2.) Cell-mediated immunity is important in controlling infection - May also contribute to symptoms 3.) Have the ability to cause latent (persistent) infection - Infection will be maintained for the life of the host - Latent period is asymptomatic and virus is dormant (no viral replication so no infectious viral particles being produced) 4.) Reactivation results in viral replication and disease can occur - With some herpesviruses (e.g. HSV), the symptoms of the subsequent episodes are similar to those of the initial infection - With other herpesviruses, (e.g. VZV), the symptoms of reactivation are different than the initial infection - Periodic reactivation provides a constant source of new infections in the population 5.) Both primary infection and reactivation are likely to be more serious in immunocompromised patients

Papillomaviruses Prevention

1.) High-grade lesions and cancer confined to cervix can be completely excised, so early identification of cervical disease is required for a favorable prognosis - Cure rate for high grade lesions: ~98% Cure rate for cervical cancer that hasn't spread: ~80% Localized spread (hysterectomy + lymph node removal) ~60% to 80% Spread to distant organs (>5 year survival) ~20% 2.) Cancer screening: - Co-testing with cytology and HPV for 30 to 65: - HPV infection detected in women over 30 is more likely to reflect persistent infection. - Frequency of screening changed to 5 years with normal or ASC-US Pap if HPV negative 3.) HPV vaccine: a. Gardasil 9: protects against HPV types 16 and 18, types 6 and 11 (the types responsible for 90% of genital warts), and 5 additional high-risk HPV types (31, 33, 45, 52 and 58). - CDC recommends routine vaccination of males and females starting at ages 11 or 12. - Vaccination series can be started at age 9.

Papillomaviruses Diseases

1.) Human papillomavirus (HPV) causes papillomas, benign tumors of squamous cells (warts). 2.) There are at least 111 types of human papillomaviruses: a. HPVs are epitheliotropic, with a tropism resulting in infections of the skin and mucous membranes: - Skin warts are caused primarily by cutaneous HPVs: HPV-1 through HPV-4 - Genital warts (condylomas) are caused by mucosal HPVs: especially HPV-6 and HPV-11 b. Approximately 40 types of HPV infect the genital tract (mucosal HPVs). c. Infections of the genital tract are largely asymptomatic d. At least 90% resolve completely in 1 to 2 years e. Genital HPV infections in women may display benign cervical cell changes. f. Mild dysplasias (abnormal cells) are associated with viral replication and shedding - Most lesions spontaneously regress, indicating self-limited HPV infection - Clearance of HPV precedes clearance of cervical lesions g. Time required to resolve low risk (LR) HPV infection is ~5 months. - LR HPVs have not been associated with the development of cervical cancer h. Some HPV types (especially 16 and 18), classified as high risk (HR) HPVs, are known to cause carcinoma of the cervix, penis and anus - For infections with HR HPV, time to resolve infection is ~14 months.

Coronaviruses Immunity

1.) Immune response to CoV-2 virus includes a combination of cell-mediated immunity and antibody production 2.) Since SARS-CoV-2 has only been in the human population since December, 2019, it remains unknown if immunity is long lasting in individuals recovering from disease 3.) The presence of neutralizing antibodies strongly correlates with protection from infection, but the level of neutralizing antibodies appears to decline with time - With asymptomatic patients, antibodies may be undetectable two months after infection - Studies indicate neutralizing antibodies demonstrated a 4-fold decrease 1 to 4 months after the onset of symptoms - Lack of detectable antibody does not mean that antibody won't be rapidly produced upon reexposure 4.) Cases of reinfection can occur but are not common - Herd immunity (a reduction in the risk of infection that can occur when a large percentage of the population is immune through vaccination or natural exposure) will not eliminate virus if reinfection is common

Coronaviruses Clinical Findings

1.) Infection may present as the common cold, characterized by rhinorrhea (runny nose), scratchy, sore throat and low-grade fever - Illness typically lasts several days 2.) The first SARS-CoV infection was a severe atypical pneumonia: a. Fever (at least 100°) b. Nonproductive cough c. Dyspnea (difficult or labored breathing) leading to acute respiratory distress syndrome d. Sore throat and rhinorrhea are uncommon e. Diffuse edema results in hypoxia (tissues are deprived of adequate oxygen) f. Up to 20% also develop diarrhea g. Mortality is at least 10% of symptomatic patients 3.) The MERS-CoV clinical findings were similar to SARS a. Also causes acute respiratory distress syndrome b. Up to 50% mortality of those identified as infected with MERS 4.) COVID-19 caused by SARS-CoV-2: - Reported illnesses have ranged from asymptomatic to severe illness, which may be associated with fatalities. - Symptoms, which can occur within 14 days of exposure, include: Mild symptoms (81%): Fever Cough Nasal congestion Breathing difficulties Loss of smell and taste - Severe symptoms (14%): Dyspnea Hypoxia, with more than 50% lung involvement - Critical symptoms (5%): Respiratory failure Shock Multiorgan dysfunction - Approximately one third of individuals infected with the virus remain asymptomatic, but can still spread the disease

Herpesvirus Replication

1.) Initiated by interaction of viral attachment proteins (glycoproteins on the surface of the envelope) with cell surface receptors 2.) Virus can fuse envelope with cell membrane 3.) Nucleocapsid delivers DNA genome to nucleus 4.) Immediate early genes are transcribed/translated: regulatory proteins 5.) Early genes are transcribed/translated: enzymes, including DNA polymerase 6.) DNA genome is replicated 7.) Late (structural) proteins are transcribed/translated - Capsid and envelope glycoproteins 8.) Virion assembles in nucleus - Nucleocapsid associates with nuclear membrane where viral glycoproteins have been inserted 9.) Virion obtains envelope as it buds through the nuclear membrane 10.) Exits cell by exocytosis (releases contents of vacuoles to exterior) or by cell lysis

Hepadnaviridae Immunity

1.) Lifelong immunity occurs after the resolution of the natural infection. a. Antibody to HBsAg (anti-HBs) is protective, neutralizing infectivity of HBV. - The HBsAg disappears as anti-HBs becomes detectable in serum b. Antibodies to HBcAg and HBeAg may be present in the serum but are non-protective. - The HBeAg disappears as anti-HBe becomes detectable in serum - Core antigen (HBeAg) does not disappear as never present in the blood

Hepadnaviridae Clinical Findings

1.) Many HBV infections are asymptomatic, especially in children 2.) Clinical manifestations of acute hepatitis B infection: a. Non-specific, mild, flu-like symptoms b. Jaundice c. Dark urine d. Pale feces e. Elevated liver transaminase enzymes f. Fatality rate up to 1% - Fatality most likely to occur if co-infected with hepatitis D virus 3.) Chronic hepatitis B: a. Most chronic carriers are asymptomatic (chronic persistent hepatitis) b. Some patients have chronic active hepatitis - Can see symptoms similar to acute stage - Can result in cirrhosis and death

Coronaviruses Lab Diagnosis

1.) Nucleic Acid Amplification Tests (NAATs): - Test for SARS-CoV-2 viral RNA: a. Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) b. Transcription-Mediated Amplification (TMA) c. Typically testing is done on respiratory specimens obtained using nasopharyngeal swabs - May also test sputum samples 2.) Viral Antigen Tests: a. Detects viral proteins but may not be as sensitive as NAATs - May use NAATs as confirmatory test 3.) Antibody or Serology Tests: a. Tests for antibodies may indicate a past infection with SARS-CoV-2 b. Should not be used to diagnose a current infection - May take 1 to 3 weeks after exposure to develop antibodies

Orthomyxoviruses Diseases

1.) Pandemics caused by influenza A virus occur infrequently, but major outbreaks due to influenza A can occur virtually every year. 2.) Influenza B virus may also result in major outbreaks, but it does not cause pandemics. 3.) Influenza C virus causes mild respiratory tract infections but does not cause pandemics or major outbreaks of influenza.

Flaviviruses Transmission

1.) Primarily parenteral (bloodborne) a. Injection drug use is a primary risk factor (current and former) - High prevalence in older individual due to sharing needles in 1970s and 1980s b. Body piercing and tattoos c. Chronic hemodialysis patients - Recipients of clotting factor concentrates made before 1987 2.) Perinatal transmission 3.) Transmission through sexual contact is possible but inefficient - High-risk sexual activity with multiple sexual partners 4.) Mechanism of transmission unknown for some infections

Hepeviruses Replicative Process

1.) Replication takes place in the cytoplasm 2.) Positive sense ssRNA strand functions as mRNA 3.) Translated into a polyprotein which yields mature viral proteins. 4.) A dsRNA intermediate is synthesized to create positive sense ssRNA genomes.

Laboratory Diagnosis

1.) Specimens a. Throat or nasopharyngeal swabs b. Bronchoalveolar lavage or sputum c. Stool specimens d. Conjunctival swabs and urine may also be appropriate 2.) Laboratory Testing a. Isolation of virus in cell culture - CPE usually apparent in 2 to 5 days - Grapelike clusters - Intranuclear inclusions b. Shell vial: less time than traditional cell culture c. Direct fluorescent antigen staining of nasopharyngeal aspirate cells - Dense intranuclear inclusion d. Real-Time PCR - Quantitative viral DNA testing (viral load) - Can detect all known serotypes Sequencing allows serotype identification - Can predict progression to disseminated disease in transplant patients - Can determine response to therapeutic interventions e. Serology testing - Indirect fluorescent antibody (IFA) - Enzyme immunoassay (EIA)

Orthomyxoviruses Epidemiology

1.) The ability of influenza A virus to cause pandemics is dependent on antigenic changes in the hemagglutinin and neuraminidase glycoproteins. a. Influenza A undergoes major antigenic shifts. - Genetic diversity that produces numerous strains of influenza A is fostered by the segmented genomic structure and ability to infect many species (zoonosis). 2.) Pandemics can occur when the antigenicity of the virus has changed sufficiently that preexisting immunity is no longer effective. 3.) Influenza B virus undergoes antigenic drift but not antigenic shift. 4.) Influenza occurs primarily in the winter months in the northern hemisphere. 5.) The morbidity of influenza in children younger than 2 is high, second only to the morbidity in the elderly.

Retroviruses Clinical Findings

1.) The clinical picture of HIV infection can be divided into three stages: a. An early, acute stage - Begins 2 to 4 weeks after infection - Mononucleosis-like symptoms - High-level viremia typically occurs, and virus is readily transmissible. - Typically resolves spontaneously in about 2 weeks. - Resolution of acute stage usually includes a decrease in viremia and a rise in CD8+ (cytotoxic) T cells directed against HIV. b. A middle, latent stage - In untreated patients, a long latent period (typically 7 to 11 years) ensues - The patient is asymptomatic during this period. - Although viremia is low or absent, HIV is produced but remains sequestered in the lymph nodes. - During this clinical latency, the virus itself is not latent. - A syndrome called AIDS-related complex (ARC) can occur. - Persistent fever, fatigue, weight loss, and lymphadenopathy. c. A late, immunodeficiency stage (AIDS) - Manifested by a decline in CD4 cells to below 200 cells / μL - Predisposes host to opportunistic infections and cancers such as Kaposi's sarcoma and lymphoma. - See an increase in the frequency and severity of opportunistic infections: - The two most characteristic manifestations of AIDS are Pneumocystis pneumonia and Kaposi's sarcoma. - May severe neurologic problems (e.g., dementia and neuropathy), caused by HIV infection or by opportunistic organisms.

Retroviruses Pathogenesis

1.) The initial infection occurs in dendritic cells and macrophages, and then the local CD4+ helper T cells become infected. 2.) HIV appears in blood about 7 days after infection. 3.) CD4+ cells decreases by 20% to 40% in early stage of infection - HIV-infected CD4+ cells are subject to attack by cytotoxic CD8 lymphocytes. 4.) Immune response reduces HIV levels in the blood - CD4+ count rebounds to 80% to 90% of original level 5.) Lymphoid tissue remains a site of ongoing HIV infection. - Latent infection as no HIV is detectable in the blood. 6.) The latent period can last for years. 7.) Late in disease, CD4+ levels decrease and cannot maintain antiviral action of CD8+ - HIV viremia increases, CD4+ levels drop and patient becomes immunodeficient

Retroviruses Immunity

1.) The main immune response to HIV infection is cytotoxic CD8+ lymphocytes. 2.) The ultimate failure of cytotoxic T cells results in the clinical picture of AIDS. 3.) Antibodies to various HIV proteins (p24, gp120, and gp41) are produced. - Typically appear 10 to 14 days after infection, and most patients will have seroconverted by 3 to 4 weeks after infection. 4.) HIV has several mechanisms to evade the immune system: a. Integration of viral DNA into host cell DNA b. A high rate of mutation of the env gene c. The ability of HIV to infect and kill CD4+ T cells enhances its capacity to avoid destruction by immune system.

Retroviruses Lab Diagnosis

1.) The p24 major core antigen is one of first markers detected in acute HIV infection. 2.) Detection of antibodies to HIV antigens (p24, gp41, gp120, etc.) using enzyme-linked immunosorbent assay (ELISA) test. a. During the first month after infection, antibody tests may be negative. b. Average time for seroconversion is 10 to 14 days; many will have seroconverted by 4 weeks. 3.) HIV antigen/antibody "Combo" test detects p24 antigen and antibodies to both HIV-1 and HIV-2. a. Useful for early infections because p24 antigen is typically detectable earlier in infection than antibody. 4.) RT-PCR can detect HIV DNA within infected cells. a. The amount of viral RNA guides treatment decisions and the prognosis. 5.) The number of CD4+ T cells is another measure to guide patient management a. A CD4 count of 200/μL or below makes the diagnosis of AIDS. - The frequency and severity of opportunistic infections increases when CD4 counts fall below 200/μL.

Orthomyxoviruses Replicative Process & Transmission

1.) The virus adsorbs to cell when viral attachment protein hemagglutinin interacts with sialic acid on the host cell surface. - The different hemagglutinins (H1, H2, etc.) bind to different sialic acid structures, which determines the species and tissue that can be infected 2.) The nucleocapsid enters the cytoplasm and migrates to the nucleus. 3.) The virion RNA polymerase transcribes eight genome segments into mRNAs. 4.) Most mRNAs move to cytoplasm, where they are translated into viral proteins. 5.) Some mRNAs remain in nucleus, and serve as template for synthesis of negative-strand RNA genomes for the progeny virions. 6.) The helical ribonucleoprotein assembles in cytoplasm and virion is released by budding from cell membrane at site where hemagglutinin and neuraminidase glycoproteins have been inserted. Transmission: 1.) The virus is transmitted by airborne respiratory droplets. - Low humidity and cool temperatures stabilize the virus, and close proximity during winter months promotes spread

Hepadnaviridae Transmission

1.) Three modes of transmission: a. Parenteral exposure to blood or blood products: - Intravenous drug users - Can be transmitted through tattooing and body piercing b. Sexual transmission - HBV is found in semen and vaginal fluids c. Perinatal transmission from mother to newborn - Transmission occurs during birth (perinatal) 2.) May be stable in drying blood or other body fluids for a brief period - More efficiently transmitted by intimate contact

VZV Transmission & Epidemiology

1.) Transmission a. Transmitted by respiratory droplets - Spread 1 to 2 days before rash until lesions have crusted over b. Direct contact with lesions - VZV in zoster (shingles) lesions can be transmitted by direct contact to susceptible individual causing varicella (chickenpox) 2.) Epidemiology a. Varicella is highly contagious disease typically seen in childhood - 90% of population have antibody by the age of 10 b. Occurs worldwide

Retroviruses Treatment

1.) Treatment has resulted in a reduction in mortality and improvement in quality of life of infected individuals. 2.) The two specific goals of treatment are: a. To restore immunologic function by increasing the CD4 count b. To reduce viral load 3.) Starting drug therapy immediately after diagnosis of infection helps achieve goals. 4.) No drug regimen results in "cure", but long-term suppression can be achieved. 5.) Treatment of HIV infection typically involves multiple antiretroviral drugs. 6.) Choice of drugs is complex, depends on several factors. a. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) - Do not have a 3′ hydroxyl group on the ribose ring and therefore are chain-terminating drugs. - Inhibit HIV replication by interfering with proviral DNA synthesis by reverse transcriptase. b. Nonnucleoside Reverse Transcriptase Inhibitors - Attach to binding site of reverse transcriptase and inhibit function c. Protease Inhibitors: - Protease inhibitors combined with nucleoside analogues are effective in inhibiting viral replication and increasing CD4 cell counts - Commonly used in HAART regimens. - With this regimen, the viral load drops below the level of detection, CD4 cell counts rise, and CD8 activity increases.

Clinical Findings

1.) Upper respiratory tract: a. Pharyngitis b. Nasal congestion c. Conjunctivitis: "pink eye" 2.) Lower respiratory tract a. Bronchitis b. Pneumonia - Mortality up to 10% in very young 3.) Gastroenteritis a. Nonbloody diarrhea Mainly seen in children under 2 years of age 4.) Hemorrhagic cystitis a. Hematuria (presence of blood in the urine) b. Dysuria (painful or difficult urination) 5.) Most adenovirus infections resolve spontaneously 6.) Approximately half of all adenovirus infections are asymptomatic 7.) May see severe disseminated disease in immunocompromised patients a. Bone marrow transplant patients: up to 30% b. AIDS patients: infections of the intestinal tract

Hepadnaviridae Prevention

1.) Vaccine Recombivax contains HBsAg - Seroconversion following vaccination is approximately 95% in healthy adults. 2.) Hepatitis B immune globulin contains a high dose of anti-HBs - Provide immediate protection to individuals exposed to HBsAg-positive blood 3.) Recommended to give both vaccine and HBV immune globulin to a newborn whose mother is HBsAg-positive.

Reoviruses Replicative Process

1.) Viral RNA-dependent RNA polymerase synthesizes mRNA from each of the 11 RNA genome segments, 2.) The mRNAs are translated into structural and nonstructural proteins. 3.) RNA polymerase synthesizes positive sense RNA strands used as the template for dsRNA genome replication. 4.) The virus is released from the cytoplasm by cell lysis.

Coronaviruses Replicative Process & Transmission

1.) Virus adsorbs to cells via its surface spikes (hemagglutinin). 2.) Enters the cytoplasm, where replication occurs. 3.) The positive sense RNA genome functions as mRNA, translated into two large polypeptides, cleaved by virus-encoded protease enzyme. 4.) Viral proteins include the RNA-dependent RNA polymerase that replicates the genome. 5.) The mRNAs are translated into the structural proteins. 6.) The virus is assembled and obtains envelope from the endoplasmic reticulum, not the cell membrane. Transmission: An infected individual can transmit virus up to two days before they show symptoms. - Individuals may remain infectious for 7 to 12 days with a moderate case and up to 2 weeks with a severe case 1.) Respiratory aerosol spread from infected person's nose and mouth 2.) Direct contact has been suggested as a source of transmission by touching a contaminated surface, then touching mouth, nose or eyes - Respiratory droplets can land on surfaces - Not thought to be a common source of transmission

Flaviviruses Epidemiology

1.) Worldwide, it is estimated 180 million people are infected with HCV (approximately 3% of world population) 2.) Approximately 4 million people in US are chronically infected - High incidence of chronic asymptomatic infections promote spread of virus 3.) Genotype 1 causes approximately 75% of infections in US 4.) Baby boomers (born 1945 to 1965) considered to be at higher risk - Many infected in 1970s and 1980s - HCV was not discovered until 1989 - Donated blood not screened for HCV until 1992

CMV Clinical Findings

A. CMV can cause heterophile-negative mononucleosis, characterized by: 1.) Fever 2.) Lymphadenopathy 3.) Splenomegaly 4.) Presence of abnormal lymphocytes in peripheral blood smears B. In immunocompromised patients, systemic CMV infections common 1.) Renal and bone marrow transplant patients: a. Pneumonia b. Esophagitis c. Hepatitis 2.) Patients with AIDS: a. Colitis b. Encephalopathy c. Retinitis, which can lead to blindness C. CMV infection in neonates: 1.) Up to 90% of newborns asymptomatic at birth 2.) May demonstrate symptoms later (hearing loss and intellectual disabilities) 3.) Cytomegalic inclusion disease (CID): a serious congenital infection - Approximately 10% show manifestations: 1. Microcephaly 2. Sensorineural deafness 3. Chorioretinitis 4. Hepatosplenomegaly is common: may demonstrate jaundice 5. One of the leading causes of intellectual disability in the US - 10% of babies with CID will die 4.) Infected infants continue to excrete CMV in the urine

EBV Laboratory Diagnosis

A. Diagnosis of Infectious Mononucleosis 1.) Hematological testing: a. Lymphocytosis: increased lymphocytes in peripheral blood - Leukocytosis (increased WBCs) due to increase in lymphocytes b. Atypical lymphocytes: - Enlarged, with expanded nucleus and abundant cytoplasm - >10% atypical lymphocytes on peripheral blood smear - Cytotoxic T cells reacting to EBV-infected B cells 2.) Immunologic testing: a. Heterophile antibody test: - Production NOT stimulated by EBV antigens - IgM antibody that recognizes Paul-Bunnell antigen on erythrocytes - Screening tool for early diagnosis of infectious mononucleosis - Usually positive by week 2 of illness - Heterophile antibody titer declines after recovery -Usually drops below detectable limits in 3 to 6 months - Incidence of heterophile-negative infectious mononucleosis increases in children - 80% to 90% of adults have heterophile antibodies, so 10% to 20% are classified as heterophile negative - >50% under age of 6 are heterophile negative - >75% under age of 3 are heterophile negative b. EBV-specific antibody tests: - Used for cases in which heterophile antibody test is not helpful (negative) - Diagnosis of infectious mononucleosis indicated by: a. IgM VCA antibody identifies acute infection b. Presence of IgG VCA antibody - seroconversion - Significant (4-fold) increase in antibody titer c. Absence of EBNA antibody 3.) Prior EBV infection indicated by: a. Presence of IgG VCA antibody b. Presence of EBNA antibody 4.) Antibodies to early antigens (EA) may also be useful diagnostically B. Diagnosis of other EBV diseases (lymphoproliferative disorders): 1. PCR 2. Fluorescent antigen detection in clinical samples

HSV laboratory Diagnosis & Treatment

A. Direct analysis of clinical sample 1.) Tzanck smear - Presence of multinucleated cells (syncytia) in vesicles. 2.) Smear of infected cells. - Can demonstrate presence of viral antigen using immunofluorescence - Can also detect viral DNA using in situ hybridization or PCR B. Detection of virus in cell culture 1.) Typical CPE in 1 to 2 days - Intranuclear inclusion bodies - Enlarged and rounded cells - Syncytia 2.) Fluorescent antibody staining of infected cells - Type specific monoclonal antibodies can differentiate HSV-1 from HSV-2 C. Enzyme-Linked Immunosorbent Assay (ELISA) test using monoclonal antibody to glycoprotein G 1.) Can distinguish HSV-1 from HSV-2 D. Real-Time PCR 1.) More sensitive than viral culture. 2.) Can be used to identify serious disseminated infection in immunosuppressed patients and neonates. 3.) Detection of HSV DNA in cerebrospinal fluid can be used to diagnose herpes encephalitis. E. Serologic tests 1.) Can be used for diagnosis of primary infection. - Significant rise in antibody titer or seroconversion (absence to presence of antibody). - Should not be used for diagnosis of reactivation. 2.) Rarely see rise in antibody titer with recurrences. Treatment: a. Acyclovir: treatment of primary and recurrent genital herpes b. Treatment of primary infection has no effect on latent infection

EBV Immunity

A. Humoral immune response: - During acute EBV infection, antibodies develop as follows: 1.) IgM antibody to VCA first to be produced after exposure - Transient antibody b. Useful in diagnosing acute infection 2.) IgG antibody to VCA appears next a. Persists for life b. Best for revealing prior infection c. Can diagnose current infection with acute/convalescent paired specimens: - Seroconversion - Significant (4-fold) increase in antibody titer 3.) Antibody to EBNA appears during resolution of infection (convalescence) 4.) Lifetime immunity to second episode of infectious mononucleosis: - Based on production of neutralizing antibody to viral membrane 5.) Heterophile antibodies: - EBV overrides normal regulatory mechanisms in the B lymphocyte, so EBV-infected B lymphocytes are stimulated to produce polyclonal antibodies, including heterophile antibodies - Nonspecific antibody 1. IgM class 2. Do NOT react with any of the EBV antigens - Transient (temporary) antibody: usually disappear within 6 months - Agglutinate sheep or horse red blood cells B. Cell-mediated immune response 1.) B-cell infection is controlled by a normal T-cell response 2.) Activated T cells become cytotoxic T lymphocytes (CTLs) that react to and destroy infected B cells - Atypical lymphocytes in peripheral blood are CTLs 3.) Lifelong carrier state with dormant infection held in check by cell-mediated immune response - T cells are essential for limiting the proliferation of EBV-infected cells and controlling the disease

VZV Clinical Findings

A. Varicella (chickenpox): 1.) First macules: small, flat, discolored areas 2.) Become papules: small, rounded bumps with no visible fluid 3.) Evolves to vesicles (small blisters filled with clear fluid), pustules (bumps filled with pus), and finally crusts 4.) Itching (pruritis) is a prominent symptom 5.) Varicella is mild in children - More severe in adults B. Zoster (shingles): 1.) Painful vesicles along course of a sensory nerve - Dermatome: an area of skin supplied by single spinal nerve. - Rash results in stripe or belt-like pattern on one side of the body, does not cross midline. - ("Zoster" from Greek for "belt" or "girdle") 2.) Initial rash becomes vesicular, with small blisters filled with serous fluid. 3.) Skin heals, may see scarring 4.) Postzoster neuralgia (postherpetic neuralgia) - Chronic condition: nerve damage caused by herpes zoster. 5.) Immunocompromised patients: life-threatening disseminated infections.

Flaviviruses Clinical Findings & Lab Diagnosis

Clinical Findings 1.) Acute infection is most often asymptomatic (70% to 80%) 2.) If infection is symptomatic: Fever Abdominal pain Jaundice Dark urine Clay-colored stool 3.) Chronic infection: a. Chronic infection occurs in 80% b. Many chronic infections are asymptomatic - Predominant symptom, if present, is fatigue c. 20% of chronic infections result in cirrhosis d. 25% of cirrhosis patients result in liver failure e. Cirrhosis resulting from chronic infection is common cause for liver transplant f. Development of hepatocellular carcinoma due to HCV is seen in 5% of chronically infected after 30 years Laboratory Diagnosis 1.) Antibodies to HCV detected 6 to 24 weeks after exposure - Enzyme-linked immunosorbent assay (ELISA) - Chemiluminescent immunoassay (CIA) - If positive, RT-PCR is performed to determine if infection is active 2.) Viral load (HCV RNA in serum) is detectable ~2 weeks after exposure 3.) Liver enzymes (transaminases such as ALT) fluctuate

Caliciviruses Clinical Findings, Lab Diagnosis, Prevention

Clinical Findings: 1.) Characterized by: a. Sudden onset forceful vomiting and watery, non-bloody diarrhea b. Low-grade fever c. Abdominal cramping 2.) Illness lasts 2 to 3 days 3.) No long-term sequelae except in immunocompromised patients 4.) Some patients manifest signs of CNS involvement Laboratory Diagnosis: 1.) Collect stool during acute phase of illness 2.) RT-PCT most widely used Prevention: 1.) No vaccine available 2.) Oral rehydration or intravenous fluids if dehydration occurs 3.) Handwashing, proper sewage disposal and disinfection (freshly made bleach solutions or steam cleaning)

Orthomyxoviruses Clinical Findings, Lab Diagnosis, Prevention

Clinical Findings: 1.) Fever, myalgias, headache, sore throat, and cough develop suddenly. 2.) The symptoms usually resolve spontaneously in 4 to 7 days. 3.) Reye's syndrome, with encephalopathy and liver degeneration, is a rare, life-threatening complication in children that may occur with influenza B infections. Laboratory Diagnosis: 1.) Most diagnoses of influenza are made on clinical grounds. 2.) A laboratory test that may be used is an enzyme-linked immunosorbent assay (ELISA) for viral antigen in respiratory secretions. 3.) Influenza can also be diagnosed by the detection of antibodies: - The hemagglutination inhibition test, which can be used to determine influenza antibody titer, is based on the ability of hemagglutinin in the influenza viral envelope to spontaneously agglutinate red blood cells (hemagglutination). - Hemagglutination Inhibition Results - Patient antibody will bind to the viral hemagglutinin antigen, preventing the viral particles from spontaneously agglutinating red blood cells (hemagglutination inhibition). - The antibody titer is defined as the reciprocal of the highest dilution of the patient's serum that still demonstrates the ability to prevent (inhibit) the viral particles from spontaneously agglutinating red blood cells. - If the highest serum dilution that still inhibits the red blood cell agglutination is 1:160 (or 1/160) as in the diagram to the right, the antibody titer is expressed as 160. - Seroconversion or a rise in antibody titer of at least fourfold in paired serum samples taken early in the illness and 2 weeks later is sufficient for diagnosis. Prevention: 1.) The flu vaccine contains two A strains and two B strains. The vaccine is usually reformulated each year to contain the current antigenic strains. 2.) The vaccine is a killed vaccine containing purified protein subunits of the virus (hemagglutinin and neuraminidase).

Reoviruses Clinical Findings, Lab Diagnosis, Prevention

Clinical Findings: 1.) Infections characterized by: a. Nausea b. Vomiting c. Watery, nonbloody diarrhea 2.) Gastroenteritis is most severe in young children. - May require hospitalization for dehydration 3.) Adults usually have minor symptoms and may be asymptomatic Laboratory Diagnosis 1.) Rotavirus detection in fecal samples: a. Enzyme-linked immunosorbent assay (ELISA) for antigen detection b. Latex agglutination c. RT-PCR Prevention 1.) Live attenuated virus (Rotarix): contains most common type (Rotavirus A) 2.) Live reassortant vaccine (RotaTeq): contains 5 rotavirus strains

Picornaviruses Clinical Findings, Lab Diagnosis, Prevention

Clinical Findings: 1.) Most HAV infections are asymptomatic and detected only by presence of antibody. 2.) Symptomatic infection is similar to other viral hepatitis infections: Fever Jaundice Dark urine Clay-colored stool Elevated liver enzymes (transaminases such as ALT) 3.) Most cases resolve spontaneously in 2 to 4 weeks. 4.) No chronic hepatitis or chronic carrier state occurs. 5.) No predisposition to the development of hepatocellular carcinoma Laboratory Diagnosis 1.) Virus isolation is not performed as it is difficult to grow in culture 2.) The detection of IgM antibody confirms diagnosis of acute HAV infection a. IgM is transient antibody that drops below detectable limits at 3 to 6 months 3.) Fourfold rise in IgG antibody titer (or seroconversion) can diagnose acute infection Prevention 1.) Active immunization with vaccine containing inactivated HAV recommended for travelers to developing countries and children ages 2 to 18 years.

Retroviruses Diseases & Transmission

Diseases: 1.) HIV is cause of acquired immunodeficiency syndrome (AIDS). 2.) Both HIV-1 and HIV-2 cause AIDS, but HIV-1 is found worldwide, whereas HIV-2 is found primarily in West Africa. Transmission: 1.) Infection occurs through HIV-infected cells or free HIV 2.) Transmission can occur by contact with infected body fluids: a. Sexual contact b. Transfer of infected blood, (shared needles with intravenous drug use) c. Transmission from infected mother to neonate, across placenta, at birth, or via breast milk. 3.) Although small amounts of HIV may be found in other fluids (e.g., saliva and tears), there is no evidence they play a role in infection. 4.) There is no evidence for airborne, waterborne, or insect transmission of HIV.

Epstein-Barr Virus (EBV): Human herpesvirus 4 (HHV-4): Lymphocryptovirus (DNA Virus)

General Properties a. Gamma Herpesvirinae b. Structurally and morphologically similar to other herpesviruses c. Antigenically different from other herpesviruses d. Humans are natural host e. EBV infects mainly lymphoid cells, primarily B lymphocytes - Can infect epithelial cells in nasopharynx - EBV has tropism for both human B lymphocytes and epithelial cells f. Important antigens that can be used diagnostically: 1. Viral capsid antigen (VCA) 2. Early antigens (EA) 3. Epstein-Barr nuclear antigen (EBNA) Overview of Diseases a. EBV enters the B lymphocytes using receptor for complement b. Can replicate in B lymphocytes and epithelial cells, producing viral particles c. Associated with immortalization and proliferation of the B cell d. Can cause latent infection of B lymphocytes

Hepadnaviridae (DNA Virus)

General Properties ***Baltimore Class VII 1.) Primary member is Hepatitis B Virus (HBV) 2.) Icosahedral symmetry 3.) Enveloped 4.) Partially double-stranded circular DNA genome 5.) Viral core contains RNA-dependent DNA polymerase: - Virus provides this enzyme to reverse transcribe RNA into DNA as part of viral genome replication 6.) Antigens present in viral core: a. Hepatitis B core antigen (HBcAg) - Located on the nucleocapsid protein that forms the virion core - Only present in infected hepatocytes (liver cells) - NOT present in blood during acute or chronic infection b. Hepatitis B endogenous antigen (HBeAg) - Truncated version of HBcAg - Soluble antigen released from infected cells into the blood - Not capable of self-assembly into the capsid - important indicator of transmissibility 7.) Antigen in envelope: a. Hepatitis B surface antigen (HBsAg) 8.)Three types of particles present during infection: a. Virions: 42 nm b. Spheres: 22 nm c. Long filaments: 22 nm wide d. Spheres and filaments composed of surface antigen (HBsAg) - Contain no viral DNA, so are not infectious Disease: - Virus causes hepatitis, which is inflammation of the liver

Coronaviruses (RNA Virus)

General Properties: 1.) Family: Coronaviridae - features prominent club-shaped spikes - Named for solar corona-like (halo) appearance due to club-shaped surface projections when viewed with an electron microscope 2.) Genus: Coronavirus (CoV) 3.) Enveloped - Unlike most enveloped viruses, the "corona" formed by the glycoproteins may allow the virus to endure conditions in the gastrointestinal tract 4.) Helical nucleocapsid 5.) Positive sense nonsegmented single-stranded RNA genome Overview of Diseases: 1.) Coronaviruses are the second most prevalent cause of the common cold (rhinoviruses are the first) - Most coronaviruses cannot replicate at body temperature: infection is therefore restricted to upper respiratory tract, causing cold symptoms - Optimum temperature for viral growth for this group is 33°C to 35°C 2.) Coronaviruses are common in many different species of animals, including bats - Outbreaks of diseases have occurred when an animal reservoir has come in contact with man 3.) SARS-CoV and MERS-CoV are zoonoses: a disease of animals that can be transmissible to humans - Additionally, they may be able to spread from person to person a. In 2002, a new atypical pneumonia called severe acute respiratory syndrome (SARS) emerged. b. In 2012, another severe pneumonia called Middle East respiratory syndrome (MERS) appeared. c. In December, 2019, a new severe acute respiratory syndrome (SARS) caused by a coronavirus named "SARS-CoV-2" emerged in Wuhan City, China. - MERS and SARS coronaviruses can replicate at 37°C: in addition to causing severe pneumonias, they can also be associated with other systemic diseases - Coronaviruses have been linked to gastroenteritis with diarrhea

Hepeviruses (RNA Virus)

General Properties: 1.) Family: Hepeviridae - Species Hepatitis E Virus (HEV) 2.) Icosahedral 3.) Nonenveloped 4.) Genome is positive-sense, single stranded non-segmented RNA 5.) Four genotypes in the following regions: 1: Asia and Africa 2. West Africa and Mexico 3. Europe and North America 4. Asia (China, Taiwan and Japan) Overview of Diseases: 1.) Usually a self-limited, acute illness

Orthomyxoviruses (RNA Virus)

General Properties: 1.) Family: Orthomyxoviridae 2.) Three types can cause disease in humans: A, B and C 3.) Single-stranded negative sense segmented RNA genome 4.) Helical capsid 5.) Enveloped 6.) Virion contains RNA-dependent RNA polymerase 7.) Spikes on the envelope surface contain the viral glycoproteins hemagglutinin and neuraminidase a. Hemagglutinin (HA) has several functions: - Serves as viral attachment protein that binds to sialic acid on host cell surface to initiate infection of cell - Hemagglutinates red blood cells - Stimulates a protective neutralizing antibody response - There are 16 types of hemagglutinin, designated as H1, H2..... H16 b. Neuraminidase (NA) functions include: - Enzyme cleaves sialic acid in the host cell membrane to release progeny virus from the infected cell. - Neuraminidase also degrades mucus in the respiratory tract. - Antibodies to HA can also provide protection - There are 9 types of neuraminidase, designated as N1, N2..... N9 c. Influenza viruses, especially influenza A, show changes in antigenicity of hemagglutinin and neuraminidase proteins. d. There are two types of antigenic changes which can result in the development of new strains of influenza: - Antigenic shift is a major change based on reassortment of genomic RNA - In reassortment, entire segments of RNA are exchanged, and each codes for a single protein. A new subtype is created with a mixture of surface antigens from the original strains. ***Antigenic shifts only occur with influenza A - Antigenic drift is a minor change based on mutations in the genomic RNA - Antigenic drifts can be seen with influenza A, B and C 8.) Many species (humans, aquatic birds, chickens, swine, etc.) can serve as hosts for influenza A viruses (zoonosis). - Viruses from the various hosts are the source of RNA segments that encode antigenic shift variants that can cause influenza A pandemics among humans. - Hybrid viruses are created by coinfection of a host cell with different strains of influenza A virus, allowing the genomic segments to randomly associate into new virions which can infect an immunologically naïve human population. 9.) The outbreak of human influenza in 2009 was caused by H1N1 influenza A virus of swine origin (swine-origin influenza virus, S-OIV), which WHO declared as a level 6 pandemic. - S-OIV was a quadruple reassortant: genes of North American swine origin, Eurasian swine origin, North American avian origin and human H3N2 origin. - The disease affected primarily young people as individuals over the age of 60 were likely to have had protective antibodies from the H1N1 pandemic in 1977. 10.) Because influenza B virus is only a human virus, there is no animal source of new RNA segments. Influenza B virus therefore does not undergo antigenic shifts. - It does undergo antigenic drift. - Antigenic drift occurs with influenza A, B and C and drift variants occur virtually every year

Picornaviruses (RNA Virus)

General Properties: 1.) Family: Picornaviridae 2.) Genus: Hepatovirus 3.) Species: Hepatitis A Virus (HAV) 4.) Nonenveloped virus 5.) Icosahedral structure 6.) Positive sense single-stranded RNA genome Overview of HAV Disease: 1.) Causes acute viral hepatitis a. Childhood infections are usually asymptomatic b. Up to 50% of adult infections demonstrate symptoms, usually with jaundice 2.) Chronic infection does not occur and no carrier state develops

Retroviruses (RNA Viruses)

General Properties: 1.) Family: Retroviridae 2.) Genus: Lentivirinae 3.) Human immunodeficiency virus (HIV): important human T-cell lymphotropic retrovirus 4.) HIV preferentially infects and destroys helper CD4+ T lymphocytes, with deterioration of immune functions and development of opportunistic infections 5.) Bullet-shaped core surrounded by envelope with glycoproteins gp120 and gp41 6.) Two identical single-stranded, positive-sense RNA strands (diploid) a. Gag gene encodes "core" proteins, including p24 protein, the major core protein. - p24: group-specific antigen b. Pol gene encodes for three viral enzymes: 1. Reverse transcriptase: RNA-dependent DNA polymerase; Synthesizes DNA from genomic RNA, creating proviral DNA 2. Integrase mediates integration of proviral DNA into the host cell DNA 3. Protease cleaves precursor polyproteins into functional viral polypeptides c. Env gene encodes gp160, a precursor glycoprotein that is cleaved to form envelope glycoproteins gp120 and gp41 1. The glycoprotein gp120 is the viral attachment protein and interacts with the CD4 receptor. - gp120 gene mutates frequently, resulting in antigenic variants. - These differences subdivide HIV into subtypes called clades. - The B clade is the most common subtype in North America. 2. Glycoprotein gp41 mediates fusion of viral envelope with cell membrane. 7.) Natural host of HIV is limited to humans 8.) Human immunodeficiency virus type 2 (HIV-2): a. Proteins of HIV-2 about 40% identical to those of HIV-1 b. HIV-2 is much less easily transmitted than HIV-1

Cytomegalovirus (CMV): Human herpesvirus 5 (HHV-5) (DNA Virus)

General Properties: a. Beta Herpesvirinae b. Structurally and morphologically similar to other herpesviruses c. Antigenically different from other herpesviruses d. Has a single serotype e. Humans are natural host f. Giant cells are formed: basis of the name - Enlargement of a cell is cytomegaly Overview of Diseases: a. Epithelial cells, granulocytes and macrophages are among the cells that can be infected with CMV b. In immunocompetent individuals: 1.) Usually asymptomatic 2.) May see heterophile-negative mononucleosis - (Heterophile-positive infectious mononucleosis is due to EBV) c. In immunocompromised patients (transplant patients) 1.) Usually symptomatic 2.) May be life-threatening d. CMV infection in neonates - Clinical picture ranges from symptomatic to fatal infection e. Most common congenital infection in the US, affecting 1% of newborns f. Monocytes and lymphocytes may be the sites of latent infection

Reoviruses (RNA Viruses)

General Properties: - REO is an acronym for respiratory enteric orphan: when the virus was discovered, it was isolated from the respiratory and enteric tracts and was not associated with any disease 1.) Family: Reoviridae - Genus rotavirus is the most important pathogen 2.) Nonenveloped 3.) Icosahedral 4.) Possesses a triple-layered capsid 5.) Double-stranded segmented RNA genome - Genome consists of 11 segments (10 code for one protein each and 1 codes for two proteins) 6.) Virion contains an RNA-dependent RNA polymerase 7.) There are six viral proteins (VPs) that form the capsid layers and six nonstructural proteins (NSPs) 8.) 9 rotavirus species: A through I -Rotavirus A causes 90% of rotavirus infections in humans. Diseases: 1.) Rotavirus is a cause of viral gastroenteritis with diarrhea - Less commonly seen in young children due to vaccine

Human herpesvirus 8 (HHV-8): Kaposi's sarcoma-associated herpesvirus (KSHV) (DNA Virus)

General Properties: a. Structurally and morphologically similar to other herpesviruses. b. HHV-8 infection in general population is 3% in the US and 50% in Africa. Overview of Disease: - Etiologic agent of Kaposi's sarcoma, common malignancy in patients with AIDS Transmission: a. Primarily sexual transmission or through saliva b. May be transmitted in transplanted organs such as kidneys Pathogenesis: - KS in AIDS patients is a malignancy of vascular endothelial cells Clinical Findings -The lesions are reddish to dark purple, and flat to nodular. - They often appear at multiple sites - Internally, lesions occur in the gastrointestinal tract and the lungs. Laboratory Diagnosis: a. Biopsy of skin lesions b. PCR detects virus in tissue and other specimens

Papillomaviruses Immunity & Clinical Findings

Immunity: 1.) Both innate and cell-mediated immunity are important in control and resolution of HPV infections. 2.) Antibody to the L1 protein neutralizes the virus. 3.) Immunosuppressed patients have more severe papillomavirus infections. Clinical Findings: 1.) Papillomas of various organs 2.) Cervical, penile, vaginal, oropharyngeal and anal carcinomas are associated with infection with high-risk HPV (particularly HPV-16 and HPV-18). 3.)Cervical cancers include squamous cell carcinoma (SCC) and adenocarcinoma. - HPV 16 and 18 infections account for approximately 73% of cervical cancers

Hepeviruses Lab Diagnosis, Treatment, Prevention

Laboratory Diagnosis: 1.) Diagnosis confirmed by IgM antibody to HEV or HEV RNA 2.) Serologic tests typically limited to reference laboratories in the US 3.) Liver function tests: Elevated bilirubin, AST and ALT at disease onset 4.) HEV infection should be considered in individuals with: Symptoms of viral hepatitis Traveled to endemic areas Negative for markers of hepatitis A, B, C and other hepatropic viruses Treatment: 1.) Infections typically resolves on its own without treatment Prevention: 1.) No vaccine 2.) Prevention relies on good sanitation and availability of clean drinking water 3.) Boiling and chlorination of water will inactivate HEV

Picornaviruses Pathogenesis & Immunity

Pathogenesis 1.) The virus replicates in gastrointestinal tract and spreads to liver via the blood. 2.) It is likely that cytotoxic T cells cause damage to hepatocytes. 3.) Typically resolves without complications or permanent liver damage Immunity 1.) Immune response consists initially of IgM antibody, which is detectable when jaundice appears. 2.) Followed 1 to 3 weeks later by IgG antibody, which provides lifelong protection.

Hepeviruses Pathogenesis & Clinical Findings

Pathogenesis: 1.) Cases of HEV infection typically present in one of two ways: a. Large outbreaks and sporadic cases where HEV is endemic b. Isolated cases in developing countries (genotype 3) Clinical Findings 1.) Similar to other types of acute viral hepatitis: Fever Abdominal pain Jaundice Dark urine Clay-colored stool 2.) Symptomatic HEV in developing countries commonly seen ages 15 to 44 - Overall case fatality rate during HEV outbreaks is about 1% - Pregnant women more likely to have severe illness with fulminant hepatitis and death - Mortality in pregnant women up to 30% in third trimester of pregnancy 3.) Acute HEV infection in solid organ transplant recipients 4.) Chronic HEV very rare, and are seen exclusively in organ transplant patients - Chronic hepatitis and cirrhosis but not hepatocellular carcinoma

Flaviviruses Pathogenesis & Immunity

Pathogenesis: 1.) HCV infects hepatocytes, but no evidence of virus-induced cytopathic effect on liver cells 2.) Death of hepatocytes probably caused by cell-mediated immune response 3.) Infection strongly predisposes the patient to hepatocellular carcinoma - For every 100 individuals infected with HCV: - 75 to 85 develop chronic infection - 60 to 70 develop chronic liver disease - 5 to 20 will develop cirrhosis over 20 to 30 years - 1 to 5 will die of consequences of chronic infection (liver cancer or cirrhosis) 4.) Prior HCV infection does not protect against infection with same Immunity: 1.) Immune response develops, but viral replication in liver hepatocytes can result in changes that evade immune response 2.) Antibodies to HCV present in 60% by 8 weeks after exposure a. >97% demonstrate antibodies by 6 months after exposure b. Antibodies to HCV are not protective 3.) Vigorous cell-mediated response: a. May be responsible for death of hepatocytes

Reoviruses Pathogenesis & Immunity

Pathogenesis: 1.) Infects and damages enterocytes lining small intestine. 2.) Diarrhea due to impaired sodium and glucose absorption can cause severe dehydration, especially in young children Immunity 1.) Antibodies to the rotavirus VP4 and VP7 proteins can help neutralize viral infectivity

Orthomyxoviruses Pathogenesis & Immunity

Pathogenesis: 1.) Neuraminidase degrades the protective mucus layer, allowing the virus to gain access to the cells of the upper and lower respiratory tract. 2.) Influenza virus infection causes inflammation of the respiratory tract mucosa, which may develop into necrosis of the superficial layers of the respiratory epithelium. 3.) Despite systemic symptoms, viremia rarely occurs. 4.) The systemic symptoms are due to the release of cytokines during the infection which circulate in the blood. Immunity: 1.) Influenza viruses have both group-specific and type-specific antigens: a. The internal ribonucleoprotein is a group-specific antigen that distinguishes influenza A, B, and C viruses. b. Hemagglutinin (HA) and neuraminidase (NA) are the type-specific antigens located on the virus surface. c. Type-specific antibody to hemagglutinin neutralizes the infectivity of the virus, whereas antibody against the group-specific antigen does not. d. Antibody against neuraminidase does reduce disease by decreasing the amount of virus released from the infected cell. e. The type-specific antibodies to HA and NA will be specific for only the strain of influenza that stimulated their production - The individual will still be susceptible to other strains of influenza 2.) Cytotoxic T cells are important for control over the infection, but also contribute to the symptoms (immunopathogenesis).

Pathogenesis & Immunity

Pathogenesis: - Primarily infects children, and less commonly, adults 1.) Adenovirus infects mucosal epithelial cells causing acute infection 2.) Adenovirus in intestinal epithelium usually result in subclinical infections 3.) May see latent infection in adenoidal and tonsillar tissues - Viremia may occur with subsequent spread to visceral organs Immunity: 1.) Immunity results in neutralizing antibody directed at fiber protein a. Type-specific b. Lifelong protection to specific serotype associated with infection

Picornaviruses Transmission & Epidemiology

Transmission: 1.) Enteric (fecal-oral) transmission -Virus can be present in feces 2 weeks before the appearance of symptoms 2.) Outbreaks can arise from fecally contaminated water a. May be associated with shellfish consumption (oysters, mollusks, clams, etc.) grown in polluted water and eaten raw b. Sewage polluted water after flood c. Swimming in contaminated water 3.) Cooked foods can transmit HAV (outbreaks associated with infected food handlers) 4.) Close personal contact with infected household member or sex partner 5.) Travelers to endemic regions 6.) Virus appears in feces 2 weeks before the appearance of symptoms 7.) HAV is rarely transmitted via the blood Epidemiology: 1.) Hepatitis A rates have declined since HAV vaccine became available in 1995. 2.) Children between the ages of 5 and 14 have the highest rate of infection. 3.) About 50% in the US have been infected, as evidenced by presence of antibody.

Reoviruses Transmission & Epidemiology

Transmission: 1.) Fecal-oral route 2.) Contact with contaminated hands, surfaces and objects - Stable in environment, surviving up to 19 days 3.) Contaminated water and foods such as raw oysters, clams and mollusks Epidemiology: 1.) Prior to vaccine, responsible for 2.7 million cases of severe gastroenteritis in children - Vaccine has significantly reduced the incidence and severity of disease in children 2.) Asymptomatic infections are common in infants younger than 6 months 3.) Most children have been infected with at least one serotype by the age of 6.

Caliciviruses Transmission & Epidemiology

Transmission: 1.) Fecal-oral route, often involving ingestion of contaminated water or seafood (especially mollusks, clams, etc. eaten raw) - Outbreaks occur in group settings such as cruise ships - One of the causative agents of traveler's diarrhea Epidemiology 1.) Infection is enhanced by: a. Only a low infectious dose is needed b. Virus is excreted in the stool before the onset of symptoms and for several weeks after recovery. c. Virus resists inactivation by drying in the environment. Resistant to routine cleaning solutions Not inactivated easily by change in pH or heat 2.) Remains infectious for several days 3.) New strains appear every 2 to 4 years and cause widespread infections.

Papillomaviruses Transmission and Epidemiology

Transmission: 1.) Primarily by skin-to-skin contact 2.) Micro-abrasions in skin and mucous membranes allow access to basal layers where infection begins. Epidemiology 1.) Warts tend to be more common in children and young adults - Tend to regress in older adults 2.) Genital warts are among the most common sexually transmitted diseases. 3.) HPV may be the most prevalent sexually transmitted infection in the world 4.) HPV responsible for 10% of cancers worldwide 5.) HPV DNA due to a persistent infection is found in 99.7% of cervical cancer specimens 6.) Data from CDC estimates of additional HPV-associated cancers: a. 90% of anal squamous cell carcinomas b. 65% of vaginal cancers c. 50% of vulvar cancers d. 35% of penile cancers e. 60% of oropharyngeal cancers (95% associated with HPV-16 or HPV-18)

Indirect Immunofluorescence

a. Antibody present in the patient's serum due to exposure to the virus is allowed to attach to viral antigen fixed to the microscope slide. b. A second fluorescently-labeled reagent antibody is used to detect the primary (or patient) antibody attached in first step. c. The reaction is visualized using a fluorescent microscope

Neutralization Tests

a. Antibody that inhibits the infectivity of a virus by blocking the attachment to the host cell receptor is called a neutralizing antibody. b. The patient's serum is mixed with a suspension of infectious viral particles, which are then inoculated into a cell culture system. c. If the patient's serum contains a neutralizing antibody, the antibody binds to the viral particles, preventing them from invading the cell culture cells

HSV Epidemiology

a. Approximately 80% of population in US infected with HSV-1 by age 30 - Current statistics indicate that approximately 25% of sexually active adults in the US are infected with HSV-2 b. Most primary HSV-1 infections occur in childhood - HSV-2 infections usually don't appear until age of sexual activity

VZV Immunity

a. Cell-mediated immunity important in controlling infection - Contributes to symptoms b. Production of antibodies limits viremic spread c. Immunity following varicella is lifelong - Circulating antibody prevents reinfection d. Zoster can occur despite the immunity to varicella e. Frequency of zoster increases with increasing age

HSV Clinical Findings

a. HSV diseases (both oral and genital lesions) can be caused by either HSV-1 or HSV-2 b. Tissue damage caused by viral pathology and immunopathology 1.) Gingivostomatitis: - Inflammation of oral mucous membranes with painful ulcers of gums, etc. - Primarily seen in children 2.) Herpes labialis (fever blisters or cold sores) - Vesicular lesions at junction of lips or nose - Recurrences occur at same site 3.) Herpetic keratoconjunctivitis - Corneal ulcers, usually limited to one eye - Recurrences lead to scarring and blindness 4.) Genital herpes (herpes genitalis): - Painful vesicular lesion - Infections may be asymptomatic - Approximately 70% of cases caused by HSV-2 - Cases caused by HSV-1 becoming more common - Recurrent genital HSV less severe than primary - Genital HSV-2 disease is more likely to recur than genital HSV-1 infection 5.) Neonatal herpes - Primarily due to contact with lesions during birth - Varies in severity: - May see disseminated systemic infection or encephalitis - May see skin, eye, mouth lesions - May be asymptomatic infection - Both HSV-1 and HSV-2 can cause neonatal infections - Serious infection more likely as a result of maternal prImary infection than recurrent infection in mother - Mother previously infected can transmit IgG antibodies across placenta to help protect infant from serious disseminated infection - Cell-mediated immunity not well developed in infant, so severe consequences may be seen in neonate c. Some specific conditions are associated with only HSV-1 or HSV-2: 1.) HSV-1: - Has been associated with encephalitis (not linked to HSV-2) - Necrotic lesion in one temporal lobe - Seen with primary or recurrent oral infection - High mortality 2.) HSV-2 - Asymptomatic meningitis - May be a complication of genital HSV-2 infection - Usually mild, self-limited disease

HSV Pathogenesis

a. HSV replicates in skin or mucous membrane at site of infection - Typical skin lesion is vesicle filled with fluid b. Can induce syncytia (multinucleated giant cells) formation c. Infects local sensory neuron, then migrates up the neuron d. Becomes latent in sensory ganglion innervating the site of infection - Trigeminal ganglia (located in the head) for oral HSV - Sacral ganglia (located in the pelvic region) for genital HSV e. Reactivated by inducers (sunlight, hormonal changes, stress, etc.) - Migrates down the neuron - Returns to initial site of infection - Replicates and may cause lesions in skin near the site of the initial infection

Herpes Simplex Viruses (HSV)

a. Herpes simplex virus type 1 (HSV-1) b. Herpes simplex virus type 2 (HSV-2) c. The two types are structurally and morphologically indistinguishable d. Can be differentiated by laboratory testing e. Humans are the natural host of both types f. Classic location of lesion: i. HSV-1 lesions typically above the waist ii. HSV-2 lesions typically below the waist Although classic locations are as described above, both types have been isolated from both locations

EBV Pathogenesis

a. Infection first occurs in oropharynx, initially infecting epithelial cells b. Viremia spreads virus and provides opportunity to infect B lymphocytes - B lymphocytes in the lymphoid tissue of the oropharynx are infected and spread to the bloodstream c. Cytotoxic T lymphocytes (CTLs) react to and destroy infected B cells - Cytotoxic T cells (CTLs)are atypical lymphocytes seen on the peripheral blood smear - Control active infection d. EBV remains in latent state within B lymphocytes e. Lifelong carrier state held in check by cell-mediated immune response f. Diseases of EBV result from either an overactive immune response or the lack of effective immune control g. Children have a less active immune response and therefore have a very mild disease, often asymptomatic (symptoms result from healthy immune response)

CMV Pathogenesis

a. Infections are milder in immunocompetent individuals (many are asymptomatic) b. More severe symptomatic infections with immunocompromised patients c. Latent state develops in white blood cells - Individuals with latent infection can shed virus in low numbers for long periods d. Reactivation occurs when cell-mediated immunity is compromised - Reactivations may be asymptomatic unless patient in immunocompromised e. CMV infections in neonates: 1.) May occur as: - Congenital infection (acquired in utero) - May be acquired during delivery (perinatal infection): Perinatal infection may result from primary infection or reactivation of CMV from latency in cervical cells - After delivery (postnatal infection), transmitted in breast milk or saliva - Perinatal infection is more common than congenital infection - Perinatal and postnatal infections are frequently asymptomatic - Congenital infection may result in cytomegalic inclusion disease (CID), with many organs affected 2.) Congenital infection most likely with maternal primary infection than reactivation - No antibodies to neutralize the virus 3.) Congenital abnormalities more common when fetus infected in first trimester - Most organ development occurs in first trimester - Cell death can result in congenital defects from cytomegalic inclusion disease

VZV Pathogenesis

a. Initially infects upper respiratory tract b. Spreads via blood (viremia) and lymphatics to skin, where vesicular rash occurs c. Multinucleated giant cells with intranuclear inclusions seen in lesions d. Virus infects sensory neurons 1.) Cranial nerve ganglia 2.) Dorsal root ganglia (in spinal cord) 3.) Trigeminal ganglia (in pelvic region) e. Virus establishes latent infection (dormant) in the neurons f. Later in life (usually after age 50) and especially with impaired cell-mediated immunity, the virus may be reactivated - 10% to 20% of patients who had varicella will experience reactivation g. Disease presentation is zoster (shingles): vesicular skin lesions and nerve pain - Damages neurons, causing very painful neuralgia (pain along path of a nerve) - Herpes zoster lesions may be responsible for varicella infection in susceptible individuals (such as children

HSV diseases

a. May cause symptomatic or asymptomatic infections or recurrences b. Productive (lytic) infections involve a number of cells, including epithelial cells c. The lesion is a clear vesicle with an erythematous (red skin) base, progresses to pustules (pimples containing pus), ulcers, then crusted lesions d. Can cause serious disseminated infection in immunosuppressed person or neonate e. Latent infection: virus infects innervating neuron (nerve supplying the region), then moves up the axon to sensory ganglia (a cluster of neuron cell bodies)

Latex Agglutination

a. Microbeads of latex are coated with viral antigens. b. The patient's serum is mixed with coated latex particles. c. Agglutination of the beads indicates the presence of pathogen-specific antibodies in patient's serum.

Regarding Human Immunodeficiency Virus (HIV), which of the following statements is NOT correct?

a. The protease enzyme mediates the integration of proviral DNA into the host cell DNA genome. b. The gp160 precursor protein is cleaved to form the envelope glycoproteins gp120 and gp41. c. During clinical latency, HIV is typically not detectable in the blood, but is produced and sequestered in the lymph nodes. d. The main immune response in an HIV infection occurs when the HIV-infected CD4+ cells are attacked by cytotoxic CD8+ lymphocytes. answer: The protease enzyme mediates the integration of proviral DNA into the host cell DNA genome. explanation: The enzyme integrase mediates integration of proviral DNA into the host cell DNA. Protease cleaves precursor polyproteins into functional viral polypeptides.

HSV Immunity

a. Type-specific (may provide limited cross protection) b. Incomplete - Reinfection and reactivation can occur c. Cell-mediated immunity limits virus - Cytotoxic T lymphocytes (CTLs) are required to kill infected cells In the absence of a functional cell-mediated immunity, HSV infections is very likely to recur and be more severe d. Immunopathologic of cell-mediated and inflammatory responses are a major cause of disease symptoms e. Antibody directed against the glycoproteins may help neutralize extracellular virus, limiting spread

The Genetic Code Chart

(picture)

Viral Nucleic Acid (Chemical Composition of Viruses)

***Either DNA or RNA (but not both) 1.) Single or double stranded 2.) Circular or linear 3.) Segmented or non-segmented 4.) Double-stranded DNA (dsDNA): "sense" and "antisense" strand, which is the template for mRNA 5.) RNA genome polarity: - Positive (+) sense: Single-stranded RNA that is functionally equivalent to mRNA - Negative (-) sense or antisense: Carries RNA polymerase to transcribe genomic negative-sense RNA into mRNA 6.) Most RNA viruses have a single copy of the genome (haploid), but retroviruses have two copies of the RNA genome (diploid) - Most DNA viruses are double-stranded

Transmission (Clinical Presentation)

***Entry of virus into body 1.)The exposure of a susceptible host to virus under conditions that promote infection - Forms of transmission include: a. Vertical transmission from mother to child can occur through: i. Transplacental (virus transferred from mother to fetus through placenta during gestation) ii. At time of birth iii. Through breast milk b. Horizontal transmission from person to person can involve the following routes: i. Respiratory (droplets or aerosols) - Inhalation is the most common route of viral infections - Depends on: Temperature Air currents Viral particle size which impacts distribution ii. Enteral (fecal-oral route) - Must be acid stable - Usually seen with nonenveloped (naked) viruses iii. Parenteral (acquisition other than through the gastrointestinal tract) - Occurs through the exchange of body fluids, such as: Through breaks in the skin or mucous membranes Arthropod (insect) or animal bites Sexual contact 2.) After entry, virus replicates in cells that express viral receptors and have appropriate biosynthetic machinery

Enzymes

***NOTE: the suffix "-ase" is used to refer to an enzyme 1.) Polymerases: a. Participates in the synthesis of nucleic acids. b. Polymerases are categorized by the nucleic acid that will be synthesized and the nucleic acid used as the template (for example: a DNA-dependent polymerase uses DNA as the template) i. DNA-dependent DNA polymerase: synthesizes DNA from a DNA template ii. DNA-dependent RNA polymerase: synthesize RNA from a DNA template (virus needs to provide). iii. RNA-dependent RNA polymerase: synthesizes RNA from an RNA template iv. RNA-dependent DNA polymerase, also called reverse transcriptase, synthesizes DNA from an RNA template, reversing the normal direction of transcription (virus needs to provide). 2.) Nucleases: a. Digest nucleic acids or nucleotides b. Can be RNA specific (RNase) or DNA specific (DNase) c. Endonuclease: site of digestion is within the strand of nucleic acid - Includes restriction enzymes; degrades in middle of strand. d. Exonuclease: site of digestion is at the end of the strand of nucleic acid Nuclease "chews off" one nucleotide at a time 3.) Ligases: a. Forms phosphodiester bonds between existing DNA strands 4.) Methyltransferases: a. Adds methyl groups to nitrogenous bases. 5.) Deaminases a. Removes amino groups from nitrogenous bases

Specimen Collection

***Selection of appropriate specimen for analysis 1.) Specimen selection depends on disease syndrome and viral etiologies suspected. a. Specimens may need to be collected from multiple body sites if a disseminated infection is involved. b. Similar clinical syndromes can be caused by many different viruses. 2.) Optimal specimen selection should include a proper specimen source, which will include infected cells and viable organisms. 3.) Specimens should be collected as early as possible after the onset of symptomatic disease when viral shedding is highest. 4.) Collection devices should be sterile, leak-proof and non-breakable. - Dacron, rayon or polyester fiber swabs with a plastic or aluminum shaft are acceptable. 5.) Viral transport medium (VTM) a. VTM contains buffered salt solution, protein and saccharide nutrients, as well as a pH indicator and antibiotics to inhibit bacterial and fungal contaminants. b. VTM is designed to stabilize viruses and the patient's infected cells. c. Swabs, nasopharyngeal aspirates and tissue biopsies should be placed in VTM for culture and antigen detection.

Regarding adenoviruses, which of the following statements is NOT correct?

- Adenoviruses have a double-stranded DNA genome, an icosahedral capsid and are nonenveloped. - Virtually all adenovirus infections are symptomatic. - Respiratory diseases and gastroenteritis are commonly associated with adenovirus infections. - Outbreaks of severe, acute respiratory disease related to adenovirus infections have been seen in military recruits. answers: Virtually all adenovirus infections are symptomatic.

With EBV infections, which of the following statements is NOT correct?

- After initially infecting epithelial cells in the nasopharynx, EBV then infects and maintains a latent state in B lymphocytes. - EBV has been associated with infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma. - The IgM antibody to the VCA antigen of EBV persists for life, and indicates prior exposure to EBV. - EBV is a common infection, with more than 90% of adults in the US demonstrating antibodies to the virus. answer: The IgM antibody to the VCA antigen of EBV persists for life, and indicates prior exposure to EBV.

Cytopathic Effects (Detection of Virus-Infected Cells)

- Cytopathic effect (CPE): the damage, morphological and functional, inflicted on the cell by the virus and includes: a. Cell lysis or necrosis b. Inclusion body formation - Concentrated regions of viral nucleic acid and protein - May be situated in: Nucleus Cytoplasm Both c. Syncytia formation - The fusion of cells to form multinucleated giant cells d. Cytoplasmic vacuolization - Presence of virus may be detected as a distinctive CPE (shrinking, swelling, rounding of cells, or syncytia) using a light microscope. - Cell culture may support growth of several viruses, each producing a distinct CPE. - The type of cell culture in which the virus grows may be used for presumptive identification. - The rate at which at which CPE progresses may help identify the virus. CPE may develop in 1 to 3 days (HSV) or may not be recognized for 5 to 20 days (RSV, VZV, CMV).

Regarding human papillomaviruses, which of the following statements is NOT correct?

- HPVs cause infections of the skin and mucous membranes. - HPV genital infections are typically asymptomatic and most resolve completely in one to two years. - There are 14 specific high-risk HPV types that have been associated with the development of cervical cancer. - Proteins encoded by the L1 and L2 genes are involved in carcinogenesis by interfering with the p53 and retinoblastoma (RB) tumor suppressor proteins. answer: Proteins encoded by the L1 and L2 genes are involved in carcinogenesis by interfering with the p53 and retinoblastoma (RB) tumor suppressor proteins. explanation:nE6 and E7 genes encode proteins that inactivate proteins from tumor suppressor genes p53 and Retinoblastoma (RB), which are an important step in the process by which a normal cell becomes a cancer cell.

When testing the serum collected from a patient with an acute hepatitis B infection, which of the following markers would NOT be likely to be detected?

- Hepatitis B surface antigen (HBsAg) - Hepatitis B core antigen (HBcAg) - IgM antibody to Hepatitis B core antigen (IgM anti-HBc) - Hepatitis B endogenous antigen (HBeAg) answer: Hepatitis B core antigen (HBcAg) explanation: The Hepatitis B core antigen (HBcAg) is never found in the serum of a patient infected with Hepatitis B Virus. The other markers can all be detected during the acute stage of the infection.

Penetration (Virus Entry)

- Internalization of virus into the cell involves interaction between multiple VAPs and host cell receptors - The mechanism of internalization depends on the virion structure and the host cell type, and includes: 1.) Endocytosis - With this receptor-mediated phagocytosis by the host cell, the virus binds to specific receptors on the host cell, which induces the formation of a pit that pinches off from the cell membrane to form a vesicle - Low pH in the vesicle promotes the release of the nucleocapsid and viral genome 2.) Fusion - This process involves fusion of the viral envelope with the host cell membrane. - It not only provides a mechanism for internalizing the virus, but can lead to the fusion between the infected host cell and additional nearby host cells, forming multinucleated cells called syncytia. - Detection of syncytia can be used to determine the presence of virus in cell cultures. - Once internalized, the nucleocapsid is delivered to the site of replication.

Regarding coronavirus, which of the following statements is NOT correct?

- It is an enveloped virus with a helical nucleocapsid containing a positive-sense DNA genome. - It is a prevalent cause of the common cold. - It is associated with severe acute respiratory syndrome (SARS). - Coronavirus appears to attach to host cells using a viral surface glycoprotein spike that interacts with the angiotensin-converting enzyme 2 (ACE2) found on host cell membranes. answer: It is an enveloped virus with a helical nucleocapsid containing a positive-sense DNA genome. explanation: Coronavirus is an enveloped virus with a helical nucleocapsid containing a positive-sense RNA genome.

Regarding CMV infection in neonates, which of the following statements is NOT correct?

- It may occur as a congenital infection (in utero), during delivery (perinatal) or after delivery (postnatal) through breast milk or saliva. - Congenital infection is most likely to occur due to reactivation of CMV from a latent maternal infection. - Congenital infection may result in cytomegalic inclusion disease. - Perinatal and postnatal infections may be asymptomatic. answer: Congenital infection is most likely to occur due to reactivation of CMV from a latent maternal infection.

Regarding HSV infections, which of the following statements is NOT correct?

- Most primary HSV-1 infections occur during childhood. - An oral HSV infection becomes latent when the virus migrates up the neuron to the trigeminal ganglia. - With reactivation, HSV migrates back down the neuron to the initial site of infection, where it replicates and may produce skin lesions. - HSV-2 infection has been linked to encephalitis, with a lesion appearing in one temporal lobe and a high mortality rate. answer: HSV-2 infection has been linked to encephalitis, with a lesion appearing in one temporal lobe and a high mortality rate.

Nucleic Acid Hybridization Probe Assays

- Nucleic acid probes are oligonucleotides (single-stranded DNA or RNA) with sequences complementary to specific regions of the viral genome - These probes are sensitive and specific tools for identifying viral infectious agents - Involves utilization of a nucleic acid probe (oligonucleotide) specific for a specific viral nucleic acid sequence in the test specimen - Nucleic acid probes, labeled with an enzyme, chemiluminescent molecule, fluorescent molecule or radioisotope, will hybridize (bind) to the complementary sequence in the patient specimen. - The hybridized products can then be detected. *In Situ Hybridization (ISH): - In situ hybridization is a direct probe technique that can be used in anatomic pathology to detect a variety of viral pathogens inside individual cells in tissue biopsy specimens. - When fluorescent detection is used by attaching a fluorochrome to the probe, it is called fluorescent in situ hybridization (FISH)

Viral Protein Synthesis

- Once the viral mRNA is transcribed (from either DNA or RNA), it is translated by host cell ribosomes into early and late proteins: a. "Early" is defined as occurring before the replication of the viral genome - The early proteins include nonstructural elements such as regulatory proteins and enzymes used to replicate viral genome - For RNA viruses, an important early protein is the RNA polymerase that will synthesize many copies of viral genetic material for progeny virus particles b. "Late" is defined as occurring after genome replication - The late proteins include structural elements such as the capsid proteins c. Some viral mRNAs are translated into precursor polypeptides (long protein strands) that must be cleaved (cut) by proteases to produce the functional structural proteins, while other viral mRNAs are translated directly into the functional proteins - At the end of macromolecule synthesis, viral proteins have been synthesized and the genome has been replicated so identical copies of the virion can be generated.

Shell Vials Cultures / Centrifuge-Enhanced Culture (Detection of Virus-Infected Cells)

- Shell vial cell culture is a modification of the conventional cell culture adapted for a few viruses - The viruses can be detected more quickly because infected cells are stained for viral antigens produced soon after infection - This modification is best used for viruses that require long incubation before the characteristic CPE is observed (e.g. CMV and VZV) - A round coverslip is added to bottom of shell vial tube, and monolayers of the cell line are grown on the coverslip. - The specimen is inoculated into the vial and the vials are incubated at 37°C. - The coverslips are removed, stained using fluorescent-labeled antibodies and viewed on a microscope. - Only a single type of virus can be detected per shell vial. - If more than one virus is present in a specimen, it would require one shell vial for each virus - A modification of the technique allows simultaneous recovery of multiple respiratory viruses using R-Mix shell vials - Inoculate specimen to two duplicate vials and incubate at 35° to 37°C for 18 to 24 hours. - Stain one vial with monoclonal antibody pool screening reagent: if result is positive, scrape second shell vial and spot an eight-well slide for specific monoclonal reagent to detect the specific virus present.

Viral Genome Detection: Molecular Methods

- The DNA or RNA genome of the virus can be used to identify the infectious agent. - The advantages of molecular techniques are sensitivity, specificity and safety. - Nucleic acid hybridization and amplification tests detect virus by targeting specific regions of the viral RNA or DNA genome. - Accurate and timely viral diagnosis optimizes patient management. - Nucleic acid-based tests can help identify viruses that propagate slowly or not at all in cell culture. - Quantitative assays can determine the number of viral genome copies in a sample, and are referred to as viral load. - With viruses demonstrating the ability to produce antiviral drug resistance, mutation detection is the method of choice for appropriately selecting antiviral therapy.

DNA Viruses (Macromolecule Synthesis)

- The genomes of most DNA viruses are transcribed by the host DNA-dependent RNA-polymerase in the cell nucleus to produce viral mRNAs, which are exported to the cytoplasm, where the host cell ribosomes are used for translation. a. Class I: Double-stranded DNA genome i. Transcription/Translation - Most dsDNA viruses replicate and assemble in the host cell nucleus - These viruses use the host cell DNA-dependent RNA polymerase to synthesize their mRNA transcripts ii. Viral Genome Replication - DNA-dependent DNA polymerase is used to replicate the viral genome b. Class II: Positive (+) sense single-stranded DNA genome i. Transcription/Translation - The positive DNA sense strand has same sequence as mRNA, and cannot be used as the source of the protein code. - The antisense or (-) sense DNA strand must be synthesized first, which can then be transcribed directly into mRNA using the host cell DNA-dependent RNA polymerase. ii. Viral Genome Replication - The host cell DNA polymerase is used to synthesize the negative (-) sense strand, which will then serve as the template to replicate the viral genome by synthesizing the positive (+) sense strand c. Class VII: Partially double-stranded DNA genome - Hepadnaviruses (Hepatitis B) have a genome that is mostly dsDNA but includes a small section that is single-stranded. - It first uses host DNA polymerase to synthesize the missing portion of the DNA genome, forming a complete double-stranded circular DNA molecule. i. Transcription/Translation - Host cell DNA-dependent RNA polymerase is used to transcribe positive (+) sense RNA strands (viral mRNA) from the negative (-) sense DNA strand of the viral genome. ii. Viral Genome Replication - The positive (+) sense RNA strands synthesized as described above will also serve as the template for replication of the genome, generating a complementary negative (-) sense DNA strand. - This process will require a viral-encoded RNA-dependent DNA polymerase as the host cell does not contain that enzyme. - The negative (-) sense DNA strand that has been generated can then serve as the template for the synthesis of the complementary positive (+) sense DNA strand. - Synthesis of the positive (+) sense strand stops when the genome and core of the viral progeny are enveloped, which results in the partially double-stranded circular DNA that is the genome of the Hepadnavirus.

Plaque Assays (Detection of Virus-Infected Cells)

- The plaque assay can be used for viruses that grow well in tissue culture, and can be used to determine virus concentration a. Monolayers of host cells are inoculated with dilutions of virus b.After adsorption, the cells are overlaid with agar to prevent virus from spreading c. Results demonstrate localized cell killing where cell has been infected with virus d. Multiple replication cycles produce a small area of cellular destruction or plaque where a single virus infects, spreads and kills surrounding cells. - A single plaque can arise from a single clonal infectious particle, termed a plaque-forming unit - Plaques can be counted macroscopically - By counting the plaques and correcting for the dilution factor, the viral titer can be calculated (plaque forming units per milliliter or pfu/mL)

Assembly (Viral Maturation)

- The progeny viral particles are assembled by enclosing the viral genome in a protein capsid. - Virion assembly is analogous to a three-dimensional interlocking puzzle that puts itself together in the box. - The capsid is self-assembled from small, easily manufactured component parts. - Assembly occurs when the concentration of the structural proteins is sufficient to drive the reaction thermodynamically, much like crystallization. - Individual protein structural subunits (protomers) are assembled in a stepwise and orderly fashion into capsomers, which are used to create the capsid

Regarding Hepatitis B Virus (HBV), which of the following statements is NOT correct?

- The viral genome is partially double-stranded DNA. - Hepatocellular carcinoma (HCC) can occur in individuals with chronic HBV. - Reverse transcriptase, an RNA-dependent DNA polymerase encoded by the host cell genome reverse transcribes a positive sense RNA strand into a negative sense DNA strand as part of the viral genome replication. - Antibody to HBV surface antigen (anti-HBs) is not detectable in the chronic HBV infection. answer: Reverse transcriptase, an RNA-dependent DNA polymerase encoded by the host cell genome reverse transcribes a positive sense RNA strand into a negative sense DNA strand as part of the viral genome replication. explanation: The human host cannot synthesize reverse transcriptase, an RNA-dependent DNA polymerase and cannot reverse transcribe an RNA strand into DNA. This enzyme must be encoded by the viral genome.

Release

- The viral nucleocapsid is released from the host cell by one of two processes: a. Lysis (rupture) of host cell membrane - Lysis may be associated with the release of nonenveloped (naked) viruses b. Budding (enveloped viruses) 1.) Nucleocapsids are enveloped as they bud through the host cell membrane modified by the insertion of viral-encoded glycoproteins 2.) The viral nucleocapsid interacts with the membrane site mediated by the matrix protein 3.) Virus exits the cell, which may or may not be damaged or destroyed in the process - Most RNA viruses bud from the cell membrane and the virus is released without killing the cell. - The HSV nucleocapsid assembles in the nucleus and buds out of the nuclear membrane into the endoplasmic reticulum. The virion is then transported to the cell surface and released. 4.) Released virions may infect adjacent cells or may be carried by fluids to distant cells 5.) Spread of infection generally occurs from virus released into the extracellular environment, but alternatively, a virus, nucleocapsid or genome can be transmitted through cell-to-cell bridges, cell-to-cell fusion or vertically to daughter cells. - These alternative routes can allow the virus to escape antibody detection.

Regarding the hepatitis C Virus (HCV), which of the following statements is NOT correct?

- There are 6 genotypes of HCV and genotype 1 causes a significant number of infections in the US. - Transmission is primarily parenteral, and includes injection drug use, body piercing and tattoos. - Acute HCV infection is often asymptomatic. - HCV rarely results in chronic infection. answer: HCV rarely results in chronic infection. explanation: Chronic infection occurs in about 80% of patients with HCV.

Regarding Hepatitis A Virus (HAV), which of the following statements is NOT correct?

- Transmission is associated with fecally contaminated water or food, such as oysters grown in polluted water and eaten raw. - Immunization with the HAV vaccine has reduced the incidence of Hepatitis A infections. - There is a strong association with HAV infection and the development of hepatocellular carcinoma later in life. - The detection of an IgM antibody to HAV is useful in the diagnosis of infection. answer: There is a strong association with HAV infection and the development of hepatocellular carcinoma later in life. explanation: An HAV infection typically resolves without complications or permanent liver damage. Chronic infection does not occur and no carrier state develops. There is no predisposition to the development of hepatocellular carcinoma.

RNA Viruses (Macromolecule Synthesis)

- Unlike the DNA viruses, most RNA viruses replicate and assemble in the host cell cytoplasm. - RNA viruses must provide RNA-dependent RNA polymerases as the host cell has no means of replicating RNA. - The RNA genome structure and polarity of the virus groups will determine how the genome is replicated and how viral messenger RNA (mRNA) is generated and proteins are processed. a. Class III: Double-stranded, segmented RNA genome - Most of the RNA segments code for a single protein i. Transcription/Translation - This process will require a viral-encoded RNA-dependent RNA polymerase as the host cell does not contain that enzyme. - For each of the double-stranded RNA genome segments, the positive (+) sense RNA (mRNA) will be synthesized from the negative (-) sense strands ii. Viral Genome Replication - Excess positive (+) sense strands synthesized as described above will serve as the template for the synthesis of the negative (-) sense strands for the replication of the genome (dsRNA segments) b. Class IV: Positive (+) sense non-segmented RNA genome i. Transcription/Translation - Positive (+) sense strand RNA genomes can serve as mRNA and can be used immediately to synthesize proteins after binding to the host cell ribosomes - Translation typically results in the synthesis of a large polyprotein that is cleaved (cut) into several smaller, functional proteins by protease enzymes ii. Viral Genome Replication - Genome replication requires synthesis of a negative (-) sense RNA intermediate strand using virus-encoded RNA dependent RNA polymerase - This intermediate serves as the template for the synthesis of the positive (+) sense genomic RNA c. Class V: Negative (-) sense RNA genomes - This group of viruses includes non-segmented and segmented genomes - Most segments code for a single protein i. Transcription/Translation - Viral-encoded RNA-dependent RNA polymerase is required to transcribe the positive (+) sense strand RNA (mRNA) using the negative (-) sense strand genomic RNA as a template ii. Viral Genome Replication - Genomic replication of the negative (-) sense strand RNA requires synthesis of a full length positive (+) sense RNA intermediate, which serves as the template for the synthesis of the negative (-) sense RNA genome d. Class VI: Positive (+) sense single-stranded diploid RNA genome - Diploid indicates these viruses have 2 copies of the single-stranded RNA genome - Although the viral genome is single-stranded positive (+) sense strand RNA, it does not serve as mRNA following viral entry into the host cell - Instead, the RNA genome serves as the template for the synthesis of a complementary DNA (cDNA) copy of the genome, manufactured in the host cell cytoplasm - It uses a virus-encoded RNA-dependent DNA polymerase - The cDNA travels to the host cell nucleus and the viral cDNA is integrated into the host cell genome, becoming a "provirus" i. Transcription/Translation - Transcription of the viral cDNA into mRNA and translation into viral proteins proceeds along with host cell processes ii. Viral Genome Replication - The positive (+) sense RNA transcripts generated as described above can be used as the new genomes - Macromolecule synthesis also includes the production of viral proteins

Which of the following statements is NOT a characteristic of the Varicella-Zoster Virus (VZV)?

- VZV can be transmitted by respiratory droplets and initially infects the upper respiratory tract. - The virus infects sensory neurons where it establishes a latent (dormant) infection. - VZV present in shingles lesions may be transmitted by direct contact to susceptible individuals, causing chickenpox. - All of the above are characteristics of VZV. answer: All of the above are characteristics of VZV.

Incubation Period (Clinical Presentation)

- Virus is replicating but has not induced sufficient damage to cause disease 1.) May be asymptomatic, resolved by host's innate immune response 2.) May see early nonspecific symptoms, referred to as prodrome: a. Fever or chills b. Malaise c. Myalgias d. Headache

Viral Replication

- Viruses multiply only in living cells. - A lytic viral response exists when productive viral replication occurs in the host cell, resulting in the release of new viral particles. - The infected host cell acts as a factory, providing energy, synthetic machinery and low molecular weight precursors for the synthesis of viral proteins and replication of the viral genome. - Processes not provided by the cell must be encoded in the genome of the virus. - The manner in which each virus accomplishes the steps of viral replication, transcription and translation is different for each type of virus. - A single round of the viral replication cycle can be separated into several phases: 1.) A single virion (infective viral particle) recognizes an appropriate target cell, attaches to the cell and penetrates the cell membrane. 2.) The viral nucleic acid (viral genome) is released into the host cell (uncoating). 3.) Uncoating of the genome initiates the eclipse period, in which no intact viral particles are present and the infectivity of the virus is eliminated. 4.) In the eclipse period, intense viral macromolecular synthesis occurs: a. Viral genome replication produces multiple copies of viral nucleic acids b. Transcription and translation are initiated, producing numerous viral proteins 5.) Some viral proteins will begin self-assembly to form the viral capsid 6.) The appearance of viral particles in the cell signals the end of the eclipse period. 7.) The virions are released to infect new susceptible host cells. - Viral replication includes the following 6 steps...

Target Amplification Assays

- With amplification techniques, viral target DNA or RNA is amplified many times a. Polymerase Chain Reaction (PCR) - The Polymerase Chain Reaction is used to amplify small amounts of specific viral DNA present in the clinical specimen - The assay uses thermostabile DNA polymerase to produce a twofold amplification of the target DNA with each temperature cycle: - In the denaturation step, the temperature is raised to 90° to 95°, which separates the two strands of target DNA - In the annealing step, the temperature is lowered to 45° to 60°, which anneals (binds) two short DNA oligonucleotides called primers to specific sequences of the target DNA. - In the extension step, the temperature is raised to 72°, which extends the primers by adding nucleotides complementary to the target DNA - The cycle is repeated 30 to 40 times to yield amplification of the target DNA by more than 1010-fold. b. Quantitative Real-Time PCR (QPCR) - Quantitative Real-Time PCR (QPCR) is a variation of a traditional PCR reaction, which is used to assess viral loads and monitor response to therapy for viruses such as HIV, CMV, HCV, HBV and BKV. - Real-TIme PCR uses a fluorescent-labeled probe which generates a fluorescent signal as viral DNA is amplified - The signal from the probes is proportional to the amount of amplified viral DNA present in the reaction - The production of DNA is measured by the increase in the fluorescent signal die to the binding of a probe labeled with a fluorescent molecule to the amplified DNA. c. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) - Another variation of traditional PCR can be performed using an RNA target, which is called Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). - The enzyme reverse transcriptase is used to reverse transcribe RNA into cDNA (complementary or copy DNA) for subsequent PCR amplification. - Once cDNA has been synthesized, DNA polymerase will amplify the DNA in a traditional PCR reaction - The concentration of the cDNA will double with each PCR cycle.

One megabase equals ____________ base pairs.

1,000,000

Transcription and Translation

1. Genes provide instructions for the production of proteins. 2. The order of base pairs in a gene determines the order of amino acids in the protein. 3. The order of amino acids in the protein determines the final three-dimensional shape and function of that protein. 4. Protein synthesis can be divided into two stages: transcription and translation

DNA Replication

1.) A parent strand of DNA serves as a template for synthesis of the new strand a. Nucleotides form complementary base pairing with the template (parent strand) in the creation of the strand being synthesized b. The enzyme DNA polymerase joins the phosphate at the 5' end of the nucleotide being added to the hydroxyl group attached to the 3' (which number?) carbon of the nucleotide already in the chain c. The covalent bond joining nucleotides to create the sugar-phosphate backbone is called a phosphodiester bond. 2. DNA is synthesized in a 5' to 3' direction, copying a DNA template strand running in a 3' to 5' direction - In the diagram to the right, the upper strand (leading strand) is in the correct orientation to be copied along its length so that the direction of replication is left to right, producing a strand oriented 5' to 3'

Viral Lipid Envelopes (Chemical Composition of Viruses)

1.) Acquired when nucleocapsid buds through the host's cell membrane. 2.) Budding occurs at sites where virus-specific proteins have been inserted. 3.) Envelope is lipid bilayer with proteins and glycoproteins 4.) Most viruses acquire envelope from outer membrane a. Herpesviruses bud through nuclear membrane 5.) Viral Glycoproteins a. Glycoproteins in viral envelope are virus encoded b. Surface glycoproteins protrude outside of the envelope - Attach virus to a target cell by interacting with cellular receptor. c. Glycoproteins serve as viral antigens d. Also function as viral attachment proteins (VAP) - VAPs that bind to erythrocytes are termed hemagglutinins (HAs) e. Neutralizing antibodies to glycoproteins in envelope may prevent infection - Damage to envelope inactivates virus 6.) Enveloped viruses vs naked (nonenveloped) viruses: a. Enveloped: - Susceptible to environment: Drying out High temperature pH below 6 and above 8 Detergents - Transmitted by direct contact: Blood or body fluids May be transmitted by insect or animal bite b. Naked (nonenveloped): - Very stable: resistant to harsh environmental conditions - Most viruses transmitted by fecal-oral route do not have an envelope - All human viruses with a helical nucleocapsid have an envelope - Icosahedral nucleocapsids can be enveloped or naked

Viral Proteins (Chemical Composition of Viruses)

1.) Capsid: protein coat surrounding nucleic acid core a. Composed of repeating protein capsomers 2.) Arrangement of capsomers determines shape of virus a. Icosahedral: 20 equilateral triangles - DNA and RNA viruses b. Helical: a hollow coil that surrounds viral nucleic acid - RNA viruses c. Complex structures d. Advantages of capsid with identical protein subunits called capsomers: - No need for complex genetic information - Promotes viral self-assembly 3.) Protects viral genome from degradation by nucleases 4.) Participates in attachment through viral attachment proteins (VAP) to specific host cell receptors 5.) Determines antigenic characteristics of virus. a. Proteins or glycoproteins exposed on surface stimulates immune response. b. Neutralizing antibodies may prevent infection. 6.) Surface antigens define subcategories of virus (serotypes) a. Antigenic variation b. Type-specific 7.) Capsid plus nucleic acid core: nucleocapsid 8.) Capsid may include enzymes essential for initiation of viral replication

Central Dogma

1.) Double-stranded DNA is copied through the process of replication. 2.) DNA also serves as the template which transcribes into mRNA which translates into protein, following the instructions that originated with the gene. - This "central dogma" was the explanation of the flow of genetic information within a biological system described by Francis Crick in 1958. - NOTE: The discovery of viruses identified biological agents that did not always follow the "central dogma" 3.) Only one strand of double-stranded DNA is template for synthesis of messenger RNA. - This strand that will be transcribed is the "antisense" or negative (-) sense strand. 4.) The other strand of double-stranded DNA is not transcribed into messenger RNA. - This non-transcribed strand is the "sense" strand or positive (+) sense strand, and has the same sequence as mRNA. 5.) The mRNA transcript created from the template antisense DNA strand is referred to as the sense RNA strand. - The sense or positive (+) sense mRNA strand can then be translated into protein. 6.) An RNA strand that would be complementary to the sense (non-template) DNA strand is called the antisense or negative (-) RNA strand - It has the same sequence as the antisense (template) DNA strand.

Acute Viral Infection (Clinical Presentation)

1.) During the acute stage, signs and symptoms of infection are displayed 2.) Initial local infection: near site of entry into host (related to mode of transmission) 3.) Virus may replicate and remain at the primary site (localized infection) 4.) Viremia: virus is present in the blood stream a. May travel free in plasma b. May be carried in specific blood cells (macrophages, lymphocytes, etc.) c. Allows inoculation of tissues and organs distant from the primary site 5.) Systemic disease: virus must cross the mucosal barrier in the respiratory, gastrointestinal or genitourinary tracts. 6.) Symptoms caused by viral replication in target tissues: a. May cause tissue damage due to the cytopathic ability of virus b. Symptoms may be due to interaction of virus and immune response c. Symptoms may continue through convalescence 7.) Specific symptoms depend on: a. Portal of entry b. Route or pathway of spread c. Tropism (propensity of virus to infect a distinct group of cells and tissues) - Availability of virus receptors on surface of host cell that interact with viral attachment proteins d. Host age e. Nutritional status of host f. Presence of genetic polymorphisms that affect susceptibility to infection g. Virulence of the virus

Viral Pathogenesis

1.) Pathogenesis is the process whereby a virus interacts with its host to produce disease 2.) Viruses cause disease when they evade the host's immune responses and either destroy cells of an important organ or trigger a destructive immune and inflammatory response. 3.) Viral and host factors determine the severity of the disease: a. The tissue targeted by the virus defines the nature of the disease and its symptoms b. Virulence is the capacity of a virus to produce disease in a susceptible host, and is dependent on viral and host factors, including: i. The rate of viral transmission, which depends on: Population density Number of susceptible individuals Weather ii. Virus strain iii. Ability of virus to escape host defenses iv. Host age and immune status v. Loss of virulence results in attenuation of the virus - The virus is altered so it becomes harmless - Some live virus vaccines use attenuated virus strains c. The host's immune response may contribute to the pathogenesis of the disease 4.) Outbreaks of a viral infection result from introduction of a virus into a new location - The outbreak originates from a common source (such as food preparation) a. Epidemics: viral outbreaks that cause a high proportion of cases in a population, community or region - Generally results from a new strain of virus introduced into a immunologically naive population b. Pandemics: worldwide epidemics (not localized to a community or region)

Deoxyribonucleic Acid (DNA)

1.) Structure: DNA is a double-stranded structure composed of building blocks called deoxyribonucleotides which contain: a. Deoxyribose: the ringed 5-carbon sugar in the DNA polymer - The 5 carbons are numbered sequentially - The five carbons are designated as 1' to 5' - A nitrogenous base is attached to the 1' carbon, a hydroxyl group is attached to the 3' carbon and a phosphate group is attached to the 5' carbon b. Nitrogenous bases in DNA: - Guanine and adenine are purine bases - Cytosine and thymine are pyrimidine bases c. Phosphate group: composed of three phosphate molecules in a string d. Deoxyribonucleoside: consists of the nitrogenous base attached to the deoxyribose sugar e. Deoxyribonucleotide: the nitrogenous base is attached to the 1' carbon of the sugar and phosphate group is bound to the 5' carbon. - The deoxyribonucleotide is the basic structural unit in DNA polymer

Ribonucleic Acid (RNA)

1.) Structure: Ribonucleic acid is a single-stranded structure composed of ribonucleotides which contain: a. Ribose: the ringed 5-carbon sugar in the RNA polymer - The 5 carbons are numbered sequentially in a similar manner to deoxyribose (see above) - RNA differs from DNA due to the presence of a hydroxyl group at the 2' C of the 5-carbon sugar in the ribonucleotide b. Nitrogenous bases in RNA: - Guanine and adenine are purine bases - Cytosine and uracil are pyrimidine bases. - RNA has uracil instead of the base Thymine found in DNA c. Phosphate group: composed of three phosphate molecules in a string d. Ribonucleoside: consists of the nitrogenous base attached to the ribose sugar e. Ribonucleotide: nitrogenous base is attached to the 1' carbon of the sugar and the phosphate group is bound to the 5' carbon - The ribonucleotide is the basic structural unit in the synthesis of the RNA polymer

DNA Strands

1.) The 5' end of a strand of DNA strand has an open and available phosphate group 2.) The 3' end of a strand of DNA has an open and available hydroxyl (OH) group 3.) A strand of DNA can be described as having a 5' (ending in a phosphate group) to 3' (ending in a hydroxyl group) orientation 4.) Complementary base pairing occurs in the creation of a double-stranded DNA polymer: a. When one strand has an adenine nucleotide, the opposite strand has a thymine nucleotide - Two hydrogen bonds form b. When one strand has guanine nucleotide, the opposite strand has a cytosine nucleotide - Three (how many?) hydrogen bonds form c. The two DNA strands anneal or hybridize to each other through hydrogen bonds, referred to as base pairing. d. In the complementary double-stranded DNA helix, one strand runs 5' to 3', while the other runs antiparallel, or 3' to 5' - Example of double-stranded DNA sequence representation: 5' GTCGGATCGTAGCTAT 3' 3' CAGCCTAGCATCGATA 5' e. The double-stranded DNA structure (dsDNA) can be described as having: i. A sugar-phosphate backbone ii. Complementary base pairing iii. Two strands that are antiparallel (oriented in opposite directions) f. Genome: the entire content of DNA contained within a cell nucleus is referred to as genomic DNA g. The length of a double-stranded DNA (dsDNA) polymer is measured in base pairs (bp) - One kilobase (kb) = 1,000 bp. - One megabase (Mb) = 1,000 kb h. Oligonucleotide: is used to describe a short, single-stranded nucleic acid chain (can be DNA or RNA) of at least 20 nucleotides in length - An oligonucleotide can be made synthetically to a specified sequence, in order to perform a specific function

Uncoating

1.) Uncoating is the process by which the capsid is removed. This can be accomplished by: a. Enzymatic degradation (viral or host cell enzymes) b. Simple dissociation 2.) Uncoating releases the viral genome for delivery of the viral DNA or RNA to its intracellular site of replication in the host cell nucleus or cytoplasm.

Attachment (Adsorption)

1.) viral attachment proteins (VAPs) which are proteins or glycoproteins on the surface of the capsid (for naked viruses) or envelope - Viral attachment proteins are conspicuous features on the viral surface and typically extend from the surface of the virus in the form of spikes. 2.) The viral attachment proteins bind to protein or carbohydrate receptors on the host cell surface. 3.) The ability of the VAPs to recognize receptors on the surface of a suitable host cell determines the host range (host specificity), which is the host species a particular virus is capable of infecting - A few viruses can infect cells from different species (a broad range of hosts) but most have a narrow range, and may be limited to a single species. 4.) The specific receptor-mediated reaction of a VAP binding to a cell surface receptor also determines the specific cells and tissues of the host that will support the growth of a particular virus (may be referred to as tissue tropism or organ specificity) 5.) Differences in host range and tissue tropisms are due to the presence or absence of receptors for the viral attachment proteins. - For example, Epstein-Barr virus (EBV) has a very limited host range and tropism because it binds to the CD3 receptor (CR2) present on human B cells.

Basis of Viral Classification

A. Morphology: 1.) Size 2.) Shape (viral capsid) 3.) Presence of lipid envelope B. Genome: 1.) Type of nucleic acid 2.) The strategy used in replication and transcription C. Viral proteins: 1.) Functions 2.) Antigenic properties (ability to stimulate an immune response in the host) D. Physiochemical properties: 1.) Susceptibility to physical and chemical agents 2.) Heat stability 3.) pH stability (some viruses can be destroyed by alkaline conditions) E. Biological properties: 1.) Natural host range 2.) Mode of transmission 3.) Pathogenicity 4.) Tissue tropisms F. Taxonomy: 1.) International Committee on Taxonomy of Viruses (ICTV) Classification - Order ends with the suffix -virales - Family ends with the suffix -viridae - Subfamily ends with the suffix -virinae - Genus ends with the suffix -virus - Species name provides unambiguous identification - The Human Immunodeficiency Virus taxonomy as specified by the International Committee on Taxonomy of Viruses (ICTV) 2.) Baltimore classification, based on: a. Nucleic acid type b. Nucleic acid number of strands c. Nucleic acid strand sense d.Methods used to generate mRNA

Cultivation and Detection of Viruses

A. Preparation of Inoculum (substance used for inoculation): 1.) Sterile specimens may be inoculated onto cell cultures directly. 2.) Tissue is washed in media or sterile water and ground into paste. Diluent is added, the tube is centrifuged and the supernatant inoculated. B. Biological Methods: - Requires specimens collected during active viral replication 1.) Embryonated Eggs: a. Still may be used for growth of certain viruses such as influenza (in association with vaccine development) b. Also used for research in specialized laboratories 2. Animal Inoculation: a. Studies of pathogenesis of viral diseases, epidemiology, immune responses and viral oncology b. To test vaccines safety 3.) Cell Cultures: - Cell cultures are host cells grown in a monolayer on the sides of glass or plastic test tubes or petri dishes - Many viruses can be grown in specific types of tissue culture cells - Cell cultures are classified as: a. Primary cultures: prepared directly from parent organ - Mixture is transferred to tubes vials, where cells attach in a monolayer on the glass surface. - Cells are overlaid with a nutrient buffer solution. - Primary cultures have been subcultured only once or twice since harvesting. b. Diploid cells: cultures of a single cell type - Remain viable through 20 to 50 generations. - Cells retain normal diploid chromosome pattern c. Tumor cell lines and immortalized cell lines can be propagated indefinitely - Derived from malignant tissues - Can also be prepared by treatment of primary cells with oncogenic viruses or chemicals - Have altered and irregular numbers of chromosomes (aneuploid) - Unfortunately, most viruses do not grow well in continuous cell lines. d. The cells are immersed in cell culture medium to provide nutrients. e. Cell cultures are incubated at 35°C for one to four weeks.

Which of the following statements would NOT be a characteristic of a latent infection?

A. There are no clinical symptoms observed in the patient. B. The virus is present in an inactive state. C. The virus may be dormant for months or years. D. Viral replication continues to take place in the host cell. answer: Viral replication continues to take place in the host cell.

Mechanisms for the vertical transmission of infection include all of the following except:

A. Transplacental transfer B. During birth C. Through insect bites D. Through breast milk answer: Through insect bites

Introduction to Virology

A. Virus: submicroscopic, obligate intracellular parasite 1.) Do not have a nucleus or cytoplasm 2.) Not capable of independent replication a. Can only replicate in living cells through self-assembly of individual components 3.) Cannot generate metabolic energy (do not have mitochondria) 4.) Cannot synthesize proteins (do not have ribosomes) B. Features: 1.) Genome: either DNA or RNA 2.) Protective protein shell a. May be surrounded by an envelope C. Virion: Viral particle that functions as infectious unit 1.) Virion is inert in the extracellular environment 2.) Viral nucleic acid directs host cell to synthesize virus-specific macromolecules

Which of the following statements is NOT true of hemagglutination inhibition:

A. Viruses such as influenza produce an envelope protein called hemagglutinin (HA). B. Hemagglutinin has the ability to bind to the surface of red blood cells causing them to agglutinate (hemagglutination). C. Antibodies to the viral hemagglutinin neutralize the virus, preventing the attachment of the virus to the red blood cells. D. Hemagglutination is inhibited when antibodies are NOT present. answers: Hemagglutination is inhibited when antibodies are NOT present.

The start codon ________ in the mRNA strand, which codes for the amino acid methionine, initiates translation in protein synthesis.

AUG Translation begins with the AUG sequence in the mRNA strand that is the start codon for translation. The tRNA having the anticodon UAC carries the amino acid methionine (Met).

Which of the following is NOT a nitrogenous base that may be associated with a deoxyribonucleotide?

Adenine Cytosine Guanine Tyrosine Answer: Tyrosine The fourth nitrogenous base is thymine. Tyrosine is an amino acid.

The term used to describe the orientation of individual strands in a double-stranded DNA polymer is:

Antiparallel

START OF LECTURE MATERIAL

Below this card is all the outlines for Virology

A 22-year-old AIDS patient has symptoms of colitis, with several episodes of bloody diarrhea. Histological staining of a biopsy specimen collected from an ulcerated lesion during the colonoscopy revealed giant cells with large intranuclear inclusions with an owl's eye appearance. Which of the following is the most likely cause of the disease?

CMV Giant cells demonstrating large intranuclear inclusions with an owl's eye appearance is a characteristic of CMV.

A pandemic is an outbreak that results in a high proportion of infected patients in a community or localized region.

FALSE Epidemics are outbreaks in a community or localized region. Pandemics are outbreaks that are spread worldwide.

If a virus has an envelope, it is not easily inactivated by drying out and detergents.

FALSE If a virus has an envelope, it is MORE easily inactivated by drying out and detergents. Viruses that do not have an envelope are very stable and more resistant to harsh environmental conditions.

Nonenveloped viral particles are released from the host cell when they bud through the cell membrane after viral assembly.

FALSE Nonenveloped viral particles are released with host cell lysis (rupture). Nucleocapsids become enveloped when they bud through the host cell membrane modified by the insertion of viral-encoded glycoproteins.

The adenovirus fiber protein stimulates the production of neutralizing antibodies capable of providing lifelong protection from all adenovirus types capable of causing human disease.

FALSE The immune response results in the production of neutralizing antibody which is type-specific only, and does not protect against infection by other types.

With VZV infection, zoster is the primary disease typically occurring in childhood, and varicella is the recurrent infection that occurs later in life.

FALSE With VZV infection, varicella (chickenpox) is the primary disease typically occurring in childhood, and zoster (shingles) is the recurrent infection that occurs later in life.

The influenza virus, which is a Class V negative (-) sense RNA virus, has a genome that serves directly as mRNA, and can be used to synthesize viral proteins immediately after entry into the host cell cytoplasm.

FALSE mRNA is a (+) sense strand of RNA. The influenza virus, a negative (-) sense RNA virus, has a genome that cannot serve directly as mRNA.

An indirect immunoassay can be used to detect viral antigen in patient specimens.

FALSE Indirect immunoassays are immunologic techniques that can identify antibodies for a specific virus in clinical samples. Viral antigens are detected using direct immunologic techniques.

Viremia is a term used to describe a localized viral infection, in which the virus replicates and remains at the primary site of entry.

FALSE Viremia is a term that indicates the virus is present in the blood stream.

Hepatitis E Virus (HEV) is one of the most common causes of acute viral hepatitis in the US.

FALSE; HEV infection is uncommon in US, and is usually the result of travel to developing country where HEV is endemic.

HPV DNA is found in 99.7% of cervical cancer specimens, and is the only HPV-associated cancer seen in humans.

FALSE; In addition to the association of HPV in 99.7% of cervical cancers, data from CDC provided the following estimates of HPV-associated cancers: 90% of anal squamous cell carcinomas 65% of vaginal cancers 50% of vulvar cancers 35% of penile cancers 60% of oropharyngeal cancers (95% associated with HPV-16 or HPV-18)

Most HAV infections are symptomatic, usually featuring fever and jaundice.

FALSE; Most HAV infections are asymptomatic and are detected only by presence of antibody.

Specimen Transport and Storage

Specimen Transport: 1.) Specimens collected for detection of virus should be processed as soon as possible 2.) Every attempt should be made to process the specimen within 12 to 24 hours after collection 3.) Specimens for viral isolation should not be allowed to sit at room or higher temperatures 4.) Specimens are generally kept cool (4°C) and immediately transported to the laboratory. 5.) Viral Transport Media: can be used for primary specimens or swab specimens - Can stabilize viruses and infected cells for up to 48 hours Specimen Storage: 1.) For storage of up to 5 days, specimens can be held at 4°C. 2.) Storage for up to 6 days or longer should be at -20°C. Specimens to be frozen should be placed in viral transport medium.

Binding of the viral attachment protein (VAP) to the receptor on the host cell surface determines the tissue tropism of the virus.

TRUE Binding of the viral attachment protein (VAP) to the receptor on the host cell surface determines the tissue tropism of the virus.

Herpesviruses obtain their envelope by budding from the nuclear membrane.

TRUE Herpesviruses do obtain their envelope by budding from the nuclear membrane.

IgM antibodies are the first antibodies produced on initial exposure to a virus, and are usually transient (temporary), typically dropping below detectable limits in 3 to 6 months.

TRUE IgM antibodies are the first antibodies produced on initial exposure to a virus, and are usually transient (temporary), typically dropping below detectable limits in 3 to 6 months.

The early proteins are regulatory proteins and enzymes, while the late proteins may include the capsid proteins.

TRUE The early proteins are regulatory proteins and enzymes, while the late proteins may include the capsid proteins.

The host range refers to the host species a particular virus is capable of infecting.

TRUE The host range does refer to the host species a particular virus is capable of infecting.

In Real-Time PCR (QPCR), the production of viral DNA is measured by an increase in fluorescent signal due to the binding of a probe labeled with a fluorescent molecule to the amplified DNA.

TRUE In Real-Time PCR (QPCR), the production of viral DNA is measured by an increase in fluorescent signal due to the binding of a probe labeled with a fluorescent molecule to the amplified DNA.

In virus-infected cells, the cytopathic effect (CPE) is the damage, morphological and functional, inflicted on the cell by the virus.

TRUE In virus-infected cells, the cytopathic effect (CPE) is the damage, morphological and functional, inflicted on the cell by the virus.

A genital infection with one of the high-risk (HR) HPV types which persists for years is a primary risk factor for the development of cervical cancer.

TRUE; A genital infection with one of the high-risk (HR) HPV types which persists for years is a primary risk factor for the development of cervical cancer.

A portion of individuals infected with SARS-CoV-2 are asymptomatic, but can still spread the disease.

TRUE; Approximately one third of individuals infected with the virus remains asymptomatic, but can still spread the disease.

Norovirus (e.g. Norwalk virus) is a common cause of viral gastroenteritis in adults.

TRUE; Norovirus (e.g. Norwalk virus) is a common cause of viral gastroenteritis in adults.

With influenza viruses, the internal ribonucleoprotein is the group-specific antigen that distinguishes influenza A, B and C viruses.

TRUE; With influenza viruses, the internal ribonucleoprotein is the group-specific antigen that distinguishes influenza A, B and C viruses.

Electron Microscopy (EM)

a. Can play a significant role in characterizing viral morphology b. Not a standard clinical laboratory technique - used more as a research tool i. Magnetic coils use a beam of accelerated electrons as a source of illumination ii. Wavelengths of electrons allow electron microscopes to have a greater magnification and higher resolving power than light microscopes c. EM is most helpful in detecting viruses that don't grow readily in cell culture d. The cause of newly recognized viral syndromes can be recognized rapidly by identifying characteristic viral morphology in infected tissue

Antigen Detection

a. Direct immunologic techniques can be used to detect viral antigens in clinical samples - Indirect techniques are variations that can identify antibodies for a specific virus in clinical samples b. Direct antigen detection techniques use specific antisera that can detect viral protein in infected cells (such as the hemagglutinin antigen of influenza virus) c. The reagent antibodies used to detect viral antigens can be: i. Polyclonal: heterogeneous antibodies that can recognize many epitopes on a single antigen ii. Monoclonal: recognize individual epitopes on an antigen d. These antigen detection techniques allow detection of viruses that do not readily grow in cell culture e. Methods to detect viral antigen in patient specimens include: i. Fluorescent Antibody Techniques for the Detection of Viral Antigens - Fluorochromes absorb short wavelength light and emit energy at a longer wavelength - These fluorescent dyes (fluorochromes) are attached to antibodies specific for a viral antigen - Reactions are observed using a fluorescent microscope - Viral antigens will appear brightly illuminated against dark background a. Direct Fluorescent Antibody (DFA): - Used to detect viral antigen in the patient's specimen - Reagent antibody to viral antigens is tagged with a fluorescent dye, which can be visualized using a fluorescent microscope ii. Enzyme-Linked Immunosorbent Assay (ELISA) - A capture antibody specific for the virus is bound to solid support - Virus present in specimen (such as blood or body fluids) will bind to capture antibody - Virus-specific reagent antibody linked to an enzyme attaches to bound viral antigen - Substrate molecules capable of interacting with the bound enzyme are added - The enzyme will cause a biochemical change in the substrate, and the resulting color change indicates the presence of virus iii. Immunochromatographic methods - A liquid clinical specimen moves across the cartridge membrane by capillary action. - Viral antigen (Ag) in sample binds to specific conjugated antibody (Ab) in the membrane. - A visible line or dot forms as a complex of Ag-Ab coated colored particles are captured in test region

Regarding Human Immunodeficiency Virus (HIV), which of the following statements is NOT correct?

a. HIV preferentially infects and kills CD4+ T lymphocytes, causing a deterioration of immune functions. b. The envelope glycoprotein gp120 has a high mutation rate, resulting in antigenic variants. c. The three enzymes of HIV are reverse transcriptase, helicase and protease. d. Patients in the late stage of infection (AIDS) are subject to opportunistic infections and cancers such as Kaposi's sarcoma and lymphoma. answer: The three enzymes of HIV are reverse transcriptase, helicase and protease. explanation: The three enzymes of HIV are reverse transcriptase, integrase and protease. Enzymes called helicases unwind and separate the double-stranded DNA helix during DNA replication.

The period of viral replication in the host cell in which there is intense macromolecular synthetic activity producing numerous copies of viral nucleic acid and viral proteins, although there are no intact viral particles detected, is called the:

a. Infectious period b. Assembly period c. Eclipse period d. Activity period answer: Eclipse period

Which of the following is NOT one of the characteristics of a virus?

a. It is an obligate intracellular parasite. b. It cannot replicate within living cells. c. It cannot generate its own metabolic energy. d. It cannot synthesize its own proteins. answer: It cannot replicate within living cells.

Light Microscope

a. Light microscopy uses cytological or histological stains for a morphological study of infected cells or tissues in clinical samples b. The resolving power of brightfield microscopes does not allow visualization of the virus but can be used to detect cytopathic effect (CPE), which is the damage inflicted on the cell by the virus, including: i. Viral inclusions, which are the intracellular structures formed by aggregates of virus or viral components in the infected cell ii. Syncytial cells, which occur due to the fusion of cells to form multinucleated giant cells

Transcription

a. One strand of DNA is the template for synthesis of a complementary strand of messenger RNA (mRNA) using the enzyme RNA polymerase b. Ribonucleotides use complementary base pairing to synthesize RNA - As with DNA replication, RNA is synthesized in a 5' to 3' direction while copying the DNA template running in a 3' to 5' direction c. The ribonucleotides are covalently bonded together by phosphodiester bonds. d. Most genes contain non-coding regions (introns) interspersed among the coding regions (exons), as illustrated in the diagram to the right. e. Introns are excised (removed) and exons are joined together to produce mature mRNA. This process is called RNA processing. f. The mRNA molecule is divided up into codons, three consecutive mRNA bases coding for one specific amino acid. g. The mRNA travels to the ribosomes in the cytoplasm of the cell, where the proteins are assembled in a process called translation.

Viral proteins serve several important functions. Which of the following is NOT considered to be one of the functions of viral proteins?

a. They protect the viral genome from degradation by nucleases. b. They participate in the attachment of the virus particle to susceptible cells through specific host cell receptors. c. They are functionally equivalent to mRNA. d. They determine the antigenic characteristics of the virus. answer: They are functionally equivalent to mRNA.

Translation

a. Transfer RNA (tRNA) is a cloverleaf-shaped RNA structure with a loop containing three bases (anticodon) complementary to the mRNA codons. b. Translation begins with the start codon for translation, AUG. The tRNA having the anticodon UAC carries the amino acid methionine (Met). c. a. tRNAs with the appropriate anticodons pick up specific amino acids, transfer them to the ribosomes, and insert them in their proper position according to the mRNA codon "message." d. The amino acids are incorporated into the growing protein chain with peptide bonds. e. The sequential joining of amino acids to form a three-dimensional, functional protein is known as the elongation step. f. This process continues in the 5' to 3' direction until the ribosome hits a stop codon that terminates the protein chain. - UAA, UAG, and UGA are the stop codons.

Regarding influenza viruses, which of the following statements is NOT correct?

a. Viruses from other species can be the source of RNA segments that encode antigenic shift variants that can cause influenza A epidemics among humans. b. Because influenza B virus is only a human virus, there is no animal source of new RNA segments, so Influenza B virus does not undergo antigenic shifts. c. Hemagglutinin binds to cell surface receptors to initiate infection of the host cell. d. The ability of neuraminidase to spontaneously agglutinate red blood cells is the basis the neuraminidase inhibition test used in the diagnosis of influenza infections. answer: The ability of neuraminidase to spontaneously agglutinate red blood cells is the basis the neuraminidase inhibition test used in the diagnosis of influenza infections. explanation: Hemagglutinin spontaneously agglutinates red blood cells and is the basis the hemagglutination inhibition test used in the diagnosis of influenza infections.