Virology Exam

Mediums

A viral transport medium is recommended for most specimens. This medium contains buffered saline; a protein stabilizer; and antibiotics, such as penicillin, vancomycin, bacitracin, streptomycin, and amphotericin B, to suppress bacterial or fungal overgrowth. Several media are suitable for viral transport and commercially available, including Hank's balanced salt solution, veal infusion broth, sucrose- phosphate-glutamate broth, and Leibovitz-Emory medium. Collection systems consisting of a swab with a viral culture medium, such as Viral Culturettes (Becton Dickinson), are also available. Bacterial culturettes that contain Stuart transport broth also have been found to be suitable for viral transport.

Selected Medically Important Viruses

ADENOVIRUS HEPATITIS VIRUSES HERPES VIRUSES PAPILLOMAVIRIDAE PAPOVAVIRUSES POXVIRUSES ARBOVIRUSES FILOVIRIDAE PICORNAVIRIDAE ORTHOMYXOVIRIDAE PARAMYXOVIRIDAE RHABDOVIRIDAE REOVIRIDAE CALICIVIRUSES RETROVIRIDAE

ADENOVIRUS

Adenovirus is a member of the family Adenoviridae and the genus Mastadenovirus. The virus has linear double-stranded DNA, icosahedral symmetry, and a size of 70 nm to 90 nm. Adenovirus does not have an envelope, but each capsomer possesses a long fiber for attachment to the host cell. Adenovirus was first isolated from spontaneous degeneration, which occurred in adenoid tissue. The virus is commonly isolated in children, and most individuals have antibody to adenovirus by 5 years of age. It is generally transmitted by the respiratory or fecal-oral route. Adenovirus is associated with sporadic or epidemic respiratory tract infections, urinary tract infections (UTIs), gastrointestinal tract (GI) infections, and eye infections, primarily in newborns and immunosuppressed patients. Adenovirus also causes an exudative pharyngitis in children and a severe diarrheal disease in newborns and immunosuppressed persons. Adenovirus may remain latent, without causing infection, and can be isolated from the tonsils, eyes, or respiratory secretions. Specimens to be collected include throat swabs, nasal washings, conjunctival swabs or scrapings, or feces. The virus can be isolated in tissue culture, and serological detection of antibody production also is available.

Human Herpesvirus Type 6 (HHV-6)

Another member of the subfamily Gammaherpesvirinae, HHV-6 was originally known as "human lymphotropic virus" because it first was detected infecting B cells in vitro. The virus was first isolated in saliva and mononuclear cells of peripheral blood in 1986. It is now known that the virus primarily infects T lymphocytes, where it remains latent. The route of infection is most likely through respiratory contact. HHV-6 is the agent of exanthem subitum, or roseola, also known as sixth disease. Roseola is a benign childhood disease most often seen in children ages 6 months to 3 years. Symptoms of HHV-6 include a sore throat and high fever, which persist for 3 to 5 days. A nonspecific maculopapular rash of the trunk and neck develops 24 to 48 hours after the fever subsides. The virus rarely produces infections in adults, which include lymphadenopathy and a mononucleosis-type disease.

Arbovirus

Arbovirus infections are acquired from an arthropod bite, most often mosquitoes but also from ticks and mites. The mosquito vector takes a blood meal from an infected vertebrate host, and the virus multiplies in the midgut of the mosquito. The virus circulates to the mosquito's salivary glands, where it replicates in large numbers. Now, when the mosquito bites another host, such as humans, it is able to transmit the virus. The virus circulates in the blood and contacts susceptible target cells, such as monocytes, macrophages, and the endothelial cells of blood vessels. These diseases range from a mild systemic disease, encephalitis, to arthrogenic disease and hemorrhagic fevers. Infection is characterized by fever, chills, headache, backache, and flu-like symptoms. After 3 to 7 days, primary viremia occurs, which leads to a mild systemic disease. There is a second viremia during which additional virus copies are made, which then attach to target organs, such as the liver, brain, skin, or blood vessels. Flaviviruses generally attach to monocytes and macrophages. Humans are usually an accidental host, with birds, small mammals, reptiles, and amphibians serving as the reservoir hosts. Humans are the "dead end" host for many arboviruses because the virus cannot spread back to the vector unless there is significant viremia. Thus, the life cycle of the virus ends within the human host. Western equine encephalitis (WEE) is found in the western United States and Canada, eastern equine encephalitis (EEE) is found in the eastern and southern United States, and Venezuelan equine encephalitis (VEE) is found in South and Central America. All of these viruses are transmitted to animals, including horses and birds, by the mosquito vector; humans serve as accidental hosts. In most cases, the virus usually dead ends in the human host.

Blood donations and HBV

Before the screening of blood donors for HBV, the virus was the major cause of post- transfusion hepatitis. Today, HBV transmission through donor blood is very rare in the United States. Hepatitis B infection is controlled through carefully screening blood donors, sterilization of dental and medical instruments, practice of blood-borne or standard precautions, and passive immunization through the HBV vaccine. The HBV vaccine is a series of three intramuscular (IM) doses of synthetic HBV. It is included in the childhood vaccination schedule and also required for most health care occupations. Newborns are vaccinated with the hepatitis B vaccine at birth, with the second dose given at 1 or 2 months, and the third dose given at around 6 months of age. In adults, after the initial injection, immunizations are given at 1 month and then at 6 months. Hepatitis B immune globulin may be given following suspected exposure. Other preventive measures include adherence to blood and body fluid precautions; the use of personal protective equipment, including gloves and eye protection; and precautions when exposed to blood or body fluids, needles, and other sharps. Disinfection of surfaces with 10% bleach is a suitable surface disinfectant.

Blood

Blood is generally not cultured in suspected viremia because of the low virus yield and because the detectable viremic phase is usually gone by the time that symptoms become present. Blood should be collected, however, from patients with suspected viral hemorrhagic fevers caused by adenovirus and with arboviral infections. In these patients, 5 ml to 10 ml of blood should be collected into either an EDTA-, acid-citrate-dextrose-, or heparin-anticoagulated blood collection tube.

CALICIVIRUSES

Calicivirus, commonly known as Norwalk or Norwalk-like viruses, is in the family Caliciviridae. Characteristics of the Caliciviridae include single-stranded RNA, icosahedral symmetry, no envelope, and a diameter of 25 nm to 35 nm. The virus is named for Norwalk, Ohio, where an outbreak of diarrhea occurred in 1968 in schoolchildren and their teachers and families. The virus is associated with epidemics of mild gastroenteritis, predominantly in children 6 years of age or older. Calicivirus infection is spread by the fecal-oral route, with virus shedding occurring in the stool as well as through contaminated food and water. Infectious outbreaks have occurred in hospitals, nursing homes, schools, restaurants, and on cruise ships. Often, the source of infection is a food handler who has contaminated foods with the virus. Caliciviral antigens can be detected using enzyme-linked immunoassay or the polymerase chain reaction technique to identify the virus. Electron microscopy also can be used.

Cytomegalovirus

Can be transmitted through direct contact with saliva, via blood transfusions, and through organ transplants. It typically produces asymptomatic or mild infection in healthy individuals, and approximately half of the population is seropositive by age 30. Formerly known as "salivary gland virus," CMV has been isolated from saliva, urine, feces, milk, and semen. Repeated contact with the virus is necessary for infection. The virus can remain latent in white blood cells, endothelial cells, and other organs. CMV is the most common congenital viral pathogen. Congenital CMV may occur as a primary infection during pregnancy if the mother has CMV infection and lacks immunity to the virus. Reactivation of a latent viral infection in the mother also may result in congenital CMV. Although some infants are asymptomatic at birth, developmental abnormalities, mental retardation, or deafness may occur later. Symptoms of congenital CMV include jaundice, hepatosplenomegaly, growth retardation, mental retardation, microencephaly, and lung disease. Perinatal CMV may occur during delivery if the mother is infected, or the virus may be acquired through the mother's breast milk. CMV is an important pathogen in transplant patients. The virus can be acquired if a seronegative recipient receives a transplanted organ positive for CMV. Post-transplant CMV infection manifests as a pneumonia more than 1 month after the transplant and thus is known as the "40-day fever." CMV also can be acquired through transfused blood when the donor is CMV positive and seropositive and the recipient is seronegative. For this reason, blood products are screened for CMV, and only CMV-negative units are given to those at an increased risk of infection, such as newborns, the immunosuppressed, and patients with ransplants. In immunocompromised hosts, CMV may lead to pneumonia, retinitis, and esophagitis. Specimens for the identification of CMV include urine, saliva, tears, milk, and semen, and vaginal secretions also can be collected. Direct cytological examination reveals the characteristic "owl eyes"—large cells with large intranuclear, basophilic staining inclusions. These inclusions are found in all body tissues but are generally identified in urine specimens and visualized with the Pap or hematoxylin-eosin staining. The virus can be isolated in cell culture, such as diploid fibroblasts, and in shell vials. CPE is typically observed in 4 to 6 weeks and appears as the rounding of cells. Viral antigens can be detected using immunofluorescence or enzyme-linked immunoassay on blood, urine, and bronchial or other biopsy specimens. There also are polymerase chain reaction methods available to identify CMV. Serological tests often are not very helpful because they cannot differentiate among primary, reactivated, and persistent infections. The use of paired sera is more useful; latex agglutination and ELISA are available to detect CMV antibody. Prevention of CMV infection is through the screening of blood donors and organ donors. Seropositive products should not be transfused into seronegative recipients. CMV infections are treated with ganciclovir, valganciclovir, and foscarnet.

heteroploid

Cell lines with less than 75% normal cells are known as ___________________, or immortal, cell lines. More than 25% of the cells have an abnormal karyotype when compared with the normal cells of the tissue type of the primary cell culture. Immortal cell lines are generally derived from malignant tissue or other transformed cells. These cell lines can undergo continuous or unlimited subcultures or passages in vitro. Examples of heteroploid cell lines include HeLa, derived from human cervical carcinoma and named for the individual from whom the cells were isolated; Hep-2, derived from carcinoma of the human larynx; KB, derived from nasopharyngeal carcinoma; A-549, derived from human lung carcinoma; and Vero, derived from AGMK. HDFs are sensitive to HSV, VZV, CMV, adenovirus, and rhinovirus. Heteroploid cell lines are useful in the detection of HSV and the enteroviruses and adenoviruses.

SPECIMEN SELECTION, COLLECTION, AND PROCESSING

Clinical and epidemiological considerations are important in the selection of specimen type and the determination of possible viral agents. In general, it is optimal to collect the specimen as early in the course of disease as possible. For most viral infections, the viral titer is highest during the first 4 days after the onset of symptoms. Exceptions include enterovirus, adenovirus, and cytomegalovirus (CMV) because prolonged shedding of enterovirus and adenovirus into the stool and CMV into the urine occurs. It is best to sample the infected site directly. For example, for skin infections, the vesicles or rash should be sampled; for upper respiratory tract infections, the throat, nasal secretions, or nasal washing is cultured. In patients with disseminated viral infections or nonspecific clinical findings, it may be necessary to culture multiple sites. An exception is certain viral infections of the central nervous system (CNS), when it may be recommended to culture the stool or throat instead of the cerebrospinal fluid (CSF). In these patients the virus is typically shed in the stool or found in the throat. Swabs with cotton, rayon, or Dacron on plastic shafts should be used because wood is toxic to viruses. Calcium alginate may bind and inactivate the virus, and charcoal is toxic to several viruses, and thus neither should be used to collect viral specimens. Exanthems include vesicular rashes and maculopapular rashes. Vesicular rashes may be associated with HSV, VZV, coxsackievirus, and echovirus. Maculopapular rashes may result from infection with measles, rubella, the enteroviruses, CMV, EBV, parvovirus B-19, and human herpesvirus type 6 (HHV-6). Throat swabs or washings and stool cultures also may be collected to isolate these viral agents. If CMV is suspected, urine, EDTA-anticoagulated blood, and serum for acute-phase serology also should be collected. Acute and convalescent sera are recommended for the diagnosis of EBV, rubella, and measles. Ophthalmic viral infections include conjunctivitis and keratitis. Viral agents associated with these infections include HSV, adenovirus, and VZV conjunctival swabs; corneal or conjunctival scrapings are suitable specimens. Viral agents associated with lymphadenopathy include EBV, CMV, and HIV. Urine or throat swabs should be collected for CMV, and serum for serology should be collected for HBV and HIV. Viral agents associated with immunosuppressed individuals include CMV, HSV, and VZV. Urine and throat swabs are recommended for CMV, and swabs of vesicular fluid should be submitted for HSV and VZV.

Coxsackie and Echo Viruses

Coxsackieviruses are named for Coxsackie, New York, where the viruses were first isolated. Coxsackieviruses A and B are associated with aseptic meningitis, paralysis, pharyngitis, myocarditis, and rash. Coxsackievirus A also is the agent of hand-foot-and- mouth disease and hepangina, which has symptoms of fever, sore throat, anorexia, and ulcerated lesions in the mouth. Echovirus has been isolated in cases of aseptic meningitis, fever, respiratory infections, and paralysis.

cytopathogenic effect

Cultures are examined for CPE using an inverted light microscope or a phase- contrast microscope with the low-power objective (4X or 10X magnification). The type of CPE depends on the infecting virus and type of cell line used. Examples of CPE include cells rounding, clumping, vacuolation, granulation, giant multinucleate cells, cell fusion or syncytial formation, cell destruction, and lysis. CPE may be observed in discrete cells or may involve the entire monolayer. CPE is quantitated based on the percentage of cells showing cellular changes or damage on a scale of +/− to 4 +. CPE that is rated 2 + or more (50% of cell monolayer exhibiting CPE) requires identification of the virus through an immunological technique. CPE = cytopathogenic effect

HEPATITIS VIRUSES

Currently, there are five recognized hepatitis viruses: hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV), previously known as non-A, non-B (NANB); hepatitis D virus (HDV); and hepatitis E virus (HEV). Viral hepatitis, or infectious disease from the hepatitis viruses, may range from mild and self-limiting disease to acute fulminating cirrhosis; chronic disease and asymptomatic carriage also are possible. The severity and course of the disease depend on the particular virus and the state of the host. Hepatitis is diagnosed serologically through the identification of specific antigens or antibodies. Also, liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LD), and alkaline phosphatase may be increased up to 10 times the normal value in hepatitis. The serum bilirubin level also is increased in symptomatic patients.

Cytological or Histological Examination

Cytological or histological examination requires properly fixed and stained cells from an appropriate specimen. The presence of multinucleate giant cells or cytoplasmic or nuclear inclusions may aid in the diagnosis of a particular viral infection. DNA viruses usually are assembled in the nucleus and thus produce intranuclear inclusions, whereas RNA viruses, which are usually assembled in the cytoplasm, typically produce cytoplasmic inclusions. HSV and VZV produce intranuclear inclusions, which can be observed in cells from the cutaneous lesions. CMV may produce the characteristic "owl eyes" inclusions. In general, however, cytological examination is insensitive and nonspecific.

Diagnosis of HBV

Diagnosis is made through clinical signs and serological testing. Currently, six HBV markers can be used in the diagnosis of infection. Tests for HAV also may be necessary if a differential diagnosis is to be made. The serological tests available for the diagnosis of HBV infection currently are as follows: HBsAg. = HBsAg is present at the onset of infection. It is the first serological marker to appear and can be detected 30 to 60 days after exposure. HBsAg disappears as the liver enzymes return to normal and the patient recovers. Persistence of HBsAg for more than 6 months may indicate chronic infection. HBsAg is present in acute, active, chronic, and carrier states of HBV infection. HBeAg. = HBeAg is present in acute and chronic hepatitis and is a marker of infectivity. Persistence of HBeAg usually indicates chronic liver disease. It usually appears soon after HBsAg. Anti-HBsAg. = Anti-HBsAg is the total antibody to HBsAg. It appears 2 to 6 weeks after HBsAg is gone and usually persists for life. The presence of anti-HBsAg generally indicates recovery or immunity after the HBV immunization. Anti-HBcAg-IgM. = Anti-HBcAg-IgM is an indicator of recent acute infection; it is usually present for 6 months. Acute infection is indicated when anti-HBcAg-IgM is present with HBsAg. Anti-HBcAg. = Total antibody to HBcAg (IgG and IgM) appearing after HBsAg but before anti-HBsAg appears; thus it sometimes is referred to as the "core window." It is present in high levels at the onset of symptoms but drops to low levels, and levels may persist for 5 to 6 years. Anti-HBcAg indicates current or previous infection and is not associated with recovery or immunity. Anti-HBeAb. = Anti-hepatitis Be antibody does not appear until HBeAg disappears; it is usually associated with a favorable outcome, recovery, and reduced infectivity. HBV-DNA = demonstrates the presences of virus particles in the specimen. It is an indicator of infectivity.

HIV diagnosis

Diagnosis of HIV infection is based on clinical symptoms, patient history, and serological testing. Several commercially available ELISAs are available as screening tests. The confirmatory test for HIV, however, is the Western blot, which identifies antibodies for various HIV antigens. Antibodies to HIV p24, a Gag protein, and either gp4l or gp160, envelope antigens, usually confirm HIV infection. In the dot blot procedure, the patient's serum is added to a nitrocellulose sheet to which are bound HIV antigens. In a positive test, the patient's HIV antibodies bind to the HIV antigens. Next a labeled antihuman antibody is added, which will bind to the antigen-antibody complex. A positive reaction appears as a dot on the nitrocellulose sheet. HIV-reverse transcriptase PCR is used to determine viral loading and the number of copies of HIV present. Molecular diagnostic methods continue to be developed and are available to identify and quantify HIV. Treatment of HIV infections involves the use of several types of therapies. These include reverse transcriptase inhibitors, nucleotide/nucleoside analogues, and protease and fusion inhibitors. Treatment of the secondary opportunistic infections also is an important part of treatment. HIV-2 infections, although rare in the United States, are found more frequently in West Africa, South America, and certain parts of Europe. HIV-2 produces an infection similar to HIV-1, with different opportunistic pathogens seen and a milder disease course.

Electron Microscopy

Electron microscopy is a sensitive tool, although it is often not available in many microbiology laboratories. In negative staining using electron microscopy, clinical material is placed on a carbon-coated grid and then stained with potassium phosphotungstate or uranylacetate. Stain surrounds the virus, and the electron beam cannot pass through the metallic background. However, the beam can pass through the virus. The virus is seen as a light structure against a dark background. The virus particle can be examined for size and shape. Electron microscopy is the only way to detect Norwalk agent, astroviruses, caliciviruses, and coronaviruses because these viruses do not grow in conventional cell cultures and tests for viral antigens are either unavailable or inaccessible.

Epstein-Barr Virus

Epstein-Barr virus (EBV), or HHV-4, is a member of the subfamily Gammaherpesvirinae and was first isolated from malignant Burkett's lymphoma cells in African children. The virus also is the cause of infectious mononucleosis. EBV is shed in the saliva and transmitted through oral contact. The virus incubates for 1 to 2 months, during which time it disseminates to the reticuloendothelial system, including the liver, spleen, and lymph nodes. The most common signs of infectious mononucleosis are lymphadenopathy, splenomegaly, and exudative pharyngitis. Other symptoms include high fever, sore throat, enlarged tonsils, hepatomegaly, malaise, and elevated liver enzymes. Recovery usually occurs in 2 to 3 weeks; however, complications may occur and include rupture of the spleen, hemolytic anemia, and encephalitis. EBV infection can recur and cause recurrent tiredness, fever, and headache. Infectious mononucleosis is diagnosed clinically and through hematology and serological testing. The differential WBC count typically shows a lymphocytosis with over 50% lymphocytes and the appearance of several reactive or atypical lymphocytes. The presence of heterophile antibody can be detected using one of several commercially available slide tests. Heterophile antibodies are defined as those antibodies that occur in one species but that react with antigens of different species. This antibody may be present in the serum of those individuals with infectious mononucleosis or in those with serum sickness, as well as in some healthy individuals or nonaffected individuals, where they are known as Forssmann antibodies. Heterophile antibodies will agglutinate sheep and horse red blood cells in cases of infectious mononucleosis and serum sickness and in those who are not affected. Guinea pig cells are used to absorb human serum and will remove the agglutinins of serum sickness and Forssmann antibodies, leaving the agglutinins of infectious mononucleosis. There also are serological tests that detect IgM antibodies to EBV. African Burkett's lymphoma is a monoclonal B cell lymphoma of the jaw and face, which may also involve the kidneys, liver, and adrenal glands. It is endemic in children in Africa, where malaria is prevalent. It is believed that the malaria parasite acts to promote EBV involvement with the lymphoma. EBV also is associated with some types of nasopharyngeal cancer.

Hepatitis A Virus (HAV)

Hepatitis A virus (HAV) is a member of the family Picornaviridae in the genus Enterovirus. The virus possesses single-stranded RNA, + sense, icosahedral symmetry, and no envelope and ranges in size from 24 nm to 30 nm. HAV is a very stable virus, can withstand temperatures of 56°C to 60°C for 1 hour, and is acid stable. However, HAV is destroyed by autoclaving for 30 minutes, boiling for 20 minutes, or using dry heat (160°C) for 1 hour. HAV infection is spread through the fecal-oral route via ingestion of the virus present in infected food or drinks or on contaminated objects. A common route of infection is through food contaminated by an infected food handler. Infections are facilitated by poor sanitation, international travel, crowded conditions, and poor personal hygiene practices. Another route of infection is through the ingestion of contaminated shellfish, such as clams, oysters, and mussels. Raw or improperly treated sewage that enters the water supply is another source of infection. The virus can be spread through the congenital route, from mother to baby at birth, if the mother is acutely infected at this time. Children in school and day care, prisoners, and those with poor personal hygiene are at an increased risk. There were over 2,500 cases of acute HAV reported to the CDC in 2008 and almost 2,000 acute cases reported in 2009. There were approximately 1,000 deaths attributed to HAV in the United States in 2009. The HAV vaccine is credited with having decreased the number of cases of HAV in the United States; for example, there were approximately 30,000 cases of HAV reported in 1990. HAV enters the blood through oropharyngeal or intestinal epithelial cells and replicates in hepatocytes and Kupffer cells. After exposure, the virus incubates asymptomatically for 15 to 40 days, with an average incubation period of 25 days. HAV infection may be asymptomatic or symptomatic; jaundice may or may not be present. Individuals become infectious during the latter part of the incubation period and remain so until 2 weeks after the appearance of symptoms. In fact, individuals are infectious 10 to 14 days prior to symptoms, and many exhibit no symptoms but are infectious. After incubation, a period of nausea, weakness, anorexia, and vomiting occurs; the individual also may have pain in the area of the liver. Chronic carriage of HAV and chronic HAV infection do not seem to occur. However, relapse is possible. In acute and symptomatic cases, classic symptoms include jaundice and elevated liver enzymes. However, many cases are asymptomatic. Acute disease lasts for about 1 week, and the disease is often self-limited. The severity of the disease depends on the host's age and health; less than 1% of cases result in mortality. Outbreaks generally arise from a common source, such as contaminated water or food or at a particular facility. HAV particles can be detected in the stool 10 to 30 days after infection. However, viral shedding is greatest before clinical signs. Diagnosis most frequently is accomplished through clinical signs and serological testing. If acute hepatitis is suspected, a differential diagnosis must be made to identify the type of hepatitis virus. Most often, serology to detect hepatitis B surface antigen and hepatitis B core antibody, as well as antibodies to HAV, is performed. Anti-HAV is a measure of the total antibody to HAV, which includes antibodies to both IgM and IgG. It is a measure of past infection to HAV. Antibody to IgM (HAV IgM) is a measure of IgM-specific antibody to HAV. Anti-HAV IgM is present in acute HAV infection. If anti-HAV is present and anti-HAV IgM is absent, a past HAV infection is indicated. Hepatitis A vaccine is available for long-term protection to HAV. Immune globulin may be given for short-term protection during the early incubation period.

Classification of Medically Significant Viruses = DNA

Family; Genus or group; Common name; Nucleocapsid; Characteristic shape and size (nm) Adenoviridae; Mastadenovirus; Adenovirus; DNA ds linear; Icosahedral, 70-90 Hepadnaviridae; Hepadnavirus; Hepatitis B (HBV); DNA ds circular; Icosahedral, 42-47 Enveloped Herpesviridae Subfamilies; -; -; DNA, ds Genome size varies with specific virus; Icosahedral, 100-200 Enveloped Alphaheredvirinae; Simplex virus; Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) Betaherpesvirinae; Cytomegalovirus; Cytomegalovirus (CMV) Gammaherpesvirinae; Lymphocryptovirus; Epstein-Barr virus (EBV) and Human herpesvirus type 6 and 7 Iridoviridae; Iridovirus; Aftrican swine fever virus; DNA ds; Icosahedral, 150-300 Papillomaviridae; Papillomavirus; Human papilomavirus (wart virus); DNA ds circular; Icosahedral, 45-55 Polyomaviridae; Polyomavirus; Polyomavirus strains JC and BK; DNA ds circular; Icosahedral, 40 Parvoviridae; Parvovirus; Parvovirus strains B-19 and RA-1; DNA ss linear; Icosahedral, 20-25 Poxviridae; Orthopoxvirus; Variola virus (smallpox), Vaccinia virus, Molluscum contagiosum, Monkeypox virus; DNA ds linear; Complex, brick or oval shaped 225x300

Classification of Medically Significant Viruses = RNA

Family; Genus or group; Common name; Nucleocapsid; Characteristic shape and size (nm) Arenaviridae; Areanavirus; Lymphocytic chloromeningitis (LCM) virus, Lassa fever virus, Machupo virus, Junin virus, Sabia virus; RNA ss linear + Sense; Helical, 100-130 Bunyaviridae; -; -; RNA ss linear + Sense; Helical, 80-100 spherical and pleomorphic Starting from genus Bunyavirus; La Crosse virus and California encephalitis viruses Phlebovirus; Rift Valley fever virus Nairovirus; Crimean-Congo hemorrhagic fever virus Hantavirus; Hantaan virus (Korean hemorrhagic fever virus) Caliciviridae; Calicivirus; Norwalk virus, Sapporo virus; RNA ss linear + Sense; Icosahedral, 25-35 Coronaviridae; Coronavirus; Coronavirus strains; RNA ss linear + Sense; Helical, 80-200 spherical and pleomorphic Filoviridae; Filovirus; Magburg virus, Ebola virus (hemorrhagic fevers); RNA ss linear + Sense; Helical, 80x800 filamentous and pleomorphic Flaviviridae; -; -; RNA ss linear + Sense; Icosahedral, 40-50 Starting from genus Flavivirus; Yellow fever virus, Dengue fever virus, St. Louis encephalitis virus, Japanese encephalitis virus Hepacivirus; West Nile virus, hepatitis C virus, hepatitis virus Orthomyxoviridae; Orthomyxovirus; Influenza viruses A, B, and C; RNA ss linear + Sense; Helical, 80-120 pleomorphic Paramyxoviridae; -; -; RNA ss linear + Sense; Helical, 50-300 enveloped and pleomorphic Starting from genus Paramyxovirus; Paramyxoviruses, Mumps virus Morbillivirus; Measles virus Pneumovirus; Respiratory syncytial virus, Human mentapneumovirus Picornaviridae; -; -, RNA ss linear + Sense; Icosahedral, 24-30 Starting from genus Human enterovirus; Poliovirus, Coxsackieviruses A and B, echoviruses Hepatovirus; Hepatitis A virus (HAV) Rhinovirus; Rhinoviruses types A, B, C Reoviridae; Reovirus, Orbivirus, Rotavirus; Reoviruses, Colorado tick fever, Rotaviruses; RNA ds linear; Icosahedral, 60-80 Rhabdoviridae; Lyssavirus; Rables virus; RNA ss linear + Sense; Bullet shaped, 75x80 Togaviridae; Alphavirus; Eastern, Western, and Venezuelan encephalitis viruses; RNA ss linear + Sense; Icosahedral, 60-70 Retroviridae Subfamilies; -; -; RNA ss; Icosahedral, 80-100 Oncornaviridae; Oncornavirus; Human T cell lymphotrophic virus types 1 and 2 (HTLV-1 and HTLV-2) Lentivirinae; Lentivirus; Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2)

FILOVIRIDAE

Filoviruses include Marburg and Ebola viruses. These are negative sense, single- stranded RNA viruses, which are enveloped and filamentous and vary in length from 800 nm to 1,400 nm with a diameter of 80 nm. They are causes of severe hemorrhagic fevers and are endemic in Africa. Ebola virus is named for the river in the Democratic Republic of Congo, where it was first discovered. Marburg virus was first discovered in laboratory workers in Marburg, Germany, who had contracted the disease from infected green monkeys. The filoviruses are endemic in wild monkeys and bats, and disease is transmitted to humans by direct contact with infected blood or secretions. Humans also may acquire the infection through contaminated needles. These hemorrhagic fevers begin with flu-like symptoms but then quickly develop into severe hemorrhages from many body sites, especially the gastrointestinal tract. Filoviruses require Biosafety Level 4 isolation techniques, which are not always readily available. The viruses may be grown in tissue culture or through animal inoculation. There also are direct immunofluorescent techniques available to detect viral antigen. Polymerase chain reaction can detect the viral genome in specimens. Serological testing also can be performed.

Viral agents

For viral infections of the respiratory tract, including pharyngitis and the common cold, nasopharyngeal secretions, aspirates or swabs, sputum, throat swabs, or bronchial washings are collected. Viral agents associated with upper respiratory tract infections include rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), Herpes simplex virus types 1 and 2 (HSV-1 and HSV- 2), and coronavirus. Viral agents associated with croup and bronchiolitis include the influenza and parainfluenza viruses, RSV, and adenovirus. Agents of pneumonia in children include RSV, the parainfluenza viruses, adenovirus, measles, and varicella- zoster virus (VZV). For adults, viral agents of pneumonia include the influenza viruses, HSV, VZV, CMV, and RSV. For suspected cases of aseptic meningitis, CSF, a throat swab, and stool cultures should be collected. Viral agents associated with aseptic meningitis include the enteroviruses, echoviruses, HSV-2, VZV, mumps virus, and lymphocytic choriomeningitis virus (LCM). Urine also should be cultured if mumps virus is suspected. Serum for serological testing should be collected in addition to cultures for HSV, VZV, LCM, and mumps.

Dane particle

HBV has a complex structure; the double-shelled form, known as the _______ ___________, is recognized as the whole-virus particle. The Dane particle consists of a core that is surrounded by a lipid envelope. Hepatitis B surface antigen (HBsAg) is the outer lipid component. This outer envelope circulates in the blood as a viral particle either bound to protein or as a free, noninfectious protein, which is spherical or tubular. There are several antigenic variants or subtypes of HBsAg, such as adr, adw, ayr, and ayw. The inner core of the virus contains HBV core antigen (HBcAg), which surrounds the partially double-stranded DNA and DNA polymerase, which is needed for viral replication. Hepatitis B e antigen (HBeAg) also is a component of the core antigen.

HIV structures

HIV has a complex structure and possesses an envelope, viral core, and other proteins, including the following: Group-specific antigen (Gag) proteins = code for retroviral core proteins and structural proteins. These are a part of the viral nucleocapsid and provide stability for the capsule. Gag proteins are p24, p17, and p7/p9. Polymerase (Pol) proteins = important in the viral life cycle. These include RNA-dependent DNA polymerase (reverse transcriptase), integrase, and protease. Envelope (env) proteins = are glycoproteins in the retroviral coat that adhere to target cells and produce cytopathic cell fusion. The envelope includes cell wall components of the host as the virus "buds." Env proteins include gp120 and gp142. Infection with HIV-1 can lead to acquired immunodeficiency syndrome (AIDS), which is characterized by loss of immune competency, opportunistic infections, and unusual neoplasms.

Herpes Simplex Virus (HSV-1 and HSV-2)

HSV infections occur throughout the world. HSV-1 infection is common in childhood, and most adults have antibody to this virus. HSV-1 generally produces more mild infections and is less resistant to treatment when compared with HSV-2. Transmission of the virus is through active ulcerations of the mucous membranes or the genitalia. HSV-1 causes oral herpes, gingivostomatitis, ulcerative mouth lesions, and fever blisters. The virus may spread to the lips and cheeks. Primary lesions may be accompanied by fever, malaise, and cervical lymphadenopathy. Most cases are mild or asymptomatic and resolve without treatment. Approximately 90% of all cases of primary herpes gingivostomatitis are attributed to HSV-1. HSV-2 produces 80% to 90% of all cases of genital herpes, a common sexually transmitted disease (STD). It is transmitted through sexual contact or from autoinoculation. Early signs of infection include fever, malaise, and inguinal lymphadenopathy, although some primary infections are asymptomatic. Primary lesions typically appear on the vagina, cervix, glans, or penile shaft. Recurrent lesions may occur. There is an association between HSV-2 and cervical carcinoma. Neonatal HSV-2 infections may result if the infant acquires infection during delivery from an actively infected mother. The virus typically attacks the infant's central nervous system, and developmental difficulties may be seen. Other infections associated with HSV include herpetic keratitis, which can lead to corneal scarring and blindness; herpetic whitlow, which infects the fingers of those who have contact with others who excrete the virus; and central nervous system infections, including sporadic encephalitis. Specimens that may be collected for diagnosis of HSV include aspirates or swabs of lesions or vesicles and conjunctival scrapings. The virus can be isolated using cell culture or shell vial (SVCE) with HeLa cells, human embryonic fibroblasts, and rabbit kidney cells with CPE observed within 1 to 7 days. CPE is typically observed as round, clumping, syncytial, giant cells. Direct examination for the virus also can be accomplished through electron microscopy and in the Pap smear. Viral antigen can be demonstrated using immunofluorescent or immunoperoxidase techniques. Serological testing is not very helpful because uninfected individuals may possess antibody and because antibody against HSV-1 and HSV-2 cannot be differentiated. There are HSV- specific DNA probes and DNA primers that differentiate HSV-1 and HSV-2. There is no vaccine currently available for HSV-1 and HSV-2. Infections may be treated with a variety of antiviral agents, such as acyclovir, valacyclovir, penciclovir, and famciclovir.

Herpes simplex virus

HSV is an important viral pathogen in the genitourinary tract. Diseases include urethritis, cervicitis, vulvovaginitis, and penile lesions. For HSV, vesicular fluid collected using a needle aspirate or cells collected from the base of the lesion are recommended. Endocervical swabs are also acceptable.

Hepatitis B Virus (HBV)

Hepatitis B virus (HBV) is a member of the family Hepadnaviridae and is a DNA- containing virus with a complex capsid and icosahedral arrangement. HBV is a 42-nm to 47-nm envelope with circular DNA, which is partially double stranded and partially single stranded. The virus is very stable and resists freezing, heating, and acidic conditions, which facilitates its transmission. In 2008, there were over 4,000 new cases of HBV reported to the CDC in the United States and over 3,000 new cases reported in 2009. There were almost 2,000 deaths reported to the CDC from HBV in 2009. The CDC estimates that there are between 700,000 and 1.4 million persons infected with HBV in the United States. HBV causes acute and chronic and symptomatic and asymptomatic disease; it is transmitted by the sexual, perinatal, or parenteral routes. The original name for HBV infection was "serum hepatitis" because of the contamination of vaccines in World War II with serum that contained HBV. This name is no longer valid because HBV is isolated from body fluids other than blood and blood products, including urine, amniotic fluid, semen, tears, saliva, feces, and CSF. Routes of infection include transmission through the transfusion of contaminated blood or blood products, accidental needlesticks, tattoos, ear piercing, sharing of contaminated razors, intravenous (IV) drug abuse, and hemodialysis. Actively infected mothers can transmit the virus to their infants through the congenital route. Individuals at risk include nonimmunized health care personnel, including laboratory workers, physicians, dentists, and medical personnel in hemodialysis units. Others at risk include those living in crowded quarters; intravenous drug users; institutionalized persons, including prisoners and the mentally disabled; those who receive frequent intravenous procedures; and homosexuals. After exposure, HBV may incubate for 50 to 180 days, with an average incubation time of 90 days. The virus replicates in the liver. The infection can be mild and asymptomatic, symptomatic, fulminant, or chronic. HBV disease has an insidious onset, with initial signs of fever, rash, or arthritis. Jaundice may occur shortly after initial signs and usually may persist for 4 to 6 weeks. Fulminant, fatal hepatitis and chronic hepatitis are possible complications of primary infection. An individual may test positive for HBsAg for years without evidence of liver disease, whereas other chronic carriers may develop hepatitis. HBV also is associated with primary hepatocellular carcinoma if the HBV genome is incorporated into the cancerous cells, which then express the HBV antigen. Patients with HBV infection typically have abnormal liver function, as evidenced by increased levels of the liver enzymes. Serum bilirubin level also is elevated.

Hepatitis C Virus (HCV)

Hepatitis C virus (HCV) is an RNA-containing virus with a lipid envelope, positive (+) sense, and a size of 30 nm to 60 nm. HCV is in the viral family Flaviviridae and in the genus Hepacivirus. Formerly known as non-A, non-B (NANB) hepatitis, HCV was first isolated in 1984. The genome encodes for a large protein, which is cleaved into structural and nonstructural proteins. There are different HCV variants, which are classified as types 1 through 6, and then further subtyped. HCV may be acquired parenterally through contaminated blood products, organ transplantation, hemodialysis, or intravenous drug use. Other possible routes of infection include the perinatal route and through sexual contact. HCV has a variable incubation rate, which ranges from 2 to 26 weeks after exposure, with an average incubation of 6 to 8 weeks. Disease is usually milder than with HBV, and many infected persons are asymptomatic, although persistent chronic infection may occur. There is a high level of chronic asymptomatic infection with HCV, which increases its rate of transmission. In fact, approximately 70% of those infected with HCV become chronic carriers; the CDC estimates that there are between 2.7 and 3.9 million persons living in the United States with chronic HCV infection. Before the development of a marker for its detection, HCV was the most frequent cause of post-transfusion hepatitis, responsible for approximately 90% of all cases. Before 1990, elevation of the liver enzyme ALT and the presence of anti HBcAg were used as markers for HCV infection. In 1990, the first ELISA test to detect antibody to HCV was made available. This test has subsequently been modified to detect earlier antigens. The serological marker is anti-HCV, which detects antibody to HCV but cannot distinguish between acute, chronic, or resolved infection. This antibody does not usually appear until 8 weeks after initial infection. The use of HCV-RNA tests is helpful in monitoring antiviral therapy and to confirm an active or resolved infection following a positive anti-HCV test.

Hepatitis D Virus (HDV)

Hepatitis D virus (HDV), or delta virus, is a small virus with a size of 35 nm to 37 nm, which possesses single-stranded RNA and is infective only in the presence of HBV. It is an incomplete virus, which can cause acute or chronic hepatitis only when HBV also is present. It is transmitted through routes similar to those of HBV, such as percutaneous or mucosal contact with infectious blood or body fluids, and is acquired through coinfection or superinfection in those who are infected with HBV. Serological testing to detect HDV antigen, IgM antibody to HDV, and total antibody to HDV is available. HDV is uncommon in the United States. Hepatitis B vaccination can prevent HDV in those persons who are not infected with HBV.

Hepatitis E Virus (HEV)

Hepatitis E virus (HEV) possesses single-stranded RNA and has a size of 32 nm to 34 nm. It is associated with enterically transmitted NANB hepatitis, which is spread through the fecal-oral route. HEV is uncommon in the United States, and most cases that are identified in the United States are attributed to foreign travel to a developing country. HEV is most prevalent in developing countries with poorly developed sanitation and contaminated water supplies. HEV has been associated with hepatitis outbreaks in several parts of the world, including Asia, the Middle East, Africa, and Central America. Natural disasters and living in refugee camps are other risk factors. The disease resembles HAV, and symptoms include fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, and jaundice. The infection is diagnosed clinically and through demonstration of HEV RNA or by identifying antibodies to HEV. HEV also should be considered in cases of hepatitis that cannot be attributed to serologic markers for hepatitis A, B, or C in someone who has traveled to an endemic area for HEV. Other laboratory diagnostic methods include Western blot assays, PCR, and electron microscopy.

Other Human Herpesviruses

Human herpesvirus 7 (HHV-7) also is the cause of exanthem subitum (sudden rash). Human herpesvirus 8 (HHV-8) is known as the Kaposi's sarcoma virus; it is associated with Kaposi's sarcoma, a cancer found in AIDS patients, and also is the cause of primary effusion lymphoma.

cell line

Once a primary cell culture is subcultivated, it is known as a __________ _____________. Diploid cell lines must have at least 75% of cells with the same karyotype as the normal cells of the tissue from which they were derived. Diploid cell lines can retain the diploid karyotype for approximately 20 to 50 subcultures before losing their viability. Examples of diploid cell lines include WI-38 and MRC-5, which are both derived from human embryonic lung, and human diploid fibroblasts (HDFs), which may be derived from human kidney or lung fibroblasts.

Papillomavirus

Human papillomavirus (HPV) has double-stranded circular DNA and an icosahedral structure, with a size of 45 nm to 55 nm. There are over 30 genetic types of HPV, which are causes of a variety of cutaneous lesions and benign growths, including plantar warts, common warts, and sexually transmitted venereal warts. Sexually transmitted diseases from HPV may be associated with neoplastic lesions, including cervical carcinoma. HPV is an oncologic agent for squamous cell carcinoma. HPV was identified as the cause of genital warts in 1907, and in 1984 HPV strain 16 was identified in some cervical carcinoma tumors; HPV strain 18 also was identified in cervical tumors in 1985. Using polymerase chain reaction, HPV-DNA was found in almost all cervical cancers in 1999. By 2003, a screening test using hybrid capture assay to identify HPV DNA in cervical specimens was available. Today, diagnostic methods are available to detect high-risk HPV types, which are those genetic types of HPV that present the highest risk for developing high-risk squamous dysplasia. The Hybrid Capture 2 (Digene) uses a nucleic acid hybridization assay probe to detect the DNA of 13 of the high-risk HPV types. There also are polymerase chain reaction techniques, which utilize a pool of 14 of the high-risk HPV serotypes, and there are specific probes for HPV 16 and 18. It is estimated that HPV strains 16 and 18 cause approximately 70% of cervical carcinoma and that strains 6 and 11 are associated with approximately 90% of genital warts. There is a licensed quadrivalent vaccine that protects against HPV 6, 11, 16, and 18. It is highly protective and recommended for the prevention of genital warts, persistent HPV infection, and cervical carcinoma.

ORTHOMYXOVIRIDAE

Important characteristics of this family include the presence of hemagglutinin antigen (HA) and neuraminidase antigen (NA). HA allows the virus to attach to sialic acid glycoproteins on the surface of the host red blood cells and respiratory epithelial cells. HA also promotes the fusion of the viral envelope to the host cell membrane and can hemagglutinate human, chicken, and guinea pig red blood cells. HA also elicits the neutralizing antibody response. NA permits virus entry into the host cell and also cleaves the sialic acid on the viral particle, which helps to release the virus from the host cells. Influenza viruses are spread through aerosols and respiratory droplets and incubate for 1 to 4 days. The infections are characterized by an abrupt onset, fever, chills, headache, muscle aches, and a dry cough. Typical types of infections include influenza, upper respiratory tract infections, tracheobronchitis, and pneumonia. Individuals generally recover within 1 week, but complications, including secondary bacterial pneumonia, may occur. Regional or national influenza outbreaks occur in the winter and are named for the viral structure and location of the outbreak. Strains of influenza A virus are named for the virus type, place and date of original isolation, and HA and NA antigens. Strains of influenza B virus are named for type, location, and date of isolation. An annual influenza vaccine offers protection against influenza. Each year, the vaccination is developed against the three viruses that are believed to be the most common. This is based on the virus type isolated in the previous year, as well as on other researched factors. Vaccinations for high-risk people, including young children, pregnant women, those with chronic health conditions, and those older than 65 years, is recommended as well as for health care workers and those who work with those in high- risk groups. In recent years, public health officials have recommended the influenza A immunization for the general population. Specimens collected for identification of influenza viruses include throat swabs and nasal aspirates or nasopharyngeal specimens. Rapid testing, direct immunofluorescence, and polymerase chain reaction can be performed on nasopharyngeal washings. The virus also can be isolated in cell culture and hemadsorption assay. There are rapid influenza diagnostic tests to detect the virus in nasal secretions, which can detect influenza A, influenza B, or both viruses. These are rapid tests that can be performed in the physician's office but suffer from low sensitivity and may have up to 30% false-negative results. Direct fluorescent antibody (DFA) stains detect influenza A or B virus in nasal secretions. These tests have higher sensitivity and specificity than rapid test methods. Viral culture is the gold standard for diagnosing influenza but requires from 3 to 10 days for results. It is useful in confirming positive rapid tests and can differentiate influenza A from influenza B, as well as from other respiratory viruses. Shell vial culture can reduce the incubation time to 24 to 48 hours. Reverse transcription polymerase chain reaction (RT-PCR) is a molecular method used to detect viral genetic material in nasal secretions. It is the most sensitive influenza test currently available. There also are serological tests to identify the immune response to influenza infection. Acute and convalescent samples are most helpful, and a fourfold titer is usually diagnostic for infection. Serological diagnosis through hemagglutination inhibition, a measure of the patient's antibody titer against the HA, also can be used. Antibodies to NA also may be detected. Both antibodies appear 1 to 2 weeks after infection and may persist for up to 4 months. Indirect immunofluorescent methods to detect IgG and IgM in influenza A and B also are available.

disseminated diseases

In cases of disseminated diseases, throat, nasal, or eye swabs; urine; blood; or skin scrapings may be collected. Swabs or scrapings should be placed into viral transport media, and 5 ml to 10 ml of urine should be collected into a sterile screw-cap tube. Blood should be collected into an EDTA-anticoagulated blood collection tube. Viruses that may be associated with disseminated diseases include the enteroviruses (coxsackieviruses, echovirus), CMV, HSV, VZV, and HBV. HBV may be diagnosed through serological testing, and acute-phase serum should be collected for HSV, VZV, CMV, and HBV.

LABORATORY METHODS IN VIROLOGY

Laboratory methods for the detection of virus infections include cytological or histological examination; electron microscopy; virus isolation in cell or tissue culture; shell vial centrifugation-enhanced virus detection; direct detection of viral antigens or genes; molecular methods, such as target gene amplification (PCR); and serological detection of viral antibody. Inoculated cell monolayers are usually incubated at 35°C to 37°C for the recovery of most viruses, although respiratory viruses are optimally recovered at 33°C. The cultures are generally incubated for 2 weeks because recovery is variable and dependent on the type of virus. HSV may be detected in as early as 5 to 7 days, while CMV may need up to 21 days for detection.

RHABDOVIRIDAE

Lyssavirus in the family Rhabdoviridae is the agent of rabies. The virus is enveloped, bullet shaped, and ranges in size from a diameter of 60 nm to 95 nm and a length of 130 nm to 350 nm. The genetic material is negative sense, single-stranded RNA, with helical symmetry. Rabies is found in both wild and domestic animals, including skunks, foxes, coyotes, raccoons, bats, dogs, and cats. Infection is transmitted through contaminated respiratory secretions, most frequently from the bite of an infected animal. Cutaneous transmission from infected secretions and inhalation of aerosolized virus are other routes of infection. Once introduced into the host, the virus binds to neurons, where it may remain for days or months. The virus next travels to the central nervous system, where the brain becomes rapidly infected. Next, the virus is disseminated from the central nervous system through neurons to those tissues that are highly innervated, such as the skin of the head and neck, salivary glands, and eye and nasal mucosa. Symptoms include fever, headache, pain, or itching at the site of infection and fatigue and anorexia in the prodromal period. Neurological symptoms appear within 2 to 10 days and include seizures, disorientation, hallucinations, and eventually paralysis. When in the central nervous system, severe encephalitis, accompanied by coma, convulsions, and death, occurs within 1 to 2 weeks following infection. Diagnosis is usually made through clinical signs and the medical history of a bite or direct contact with secretions of a possibly infected animal. The virus can be detected using direct immunofluorescence to detect viral antigen. Characteristic Negri bodies are found in the cytoplasm of infected brain cells. However, once rabies is apparent, the disease is often fatal. The rabies vaccine is administered in animal bite cases when it cannot be determined whether the bite or contact occurred with a rabid animal or from a domestic animal that has not had the proper immunizations. Prevention of rabies requires control of rabies in wild and domestic animals. The use of attenuated oral vaccinations in the United States, which are dropped into areas where rabies is a concern, have been an effective control measure.

Molecular Techniques in Virology

Method = Principal = Purpose Restriction fragment length polymorphism (RFLP) = Comparison of DNA = Distinguish different strains of same virus, such as HSV-1 and HSV-2 Gene probes in situ hybridization = Detect and locate specific genetic sequences = Viral probes for CMV and human papilloma virus- infected cells Polymerase chain reaction (PCR) = Amplifies a single copy of viral DNA millions of times to enhance sensitivity of detection = Detect DNA viruses Reverse transcriptase polymerase chain reaction (RT-PCR) = Converts viral RNA to messenger RNA to DNA prior to PCR amplification; amplifies RNA viruses = Detect RNA viruses Real-time polymerase chain reaction (real-time PCR) = Quantification of DNA and RNA virus in samples = Quantitate HIV genomic viral load

antigenic drift vs antigenic shift

Minor antigenic changes are known as antigenic drift, and these occur every 2 to 3 years. Drifts are a result of minor mutations in HA or NA and account for local influenza outbreaks. Major antigenic changes are known as antigenic shift and involve a reassortment of genomes among different strains. Antigenic shift is generally associated with a pandemic. Influenza A pandemics occur approximately every 10 years. Antigenic shifts occur because of the diversity of influenza A virus in its ability to infect and replicate in a variety of hosts, including humans, birds, pigs, and other animals. Because influenza B is primarily a human virus, there is no antigenic shift.

Morbillivirus

Morbillivirus, the cause of rubeola (measles), possesses hemagglutinin antigen (HA) and a hemolysin but does not possess neuraminidase antigen. Once a severe, acute, highly contagious childhood disease with epidemics seen every 2 to 3 years, the incidence has dramatically decreased because of successful vaccination. The virus is spread via respiratory secretions, nasal secretions, or coughing. Measles virus incubates in approximately 7 to 13 days, multiplying in the respiratory tract, before the first symptoms of fever, nasal drainage, headache, cough, and sore throat occur. A primary viremia occurs, and the virus is spread to the RES and viscera, where it multiples a second time. This is followed by a secondary viremia and the appearance of a characteristic maculopapular rash, initially behind the ears and then on other parts of the head. The rash characteristically moves down the body and heals in the order in which it first appeared. Measles vaccination is a part of the MMR given to infants at 15 months of age and to children at 4 to 6 years of age. Prior to the vaccine, almost all children had measles by age 15, and there were almost 500 deaths from measles each year. Because of vaccination, measles is seen much less frequently; in fact, on average there are about 50 cases each year in the United States, with most cases originating in another country. Sporadic cases of measles occur in the United States, with most associated with traveling abroad or from visitors to the United States from other countries. The CDC estimates that worldwide there are 20 million cases of measles and almost 200,000 deaths each year. Complications of measles infection are common and include pneumonia and encephalitis. Measles is diagnosed through clinical signs and usually not cultured because the virus is shed very early in the infection. The virus may be isolated in cell culture. Serological testing is available to determine whether an individual is seropositive to the virus.

Oncornavirus

Oncornavirus includes the genus Oncornavirus, of which human T cell lymphotropic virus type 1 (HTLV-1) and human T cell lymphotropic virus type 2 (HTLV-2) are members. HTLV-1, the first human retrovirus to be identified, is an oncogenic virus and the cause of adult T cell leukemia, lymphoma, and tropical spastic paraparesis. T cell leukemia is a malignancy of specific subtypes of T lymphocytes, which is characterized by dermal and bone involvement, including lesions of the bone. The second retrovirus to be isolated, HTLV-2, was originally isolated from a patient with hairy cell leukemia. Prevalence of both viruses is low in the United States but higher in other areas of the world. Transmission of the HTLV viruses is believed to occur through contaminated blood or blood products or through sexual contact.

Paramyxovirus

Paramyxovirus includes the parainfluenza viruses, mumps virus, and New Castle virus. Parainfluenza viruses cause mild upper respiratory tract infections as well as pneumonia, hepatitis, and meningitis. Infections are found worldwide. Parainfluenza virus type 1 produces laryngotracheobronchitis and croup, an infection seen in infants and characterized by difficulty in breathing and a hoarse, barking cough. These infections may cause subglottal swelling, which may block the airway. Parainfluenza infections are spread by direct contact from infected persons or through respiratory droplet infection. Respiratory tract specimens are collected for identification of the parainfluenza viruses. Direct immunofluorescence and polymerase chain reaction can be performed on nasopharyngeal washings, and the virus can be isolated in cell culture. Hemadsorption is recommended for cell culture techniques because not all the viruses produce CPE. Serological diagnosis is not recommended because of recurrent infections. Mumps virus causes parotitis, a painful infection of the parotid glands characterized by swelling behind the ears and difficulty swallowing. Mumps virus possesses both HA and NA antigens and a hemolysin. The virus is extremely contagious and transmitted through respiratory secretions in contaminated saliva. Mumps virus is most contagious just before and immediately after the parotid glands swell. The virus multiplies in the upper respiratory tract and in the adjacent lymph nodes. This is followed by a viremic phase, where the virus spreads through the blood to the testes, ovaries, thyroid gland, pancreas, and meninges. A complication that may be seen in adult males affected with mumps is inflammation of the gonads, which may lead to sterility. Once a common childhood disease, mumps infections are rare in those countries where there has been consistent immunization. The mumps vaccine was first introduced in 1967; today the MMR is given to infants at 15 months of age, with a second dose given at 4 to 6 years. Most cases today are seen in those parts of the world and in communities where immunizations are not available or where there are compliance problems. There also have been cases of mumps in young adults and college students whose immune status has waned or who were not immunized. Specimens collected to diagnose mumps infection include saliva, urine, and pharyngeal secretions. Mumps virus can be isolated in cell culture. Serological detection of IgM and IgG antibodies through enzyme immunoassay, immunofluorescence, and hemadsorption also are available. A fourfold increase in IgM antibody for mumps is considered to be diagnostic for mumps infection.

Parvovirus

Parvovirus is a small (18 nm to 26 nm), DNA-containing, nonenveloped virus with icosahedral symmetry. Parvoviruses are known to infect mice, hamsters, cats, and dogs; however, only parvovirus B-19 is known to cause human infection. The virus is the agent of erythema infectiosum, or fifth disease (after measles, rubella, varicella-zoster, and roseola), a childhood illness. The virus is spread through the respiratory route and characterized by fever and a unique "slapped-cheek rash." The virus has an affinity for red blood cell precursors, which may lead to a mild anemia; those with malignancies or other hematologic abnormalities may develop aplastic crisis or chronic anemia when infected with parvovirus B-19.

Poliovirus

Poliovirus occurs naturally only in humans and is disseminated through the fecal-oral route and through respiratory secretions. The virus is shed in oral secretions prior to symptoms and for approximately 1 month after symptoms are noted. Primary infection occurs in the respiratory tract, followed by viremia to various parts of the body, including the skin, heart, and meninges. The virus is cytolytic and infects skeletal muscle. The most common type of polio is known as asymptomatic, or abortive, poliomyelitis. Abortive polio, which is mild or asymptomatic, with a rapid recovery, accounts for approximately 90% of all cases of polio. Symptoms include headache, fever, and sore throat. Poliovirus also may cause an aseptic meningitis or nonparalytic poliomyelitis in about 1% to 2% of those affected. Polio also may extend into the central nervous system in 1% to 2% of these cases. The virus travels through the blood to the anterior horn cells in the spinal cord and motor cortex in the brain. The virus is cytolytic and attacks skeletal muscle cells as it travels along neurons to the brain, resulting in paralytic poliomyelitis, the most severe type of polio infection. It is characterized by destruction of large motor neurons in the spinal cord and is usually accompanied by paralysis of the limbs and the respiratory center. Polio vaccination began in the United States in 1955, when the first polio vaccine was approved. There are two formulations of the vaccine, both of which contain three strains of the poliovirus and induce an antibody response. The inactivated polio vaccine (IPV), first prepared by Salk, is formalin-killed virus and stimulates antibody production in the serum but not in the mucosa. The oral polio vaccine (OPV), first created by Sabin, is a live, attenuated vaccine given orally, which stimulates the production of both IgA and IgG. The OPV was recommended in the United States until 2000 because of the benefits of secondary spread and intestinal immunity. However, OPV presents a rare risk for vaccine-associated paralytic poliomyelitis (VAPP), which occurred in one child out of every 2.4 million, according to the CDC. Also, live poliovirus is shed in the stool of those vaccinated, which also presents a risk of infection for susceptible persons. Thus, the current recommendation for polio vaccination in the United States is the IPV, which is given as an injection to children at 2, 4, and 6 to 18 months, with a booster given at 4 to 6 years of age. There have not been any cases of wild polio in the United States in over 20 years, but the disease remains common in other parts of the world. OPV is used in other parts of the world, where the disease is endemic and where there is a high risk of transmission. Enteroviruses may be isolated from throat swabs, feces, CSF, urine, blood, and conjunctival swabs, depending on the site of the infection. Direct detection and serological testing are not recommended, and identification most often is accompanied through clinical signs and virus isolation in cell culture.

Storage

Prompt transport enhances the detection of the virus, but if a delay in processing the specimen is anticipated, specimens should be held at 4°C but not frozen. Freezing can destroy the infectivity of some viruses quickly and is recommended only when a prolonged delay of 24 hours or more after collection is expected. When freezing is required, viral specimens should be snap frozen to at least −70°C and transported in dry ice or with liquid nitrogen. Conventional freezing to −20°C is not suitable for storing specimens for virology.

Pneumoviruses: Respiratory Syncytial Virus

Respiratory syncytial virus (RSV), a member of the genus Pneumovirus, is a small virus with single-stranded RNA. RSV is pleomorphic and enveloped and does not contain hemagglutinin antigen (HA). Types of infections range from mild respiratory tract and ear infections to pneumonia and other severe respiratory diseases. RSV is a leading cause of pneumonia and bronchiolitis in infants and young children. RSV is spread through droplets of contaminated respiratory secretions, which enter through the eyes or nose. The virus attaches to the respiratory epithelial cells, and following cell-mediated immunity the bronchi are damaged. Necrosis and fibrin deposits clog the airways in the lung, leading to a severe respiratory infection. This acute respiratory tract infection may be fatal in infants and young children. RSV is a leading cause of hospitalization in infants and children less than 2 years old. RSV is very contagious and more common in the winter months. Almost all children have had RSV by the age of 4 years. The CDC estimates that there are between 75,000 and 100,000 hospitalizations related to RSV each year. Diagnosis is achieved by clinical signs and cultures, including nasal aspirates, throat swabs, nasopharyngeal specimens, or sputum. The RSV antigen can be detected directly using molecular assays and immunofluorescence on the specimens or in cell culture. Serological testing methods include immunofluorescence or complement fixation; the IgM antibody is present within 1 to 2 days. A fourfold increase in titer is considered to be significant. Treatment relies most on supportive care, including respiratory therapy. There is no vaccine for RSV.

Rhinovirus

Rhinovirus is the most frequent cause of the common cold. Other viruses that cause the common cold include coronavirus, adenovirus, respiratory syncytial virus, and parainfluenza virus. Rhinovirus infections are usually self-limiting, with symptoms of a mild respiratory illness, including nasal congestion. The virus incubates for 8 to 10 hours, and symptoms generally appear within 1 to 3 days following exposure. There are over 100 serotypes, and the virus is grouped into species, which are designated as species A, B, and C. Species A is found most often. Rhinovirus can survive at room temperature for 24 hours, and because it prefers a growth temperature of 33°C, the virus does not usually infect the lower respiratory tract. If rhinovirus does invade the lower respiratory tract, severe pneumonia may occur; this is more common in immunosuppressed persons. Rhinovirus is transmitted through contaminated hands and also through contaminated respiratory aerosol droplets. Those infected develop mucosal immunity, which doesn't reach the blood; thus, immunity is short lived. Because there are so many serotypes and there is short-term immunity, individuals can be repeatedly infected. Because of the large number of serotypes, immunizations have not been developed. Diagnosis is generally based on clinical symptoms, and identification is not attempted because of the large number of serotypes and the infections are most often mild. Rhinovirus infection is prevented through practicing thorough hand washing, avoidance of contact with those who have a cold, and good personal hygiene.

Shell Vial Centrifugation-Enhanced (SVCE) Virus Detection

SVCE virus detection involves centrifugation of the specimen onto virus-sensitive cells, which are grown on coverslips at the bottom of shell vials. SVCE produces a more rapid result than conventional cell culture because incubation time can be decreased to 1 to 5 days, depending on the virus. After incubation the cells are fixed, and a fluorescein- labeled monoclonal or polyclonal antibody specific for antigens of the virus is added. Detection of the antigen-antibody binding is accomplished through fluorescence microscopy. The detection time is shortened because viral gene products, or antigens, rather than viral CPE are detected. SVCE was originally developed to detect CMV early antigens, and methods are currently available for HSV, adenovirus, VZV, influenza A and B viruses, and RSV.

SEROLOGICAL DETECTION OF VIRAL ANTIBODIES

Serological detection of antibodies to a virus is an indirect indicator that infection or exposure to the virus has occurred in the individual's past. Serological methods are useful when the virus will not grow in cell cultures; when viral antigen, nucleic acid probes, or amplification methods are not available; or when the virus is in a site that cannot be readily cultured, such as in brain tissue. Within 1 to 2 weeks after a primary viral infection, virus-specific immunoglobulin M (IgM) antibodies will begin to appear. The first appearance of IgG occurs 1 to 2 days later. IgM levels peak in 3 to 6 weeks and drop to undetectable levels in 2 to 3 months. IgG levels peak in 4 to 12 weeks and remain for several months. In some types of viral infections, IgG may remain for years or life. Paired sera are recommended for serological detection of viral infections. The first specimen, known as the acute-phase specimen, is collected when the clinical signs first appear. The second specimen, known as the convalescent-phase specimen, is collected 2 to 3 weeks later, depending on the virus. Traditionally, a fourfold increase in antibody titer indicates a seropositive reaction and strongly supports a diagnosis of current infection. False-positive and false-negative reactions are important points to consider in serological diagnosis. Antibody detection is not useful in the diagnosis of chronic or recurrent viral infections, such as HIV and CMV. Antibodies may be produced indefinitely in such patients and do not necessarily indicate a current infection. The specific method(s) chosen for viral detection depends on the virus suspected, host factors, and laboratory capabilities.

Variola Virus: Smallpox Virus

Smallpox is an ancient disease, which has killed millions throughout recorded history. The virus is spread through direct respiratory contact and multiplies in the lymph nodes. This is followed by the viremic phase, with dissemination to various organs and the development of pox lesions. The rash occurs in a single crop, in contrast to chickenpox. Vaccinations were administered to those who lived or worked in potentially hazardous areas. The vaccine was produced using vaccinia virus as the carrier; because variola virus is a single serotype, the vaccine is very effective. The disease was considered to be eradicated, and in 1980 the World Health Organization (WHO) declared that the world was free of smallpox. All reference stocks of the virus were destroyed by WHO in 1996. However, some countries did maintain stocks of variola virus, which were stockpiled for believed intended use as agents of biowarfare. Today, smallpox virus is listed as a category A bioterrorism agent by the CDC.

Stool

Stool specimens should be collected in cases of viral gastroenteritis. Viral agents of gastroenteritis include rotavirus, Norwalk-like agent, adenovirus types 40 and 41, and calcivirus. Rectal swabs also may be submitted, although stool specimens are preferred. Direct antigen testing using enzyme-linked immunosorbent assay (ELISA) can be performed if rotavirus or adenovirus types 40 and 41 are suspected. Norwalk agent and calcivirus must be examined through electron microscopy.

HERPES VIRUSES

The Herpetoviridae (herpesviruses) are enveloped, DNA-containing viruses with icosahedral symmetry of approximately 100 nm in size. This viral family produces lytic, persistent, and latent viral infections, which can be reactivated and cause disease months and even years later. These viruses may remain latent in various body sites, including the white blood cells (WBCs) and peripheral nerves. Reactivation generally occurs from physical stresses, such as immunosuppression, chemotherapy, and other medical disorders. Infections of the herpesviruses generally are more severe in adults than in children. Asymptomatic infection is common, and patients may unknowingly carry and shed the virus. There are currently seven recognized herpes viruses: The subfamily Alphaherpesvirinae includes herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) and varicella-zoster virus (VZV). Subfamily Betaherpesvirinae includes cytomegalovirus (CMV), and the subfamily Gammaherpesvirinae includes Epstein-Barr virus (EBV), human herpes virus type 6 (HHV-6), and human herpes virus type 7 (HHV- 7).

Lentivirinae and Human Immunodeficiency Viruses

The Lentivirinae (Latin lenti, or "slow") include human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2), which are characterized by slow viral diseases with neurological manifestations. The viruses were first named HTLV-3 and LAV (lymphadenopathy virus) by researchers who discovered the virus in the United States and France in 1983, respectively. Later, the virus was named HIV-1, and a second HIV virus (HIV-2) was subsequently isolated. After being infected with HIV, some people develop a flu-like syndrome, which may occur 6 to 8 weeks after infection and persist for a few weeks, while others may have no symptoms. Circulating virus is present at this time, and antibodies develop within a few months of infection. This is followed by a long, asymptomatic phase, which may range from months to years. However, the virus may be multiplying within the host and affecting many organs in the body. Untreated early HIV can lead to kidney, liver, or cardiovascular disease and cancer. AIDS is the late stage of HIV infection, at which time the host immune system is severely devastated. HIV attacks the CD4-positive T lymphocytes, which decreases T helper cell activity and diminishes delayed type hypersensitivity. As the level of CD4-positive T cells declines, the characteristic symptoms of AIDS infection become apparent. These include opportunistic infections, such as Pneumocystis carinii pneumonia, CMV infections, cryptosporidiosis or isosporiasis diarrhea, candidiasis, toxoplasmosis, and mycobacterial infections. Other symptoms include fever, night sweats, loss of weight, and lymphadenopathy. The development of unusual neoplasms, in particular Kaposi's sarcoma, as well as anal carcinomas and B cell lymphomas, also may occur. Neurological damage, including encephalopathies, also may be present. The clinical disease now known as AIDS was first described in 1981; HIV was identified as the cause of AIDS in 1986. Routes of infection include sexual contact with an infected individual, intrauterine infection from an infected mother to the baby, and contaminated needles used in intravenous drug use. There also is the risk of transmission for health care workers through contaminated needle-sticks and other sharps and contact with mucous membranes through splashing. Many early cases of HIV-AIDS were diagnosed in homosexual males; however, today the infection is prevalent also in heterosexual populations. No evidence indicates that the virus is spread through the aerosol route or through casual contact. Standard precautions for blood-borne pathogens have been established and must be adhered to to decrease the incidence of HIV-acquired infections in the health care setting. Before 1985, HIV was transmitted through contaminated blood; blood products, such as antihemophiliac factor (Factor VIII); and organ transplants. All donor products are screened for HIV; in addition, blood donor screening includes a rigorous donor history, which probes lifestyle activities that make an individual more likely to acquire HIV. All HIV-positive donors are notified of their status and all blood products discarded.

PICORNAVIRIDAE

The Picornaviridae are a large, diverse viral family, which includes the enteroviruses and rhinoviruses. The family is characterized by the presence of single-stranded RNA, icosahedral symmetry, and absence of an envelope. The viruses have a very small size of 20 nm to 30 nm, which is described by the prefix "pico" in their names. The Picornaviridae cause a variety of infections, which include central nervous system disorders, such as aseptic meningitis and polio; myocarditis; and mild or asymptomatic respiratory disease. Often, poor personal hygiene, overcrowding, or substandard sanitation are factors in the acquisition of these infections. Widespread vaccination against poliovirus has dramatically decreased the number of cases of polio from the wild poliovirus in the United States, as well as in many other parts of the world.

ARBOVIRUSES

The arthropod-borne viruses, or arboviruses, include the families Togaviridae, Flaviviridae, and Bunyaviridae. Most members of these families include viruses that require an arthropod vector. All three viral families have positive sense, single-stranded RNA and are enveloped. The alpha viruses, or group A togaviruses, are the agents in various types of encephalitis, which is an inflammation and infection of the brain, most often as a result of a viral infection. The arboviruses include the togaviruses and the flaviviruses. Arboviruses can be identified serologically by demonstrating the specific viral IgM antibody through hemagglutination, enzyme-linked immunoassay, and latex agglutination. A fourfold increase in the IgM titer is generally diagnostic for infection. There also are polymerase chain reaction methods that identify viral RNA in blood or tissue. Animal inoculation or cell culture also may be used to isolate the arboviruses. Arboviruses are controlled by controlling the insect vector to reduce the breeding of mosquitoes. Infections are highest in the spring and summer months, when there are increased numbers of mosquitoes.

nucleocapsid

The capsid and nucleic acid compose the ________________________. Viruses that have an outer membrane, or envelope, surrounding the capsid are described as having enveloped nucleocapsids. The envelope is a phospholipid bilayer in which glycoproteins and matrix proteins are embedded. The matrix proteins serve to connect the envelope to the capsid, and the glycoproteins act as "spikes" to aid the attachment to host cells. Viruses with enveloped nucleocapsids are more resistant to unfavorable conditions, such as drying and pH changes; they are able to remain moist and are more readily transmitted via respiratory droplets, blood, and other tissues. All negative-sense RNA viruses are enveloped. Viruses that have only a protein coat and no outer envelope are described as having naked nucleocapsids. These viruses are more susceptible to drying and other environmental conditions

Enteroviruses

The enteroviruses include poliovirus, echovirus, the coxsackieviruses, and hepatitis A virus (HAV), which has been discussed previously under the hepatitis virus section.

Bunyaviridae

The family Bunyaviridae includes the genera Bunyavirus, Phlebovirus, Nairovirus, and Hantavirus. Bunyavirus includes the La Crosse virus, or California encephalitis virus. Bunyavirus infection is characterized by a fever and rash and encephalitis. It is spread by the mosquito vector, and vertebrate hosts include rodents, birds, and other small mammals. Phlebovirus is the agent of Rift Valley fever, which is spread through the fly vector. Vertebrate hosts include sheep, cattle, and domestic animals. Rift Valley fever is associated with encephalitis, conjunctivitis, and hemorrhagic fever. Hemorrhagic fevers are characterized by a petechial rash and ecchymosis, which occurs as the blood passes from ruptured blood vessels into the subcutaneous tissue. There also is epistaxis, or bleeding from the nose; hematemesis, or the vomiting of blood; and bleeding gums. Nairovirus is the agent of Crimean-Congo hemorrhagic fever. It is transmitted through the tick vector, and animal reservoirs include cattle, goats, and hares. Hantavirus, the agent of Korean hemorrhagic fever and Hantaan virus, does not require an arthropod vector. The animal reservoir is the deer mouse. Hantavirus infection causes a severe pulmonary syndrome with high fever and muscle aches. The infection may lead to pulmonary edema, respiratory fever, and shock.

PAPOVAVIRUSES

The family Papovaviridae is characterized by having double-stranded DNA, icosahedral symmetry, no envelope, and a size of 45 nm to 55 nm. Included in the Papovaviruses are polyoma strains JC and BK, which produce mild or asymptomatic infections. These infections are most likely spread through respiratory secretions.

REOVIRIDAE

The family Reoviridae includes the genera Reovirus, Rotavirus, and Orbivirus. Characteristics of the family include double-stranded RNA, icosahedral symmetry, no envelope, and a diameter of 60 nm to 80 nm. Rotavirus is a common cause of gastroenteritis and is associated with both sporadic and epidemic outbreaks. Infants ages 6 months to 2 years are most frequently affected. In fact, rotavirus is the most common cause of gastroenteritis in this age group, and almost all children have been infected by 5 years of age. Rotavirus is named for its appearance under electron microscopy as a double- shelled capsule that resembles a wheel (Latin rota). The virus is spread through the fecal-oral route and also may be associated with food- or water-borne infections. It is stable at room temperature and resistant to treatment with detergents and pH and temperature changes. Rotavirus also can survive in the stomach's acidic environment. In the past, electron microscopy was needed to identify Rotavirus. However, rapid latex agglutination and ELISA are now commercially available for detection of the viral antigen in the stool.

RETROVIRIDAE

The family Retroviridae includes the subfamilies Oncornavirinae and Lentivirinae. The Retroviridae viruses possess single-stranded RNA, icosahedral symmetry, an envelope, and a diameter of 80 nm to 130 nm. The retroviruses possess the enzyme reverse transcriptase, or RNA-dependent DNA polymerase, which can transcribe RNA into DNA. This is the "reverse" of the normal transcription of DNA into RNA. There also is an integrase enzyme, which enables the viral genome to be incorporated into the host cell.

VIRAL REPLICATION

The host may be exposed to a virus through respiratory aerosols or contaminated food or water; through the congenital route; through contaminated body fluids, such as blood or plasma; through animal or insect bites; or through endogenous latent infections. After transmission of the virus, viral replication occurs in the host cell. Adsorption = which involves the attachment of the virus to the host receptor site Penetration = of viral genetic material into the cell. Penetration can occur in various ways, including fusion, phagocytosis, or the injection of viral material into the host cell Uncoating = The virus loses its capsid, which exposes the viral nucleic acid Eclipse = Replication and expression of genetic material, viral nucleic acid acts as a template for production of mRNA, which codes and directs the synthesis of viral proteins. Typically, protein synthesis occurs in the host cell cytoplasm, and the capsid is synthesized in the cytoplasm or nucleus Assembly = of genetically material into the protein coat occurs. In this maturation stage, virions are assembled. Finally, mature virions migrate to the host nuclear or cytoplasmic membrane and are released. The release of viruses occurs by various means. In enveloped viruses, such as the influenza virus, the cytoplasmic or nuclear membrane surrounds the nucleocapsid to form an envelope. Next, the virus is released through "budding" as the virus is pinched off from the cell. Poliovirus is gradually leaked out of the host cell, while some other viruses enzymatically lyse the host cell after replication is completed and are then released.

DNA and RNA

The nucleic acid may be either DNA or RNA; DNA may be either single stranded or double stranded and arranged in either a linear or a circular form. RNA has either a positive sense (+), such as mRNA, or a negative sense (-) and is double stranded or ambisense, with both a positive and negative region. Considerable diversity exists in the genetic makeup and size of viruses. The nucleic acid may code for as few as four genes or as many as several hundred genes. Those viruses with larger genomes are able to carry more genetic information than those with smaller genomes.

Other Pox Viruses

The other pox viruses are primarily pathogenic for animals other than humans and humans becoming infected as incidental hosts. The orf virus is a pox virus that infects goats and sheep; vaccinia virus infects cattle and causes cowpox. There also is the monkeypox virus, which infects monkeys and squirrels, with its origin in Africa. All of these viruses pose occupational hazards to those with contact with animal pox lesions. After direct contact with infected lesions, humans develop nodular lesions on their hands or fingers, which form vesicles.

PARAMYXOVIRIDAE

The paramyxoviruses include the genera Paramyxovirus, Morbillivirus, and Pneumovirus. The family possesses negative sense, single-stranded RNA, helical symmetry, an envelope, and an average size of 150 nm to 300 nm.

POXVIRUSES

The poxviruses are the largest viruses known, with a size of 225 nm × 300 nm. These are DNA-containing viruses that are enveloped with complex coats with a brick shape and complex morphology. The family includes variola, the smallpox virus, and vaccinia virus, as well as the agents of cowpox, monkeypox, and canarypox.

virion

The virus particle is known as the ___________. The basic structure of the virion consists of a capsid, or protein coat, which encloses the genome or genetic material of either DNA or RNA. The capsid is composed of repeating, identical subunits arranged in a precisely defined fashion. Each protein subunit is known as a capsomer. The capsid functions to protect the nucleic acid and enables the virus to attach to and enter the host cell. Capsids vary in size and shape among the various types of viruses. Some capsomers assemble to form rod-like capsids, resulting in a helical structure. Other capsids that assemble in a cubic manner create an icosahedral arrangement.

Direct Detection of Viral Antigens or Genes

This method can identify a virus through the detection of specific antigens present in patient cells. A sufficient number of cells must be recovered from the specimen. The specimen is fixed onto a microscopic slide and then stained with a virus-specific fluorescein-labeled monoclonal or polyclonal antibody. This process is known as direct fluorescent antibody (DFA) and is used to detect viruses in patient specimens. Alternatively, viral antigens may be detected in specimens through antigen capture techniques. In these methods, viral antigen is captured through complexing with specific antibodies; ELISA is used to detect the reaction end point. Latex agglutination methods also are available for the direct detection of viruses in clinical specimens. One example is the latex agglutination products that are available to detect rotavirus in stool specimens. Latex beads are coated with antibodies to the virus, and agglutination indicates a positive reaction. Antigen detection is limited by the range of detection because the antibodies are highly specific and the range depends on the antibody used.

viremia

Upon release, the virus may spread to local tissues or through the blood, which is known as ____________. Primary viremia involves viral invasion of cells of the reticuloendothelial system (RES). Replication in the RES system can result in a secondary viremia that spreads the virus to distal visceral cells. Clinical symptoms of viral infection may occur early in the disease, as is exhibited with rhinovirus, the cause of the common cold. In other types of viral infections, such as polio, the clinical signs may not be evident until the second viremic phase. Other viruses, such as hepatitis A virus (HAV) and hepatitis B virus (HBV), may incubate for weeks or months until clinical signs become apparent.

Varicella-Zoster Virus (VZV).

VZV is the agent of varicella, or chickenpox, and herpes zoster, or shingles, which is the reactivation of a latent varicella infection. Chickenpox is generally a childhood disease, with most cases occurring in children less than 10 years of age. The infection is characterized by a generalized skin rash with raised, fluid-filled lesions. VZV infection is transmitted through the respiratory route or fluid from the lesions; the virus may incubate for 10 to 20 days. VZV multiplies in the respiratory tract and regional lymph nodes and is then disseminated through the blood to the skin. In addition to the rash, there also may be headache and fever. The lesions first appear on the scalp and trunk and other warm areas of the body and later on the arms and legs. Lesions pass through several stages before healing. The lesion first appears as a vesicle with clear fluid, resembling a "dewdrop on a rose petal." The lesions then develop into pustules with purulent fluid, which eventually rupture. In the final stage, the lesions form scabs or crusts. Individuals are contagious 24 to 48 hours before the eruption of the rash and remain infectious until all lesions have scabbed. Chickenpox is very contagious and may be transmitted through the respiratory route and also through skin vesicles. Primary varicella is more serious in immunosuppressed children, including those with leukemia or solid tumors and transplant recipients. In these individuals, the rash may be more severe, and pneumonitis, hepatitis, and encephalitis may occur. Primary varicella often is more serious in adults than in children, and complications such as pneumonitis may occur. Reactivation of VZV occurs in the form of herpes zoster. It is believed that the virus remains latent in the dorsal root ganglia of peripheral or cranial nerves after the primary infection. Reactivation occurs during periods of physical or emotional stress. Shingles occurs in the elderly, immunosuppressed patients, patients with transplants, and those individuals with other illnesses. Factors involving reactivation are not entirely understood, but trauma and an altered host immune system seem to play a role. Herpes zoster is accompanied by fluid-filled skin vesicles and pain along the areas of the rash. Complications include neuralgia, keratitis, ophthalmia, hearing loss, facial paralysis, and aseptic meningitis. Diagnosis of VZV infections is usually made from clinical signs. The vesicular lesions can be examined directly for the appearance of intranuclear inclusions and multinucleate giant cells. Cell culture and serological tests to determine prior immunity to VZV also are available. Serological tests to detect antibody to VZV are mainly used to determine an individual's immune status and not to diagnose infection. A live attenuated vaccine for VZV is available and given on the same schedule as the measles-mumps-rubella (MMR) for infants and children. The first dose is given at 12 to 15 months and the second dose given at 4 to 6 years of age. For those older children and young adults who have not had chickenpox or the vaccine, two doses administered 28 days apart is recommended. There also is a vaccine for shingles, which is recommended for those over 60 years of ages and is administered to prevent herpes zoster. Vaccination with VZV immune globulin is also available. Varicella infections are treated with acyclovir, famciclovir, and valacyclovir.

Characteristics of the Hepatitis Viruses

Viral type; Route of infection; Incubation period (days); Characteristics; Lab diagnosis; Vaccine available HAV = Picornavirus, RNA; Fecal-oral; 15-50; Abrupt onset, milder disease, <0.5% mortality; Anti-HAV (IgM); Yes HBV = Hepadnavirus, DNA, Enveloped; Parenteral, sexual; 45-160; Insidious onset, occasionally severe, 1-2% mortality, chronic carriers; HBsAg, HBeAg, anti-HBcIgM; Yes HCV = Flavivirus, RNA, Enveloped; Parenteral, sexual; 14-80; Insidious onset, often subclinical, 70% become chronic carriers, ~4% mortality; Anti HCV HCV-RNA; No HDV = Deltavirus, RNA, Enveloped; Parenteral, sexual; 14-64; Abrupt onset, coinfection with HBV, high mortality fulminant hepatitis, chronic carriers; Anti-HDV; No HEV = Calicivirus RNA; Fecal-oral; 15-50; Abrupt onset, mild disease, more severe infection in pregnant females; Anti-HEV HEV RNA; No

Virus Isolation in Cell or Tissue Culture

Virus isolation in culture remains the gold standard for the isolation of many viruses. When performed correctly, few, if any, false-positive results occur because infected target cells definitely indicate that the infectious agent is present in the specimen. Three types of cell cultures exist, including traditional cell cultures, shell vial centrifugation- enhanced (SVCE) cultures, and multiwell microplate cultures. Cell cultures are animal or human cells grown in vitro that have lost their differentiation. The cells are grown as a single layer on the internal surface of glass or plastic containers to form a cell monolayer. As an alternative, the cells also can be grown in multiwell microtiter plates. These cells are sensitive to the effects of viruses, and after inoculation and incubation with the specimen are examined for cytopathogenic effect (CPE), which refers to cellular damage or changes in the cellular structure. Examples of CPE include a rounding of the cells, forming giant multinucleate cells, or other injurious effects to the original culture cells.

Table of Contents

Viruses virion DNA and RNA nucleocapsid Classification VIRAL REPLICATION viremia Classification of Medically Significant Viruses = DNA Classification of Medically Significant Viruses = RNA SPECIMEN SELECTION, COLLECTION, AND PROCESSING Mediums Storage Viral agents Encephalitis Herpes simplex virus Stool Congenital or neonatal disseminated diseases Blood LABORATORY METHODS IN VIROLOGY Cytological or Histological Examination Electron Microscopy Virus Isolation in Cell or Tissue Culture Primary cell cultures cell line heteroploid cytopathogenic effect hemadsorption Shell Vial Centrifugation-Enhanced (SVCE) Virus Detection Direct Detection of Viral Antigens or Genes Molecular Diagnostic Methods SEROLOGICAL DETECTION OF VIRAL ANTIBODIES Molecular Techniques in Virology Selected Medically Important Viruses ADENOVIRUS HEPATITIS VIRUSES Hepatitis A Virus (HAV) Hepatitis B Virus (HBV) Dane particle Diagnosis of HBV Blood donations and HBV Hepatitis C Virus (HCV) Hepatitis D Virus (HDV) Hepatitis E Virus (HEV) Characteristics of the Hepatitis Viruses HERPES VIRUSES Herpes Simplex Virus (HSV-1 and HSV-2) Varicella-Zoster Virus (VZV). Cytomegalovirus Epstein-Barr Virus Human Herpesvirus Type 6 (HHV-6) Other Human Herpesviruses Papillomavirus PAPOVAVIRUSES Parvovirus POXVIRUSES Variola Virus: Smallpox Virus Other Pox Viruses ARBOVIRUSES Arbovirus flavivirus West Nile virus (WNV) Rubivirus Bunyaviridae FILOVIRIDAE PICORNAVIRIDAE Enteroviruses Poliovirus Coxsackie and Echo Viruses Rhinovirus ORTHOMYXOVIRIDAE antigenic drift vs antigenic shift PARAMYXOVIRIDAE Paramyxovirus Morbillivirus Pneumoviruses: Respiratory Syncytial Virus RHABDOVIRIDAE REOVIRIDAE CALICIVIRUSES RETROVIRIDAE Oncornavirus Lentivirinae and Human Immunodeficiency Viruses HIV structures HIV diagnosis

Molecular Diagnostic Methods

Viruses also can be directly identified through the detection of viral DNA or RNA in specimens. Molecular diagnostic test methods continue to expand in both the variety of methods and the types of viruses that can be identified. These methods include restriction fragment length polymorphism (RFLP), DNA and RNA genetic probes, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), and real-time polymerase chain reaction (real-time PCR). Commercially available viral gene probe kits can be used for the in vitro detection of HSV and CMV. Gene amplification techniques, including the application of the principle of target amplification in a PCR, are now available. Target amplification can increase the sensitivity of the method by amplifying one viral genome into several genomes. The viral DNA or RNA can be amplified 1 million times or more, thus enhancing the detection of the virus.

Classification

Viruses are classified according to the type and arrangement of nucleic acid as well as by the shape of the capsid. The presence or absence of an envelope also is used in classification

hemadsorption

Viruses, such as the influenza, parainfluenza, and mumps viruses (orthomyxovirus and paramyxovirus), that produce little or no detectable CPE may be identified through _______________________. These viruses produce virus-specific hemagglutinins in the cell monolayer, which can combine with erythrocytes of certain animals. This permits the detection of the virus in the infected monolayers. Hemadsorption is characterized by the presence of plaques of red blood cells (RBCs), which adhere to the cell monolayer, and hemagglutination is visible as a clumping of RBCs. Influenza A and B viruses are able to hemadsorb and hemagglutinate guinea pig RBCs, whereas parainfluenza and mumps viruses produce only hemadsorption.

Rubivirus

also a togavirus, is unique in this viral group because it is a respiratory virus with no arthropod vector. The virus is the agent of rubella, or German measles, an acute febrile disease first discovered by German physicians. Rubella once was responsible for epidemics every 6 to 8 years, with cases occurring each spring in school- age children. The virus has single-stranded RNA; icosahedral symmetry; a diameter of 60 nm to 70 nm; and surface projections, including hemagglutinins. Humans are the only host for rubella. Rubella infection is spread through respiratory secretions, incubates for 2 to 3 weeks, and then invades the nasopharyngeal mucosa. The virus multiplies in the lymph nodes, followed by viremia and dissemination through the blood. Other symptoms include fever, lymphadenopathy, and a maculopapular rash, which has a duration of 3 to 4 days. Hence, the name "3-day measles" is commonly used to describe rubella infection. The virus is shed for approximately 1 week before clinical signs of infection and for up to 2 weeks following onset of the rash. The disease is more serious in adults, who may experience bone or joint pain also. Pregnant women who have rubella infection may infect the baby in utero. The virus multiples in the placenta and enters the fetal blood, where it can infect most tissues in the fetus. Congenital infection may be mild and asymptomatic or severe, causing cataracts, glaucoma, deafness, heart abnormalities, mental retardation, or even death. The rubella vaccine is given to children at 15 months of age and the second dose at 4 to 6 years of age as a part of the MMR (measles-mumps-rubella) immunization. Unvaccinated adults also should get the rubella vaccine if they have negative antibody titers for rubella and were born after 1957. Immunization is especially important for college students, those who work in health care facilities and travel internationally, and for women of child-bearing age. The vaccine is live and attenuated and confers lifelong immunity in approximately 95% of those immunized. There is only one serotype of rubella virus, so immunization is very effective. Before the 1962 to 1965 global pandemic, there were over 12.5 million rubella cases in the United States, with over 2,000 cases of encephalitis. In 1969, the live attenuated rubella vaccine was licensed in the United States, and the number of cases of rubella in the United States has declined from over 50,000 cases in 1969 to 10 cases in 2005, according to the CDC. Tests to identify rubella include viral cultures and serologic testing. Acute infection is confirmed by the presence of rubella IgM or a significant increase in the rubella IgG titer between the acute and convalescent specimens. Serologic methods include enzyme immunoassay, hemagglutination inhibition, and immunofluorescent antibody. Rubella also may be isolated from nasal, throat, blood, urine, or cerebrospinal fluid specimens and grown in tissue cultures. There also is molecular typing, which assists in determining the origin of the virus and its frequency of isolation in the United States.

Viruses

are obligate intracellular parasites, which are unable to multiply by binary fission. Viruses lack ribosomal ribonucleic acid (RNA) and require host cell biochemical mechanisms for replication. Viruses contain either deoxyribonucleic acid (DNA) or RNA but never both. Because they lack ribosomal RNA, viruses are not able to make their own proteins. The viral RNA or DNA is transcribed into messenger RNA (mRNA) through use of the host cell ribosomes. Viruses cannot generate adenosine triphosphate (ATP) and thus depend on host cells to provide these missing components. Viruses may be small with a simple structure or large with a complex structure. A virus may have a definite host range and may specifically infect animals, plants, or humans. The size of viruses ranges from 18 nm (parvovirus) to 300 nm (poxviruses), which is below the limits of light microscopy. Viruses may be classified by type of disease but are most commonly categorized based on the type of nucleic acid as either RNA-containing viruses or DNA-containing viruses. There are currently 21 viral families associated with human infection; 14 of these viruses are RNA-containing viruses, and the remaining seven are DNA-containing viruses.

Primary cell cultures

derived directly from parent tissue. The tissue is minced, treated with a proteolytic enzyme, filtered, and added to a nutrient medium. The cells are transferred next to a container with a flat surface, which permits the cell to multiply, forming a monolayer. In a primary culture the cells have the same karyotype and chromosome number as the original tissue. Examples of primary cell cultures include human embryonic kidney (HEK), rabbit kidney (RK), primary monkey kidney (PMK), Rhesus monkey kidney (RMK), cynomolgus monkey kidney (CMK), and African green monkey kidney (AGMK). Primary cell lines have a broad sensitivity to a number of human viruses, including influenza viruses, parainfluenza viruses, mumps viruses, enteroviruses, and adenoviruses.

West Nile virus (WNV)

first detected in the western hemisphere in 1999, and it quickly spread across North America. The virus is transmitted by infected mosquitoes, which have fed on infected birds, including crows, blue jays, and other wild birds. Humans are an incidental host. In 2007, there were 3,600 cases of WNV reported in the United States; this number declined to approximately 1,300 cases in 2008. WNV infection can range from a mild fever to a fatal encephalitis. Because WNV causes a significant viremia, the infection does not dead end in the human host but instead can be transmitted to the mosquito vector during the blood meal. There have been increased isolate serotypes, which are associated with a more severe neurological disease. WNV is identified by detecting IgM or IgG antibodies, using enzyme immunoassay or immunofluorescence. Viral RNA also can be detected through reaction with viral- specific IgM.

Congenital or neonatal

include rubella, HSV, and CMV. CMV is isolated from the urine or throat. When rubella is suspected, throat swabs placed into viral transport media are recommended for collection. Serum also should be collected during the acute phase of the illness for CMV and rubella.

Encephalitis

may result from infection with HSV-1, VZV, and the arboviruses. Brain biopsy and blood should be submitted for analysis, and serum also should be collected when arbovirus, HSV, or VZV are suspected.

flavivirus

the agent of St. Louis encephalitis (SLE), which is found sporadically in the southern and south-central United States. Birds constitute the major reservoir, and mosquitoes are the vector in SLE. Epidemics seem to occur every 10 years in Texas or in the Gulf Coast area. Flaviviruses also are the cause of Japanese B encephalitis, a severe form of the disease, found in the Far East, Korea, and Japan, as well as yellow fever and dengue fever. The vector in yellow fever is the mosquito, and the disease is severe and systemic. Symptoms of yellow fever include headache, backache, nausea, jaundice, and extensive damage to the liver, kidney, and heart. The disease gets its name from the jaundice; severe gastrointestinal hemorrhages also may occur. Dengue fever, found in the Caribbean and Southeast Asia, is characterized by fever, headache, severe bone pain, backache, fatigue, and chills. Rash and arthritis also may occur. Dengue fever also is known as "break bone disease." Hemorrhagic fever and dengue shock syndrome also may occur. In these diseases, the blood vessels rupture, and there is internal bleeding of the blood vessels and other tissues, which may lead to shock.