Chapter 9 Virology Quiz 8

Products of vRNA generated post transcription

+vmRNA +vcRNA.

Clinical rapid viral testing

10-20 minutes completion time. Cheaper than virus isolation. The rapid tests are about $15... 25 tests per box. Performed with a nasal swab specimen. ElisAs or immunofluorescent assays. Antiviral for people with symptoms less than 48 hours. 30% of samples may be false-negative, but culture may be positive.

Epidemiological information

3-5billion annual episodes worldwide. 1 million children annually about 50% of acute gastroenteritis cases in hospitalized children. Reinfection is common in non immunized individuals. By age 3, 90% of children have serum in antibodies against 1 or 2 types.

How many deaths nationwide during flu season?

36000 deaths nationwide, and many more hospitalizations.

How many people has the spanish influenza killed?

675,000. 4.39 people per 1,000.

1957 Asian Influenza

70,000 deaths in US alone. Feb of 1957 in Northern China, spreading to US by June. Vaccine was created via chicken egg embryos, and made available in August.

Recommended dose for seasonal influenza cases

75mg BID for 5 days. Early treatment is critical for survival of high risk individuals.

Structure of influenza A

80-120nm in diameter Virions may be filamentous with helical symmetry immediately after isolation from cell cultures in the lab. Later becoming spherical in appearance. Host derived envelope. With the surface covered with 500 rod-shaped glycoprotein spikes. 80% of spikes are hemagglutinin, 20% mushroom-shaped neuraminidase.

Why are seasonal vaccines so important?

A vaccine against a virus works by teaching the immune system to recognize the antigens exhibited by this virus. Antibodies of older strains don't recognize the new ones, reinfection can occur.

Immunity to influenza A in immunosuppressed humans

Activated macrophages including Tc lymphocytes, and certain cytokines including interferon.

Optimal spreading environment for influenza A viruses

Aerosol likes low humidity (20%) and colder temperatures (41F).

Effectiveness of influenza vaccines depends upon several factors:

Age of the vaccine recipient Immunocompatence of vaccine recipient Degree of similarity between the viruses in the vaccine and those in circulation.

Why are waterfowl significant when it comes to influenza and subtypes?

All influenza A subtypes circulate in wild waterfowl (ducks, geese, and swans) and shore-birds (mainly gulls, terns, and waders) which are considered the natural reservoir of influenza A viruses (exception of H17, 18, N10, and 11. H17N10 and H18N11 can be found in flat-faced, fruit-eating bats. Birds may not be the exclusive reservoir.

Why no more eggs?

Allergic reactions, and organismal complications. Hybrid cell lines being used. Virus particles produced, collected, and used for making vaccination.

Nuclear Export proteins

Also found inside of the virion (few copies).

Explain why a cytokine storm response by the body during infection with influenza A virus is challenging for physicians.

Also known as the systemic inflammatory response syndrome, the cytokine storm response is defined as an immune system that is overreacting toward the pathogen. It signals immune cells like macrophages and T lymphocytes traveling to the site of infection (lungs), causing damage that ultimately results in multiple organ failure. SIRS is rare. Death rate is high.

M2 inhibitors first found

Amantadine and Rimantidine block M2 ion channel of influenza viruses, preventing uncoating during the virus replication cycle.

What is the best indicator of the scale of influenza epidemic?

An escalation in absenteeism from schools and the workplace that is accompanied by an increase in hospital admissions and deaths, especially among the elderly.

Two processes of influenza A variation

Antigenic shift and drift. Gives rise to new strains of influenza A virus.

Neuraminidase inhibitors

Approved to treat influenza A and B today. Oseltamivir phosphate (Tamiflu) Zanamivir (Relenza) Peramivir (Rapivab) Inhibits release of viral progeny during replication. "plug drugs". Sialic acid analogs blocking active site of N, preventing it from cleaving host cell sialic acid receptors. Reduction of Influenza A or B virions infecting the host.

Virion Assembly and release

Assembled on the membrane surface of host cell. Released by budding. H anchors influenza A virus to cell by binding to sialic acid receptors on the cell surface. Neuraminidase activity destroys sialic acid receptors present on the host cells during virus release or exit, preventing newly assembled viruses from aggregating or clumping together on the cell surface. N facilitates the release of viral progeny.

RotaTeq RV5

Attenuated, reassortment rotavirus of human and bovine strains, contains 10^6 infectious units/mL of each of the 5 strains. Comes reconstituted in tissue culture media with other constituents, given as 3 doses at 2, 4, and 6 months.

H9N2

Avian Hong Kong 1999 Mild. No evidence of reassortment.

Difference between human and avian influenza binding

Avian influenza binds to sialic acid bound to galactose through alpha-2,3 linkage.

Why is mutation common in RNA viruses?

Because their RNA-dependent RNA polymerase lack proofreading ability. They're 1-10 thousand times more prone to error than human or viral DNA polymerases with proofreading ability.

1918 Spanish influenza

Between 20 to 50 million deaths. Including 675,000 Americans. Spread faster than any plague in history. Charged across America in 7 days. Took 3 months to spread across the world. 60% of Alaskan Inuit population was wiped. H1N1 strains. WW1 conditions in the trenches of France fostered Influenza A virus transmission. It shaped the war by rendering much of the Army and Navy ineffective. Lungs of autopsied men who passed of influenza looked blue, swollen, and foamy as a result of congestion and hemorrhaging. Doctors used today's controlled substances to help manage symptoms including heroin, opiates, alcohol, morphine, cocaine, etc. Large percentages of alcohol.

How to identify influenza virus using serology

Blood and serum drawn from the patient to detect antibodies against the flu virus. Blood is drawn again after recovery (10-20 days later). Convalescent serum also drawn and screened for viral antibodies after recovery. Fourfold or greater increase in antibody titer is considered Dx of viral infection.

Limitation of Influenza A vRNA replication

Can only replicate in physiologically active cells containing functional DNA-dependent polymerase (RNA polymerase II) even though this enzyme does not transcribe the virus genome.

Virion Maturation

Capped viral mRNA exported to cytoplasm. Translation by ribosomes. H, N, and M2 glycoproteins synthesized on membrane-bound ribosomes. 3 membrane proteins enter ER, folded, and glycosylated while being transported to golgi network and cell surface for assembly. Only takes 1 copy of each genome segment packed into a new virion for it to be infectious.

Resistant to:

Chlorine, concentrations used to treat sewage effluent. Hand sanitizing lotions are effective in reducing hand to hand viral transmission.

Intestinal trypsin

Cleaves one of the capsid proteins to promote infection. Cell surface receptors include glycoprotein and integrin. The modified viral protein undergoes structural arrangement and promotes penetration through cell membrane (jackknifing) during which the outermost protein layer is shredded. Transcription of the viral genes occurs in double-layered viral particles.

Why do pandemic influenza A strains originate in Southern China?

Close association of humans with domestic animals and birds raised for food. Small farms in China and other Southeastern Asian countries. Genetic reassortment of influenza A between humans and other species is considerable. Dense population, factoring into evolutionary rates.

Nucleoproteins

Coats -ssRA segments. Each segment contains an RNA-dependent RNA polymerase or transcriptase complex that consists of three additional viral polymerase proteins PB1, PB2, PA.

Trivalent vaccines

Cocktail of 3 anticipated, inactivated influenza viruses. 2 are influenza A strains, 1 is B. Sometimes quadrivalent vaccines are needed.

Seasonal infection in children

Croup (laryngitis with a barking cough and dyspnea). Secondary bacterial pneumonia Middle ear infection.

Why is proofreading important in DNA?

DNA polymerase proofreading is 3' to 5' exonuclease activity that removes misincorporated nucleotides during DNA replication.

How are they released?

Damaged cells slough off into the lumen, release large numbers of viral particles.

Detection of rotavirus

ELISA for viral antigen in stool sample, rectal swab, PCR, electron microscopy.

How is it possible from more segments to produce less or more viral proteins.

Each virus has unique proteins.

Who is at the highest risk of developing the flu?

Elderly, pregnant women, very young children, and otherwise immunocompromised individuals. They account for more than 90% of all deaths. People with pulmonary, cardiac, renal, hepatic, or endocrine system is also at risk.

Upon infection, where does the influenza virus go in humans?

Entering the respiratory tract, they will attach to and penetrate ciliated columnar epithelial host cells lining the sinuses and airways. Viral replication begins. Ciliated epithelial cell is destroyed, releasing progeny influenza viruses to nearby cells. Primary site is the tracheo-bronchial tree, but nasopharynx is also involved. Loss of cilia in the cells makes mucus get built up and cough. In addition, loss of cilia and proper cleaning system in the lungs contributes to sinusitis, OM, and pneumonia.

Flyway

Entire range of a migratory bird species through which it travels on an annual bases from breeding to nonbreeding grounds. Intermediate resting and feeding places.

Describe the function of neuraminidase and Hemagglutinin. How are non-immunosuppressed humans combating them?

Envelope glycoproteins found on the surface of the virus. The host will develop antibodies against this and hemagglutinin, also found on the surface of the virus. Anti-H antibodies will neutralize influenza A virus, preventing attachment and viral infectivity. Can neutralize the virus if the host experienced a similar infection within a couple of years. Antibodies against N do not neutralize influenza A, but reduce release of virions from infected host cells.

Differentiate between antigenic drift and antigenic shift of influenza viruses and how they relate to seasonal and pandemic strains of influenza viruses.

Epidemiology of influenza viruses differs in that: A has antigenic shift and antigenic drift B and C only depend on antigenic drift. Influenza A may cause pandemics with significant mortalities in affected young people, whereas B and C do not have possibility for pandemics. B is severe, but typically seen in high risk individuals. C is mild and common in children without seasonality.

+vcRNA

Exact copy of the genome segment No 3' poly(A) tail No 5' cap Synthesis requires continuous viral protein synthesis.

What are the best model for influenza A viruses

Ferrets. Sharing similar lung physiology with humans.

Influenza vs. cold

Fever! Above 102F, slowly decreasing. Infectious dose spikes 2 days after 2 days of symptoms (4 days after infection) and continues for about a week.

Compare and contrast the clinical signs and symptoms of influenza and the common cold.

Fever: Only in children with a cold, 3-4 days in everyone with the flu. Headache: Sometimes in cold, always in flu Generalized myalgia: slight/mild in cold, common and often severe in flu. Sore throat: Sometimes in cold, common in flu Cough and chest discomfort: mild hacking cough with cold, common and severe in flu. Sneezing: usual in cold, sometimes in flu Congestion: Common in cold, sometimes in flu Fatigue/weakness: Sometimes in cold, extreme lasting 2-3 weeks in flu. Extreme exhaustion: never in cold, severe in flu. Complications: sinus congestion or earache in cold, bronchitis or pneumonia for the flu. Prevention: Washing hands and quarantine for cold, annual vaccination and antiviral drug for flu. Treatment: Assurance for cold, antiviral drug 24-48 hours after symptoms onset for flu.

dsRNA viruses

Few.

Vaccines that are not derived from eggs

Flublock which is a recombinant vaccine composed of purified H protein made in insect cultures Flucelvax is an inactivated vaccine produced in MDCK cells in culture.

Lab diagnosis

Fluorescent Ab or ELISA Haemagglutination Adsorption inhibition RT-PCR Viral isolation

Vaccine effectiveness of rotavirus

For any rotaviral gastroenteritis: 74-85% Severe gastroenteritis 85-98% Both vaccines significantly reduce hospitalization and visits to pediatricians.

W-shaped curve

Graphical analysis of the 1918 influenza pandemic shows a "W-shaped" curve, in which mortalities peaked among the very young, the elderly and those ages 20-40 years. It was unique in that it killed healthy adults between 20 and 40 years of age. Typical influenza seasons have U-shaped curves (death in young and elderly).

How are influenza vaccinations created?

Grown in fertilized chicken eggs or cell cultures for vaccination production.

Outer capsid proteins

Guanylyl transferase (capping enzyme), cell attachment, hemagglutinin, penetration, promoting protein

Hot spots

H and N genes contain hot spots coding for antigenic sites recognized by virus-neutralizing antibodies. These antibodies defend host cells from viral attack by inhibiting the effects of virus during infection. Changes in antigenic structure of H and N glycoproteins of influenza A influence epidemic and pandemic incidents.

Similarity between all H genes of human influenza A viruses

H genes code for hemagglutinin glycoproteins that contain a single basic arginine residue located at the proteolytic cleavage site between H1 and H2.

Spanish flu

H1N1

Swine flu

H1N1

Asian flu

H2N2

Hong Kong flu

H3N2

What are the scary sub-types of Influenza A that shouldn't replicate?

H5 and H7. Devastating epizootics. H5N2 identified in poultry in BC, Canada in 2014. U.S. birds started it :/

Bird flu

H5N1 1997 Hong Kong 33% of individuals died. Chickens, and controlled by cullination of chickens throughout Hong Kong. 18 people infected, 6 dyeing.

Shortened processes of the cytokine storm

H5N1 influenza viruses A attach to and replicate in type 2 pneumocytes of the alveolar epithelium and macrophages located in alveoli of the lower respiratory tract. Seasonal influenza viruses prefer cellular receptors located in the upper respiratory tract of the lung. Viral infection stimulates the infected and uninfected epithelial pneumocytes to release cytokines and chemokines. Infected macrophages secrete cox-2, an enzyme that causes inflammation, pain, and fever, and pro-inflammatory cytokines and chemokines including macrophage inflammatory protein, onocyte chemotactic protein. Leukocytes such as macrophages, dendritic cells, and lymphocytes are drawn to and activated by the cytokines and chemokines in the alveolar spaces. The dendritic cells and macrophages in the alveolar space present viral peptides to CD8+ cytotoxic T lymphocytes, leading to the proliferation of influenza A virus-specific CD8+ cytotoxic T lymphocytes in the lung. CD8+ cytotoxic T lymphocytes play an important role in controlling the viral infection, but in excessive numbers they might contribute to tissue damage and multiple organ failure via apoptosis, tissue damage, necrosis, and dilation of blood vessels.

Why is H5N1 different?

Hemagglutinin glycoprotein of avian H5N1 contains an insertion of multiple basic residues upstream of the cleavage site associated with highly pathogenic avian influenza A viruses. These additional amino acids increase accessibility of cleavage site to host cell proteases than can cleave H0 into H1 and H2 increasing its infectious nature.

Oversimplified version of the cytokine storm

Highly virulent influenza A viruses infect type 2 pneumocytes present in the alveoli of the lower respiratory tract. During infection, macrophages become activated and present viral peptides on their outside surfaces. The peptides activate T lymphocytes, resulting in an overly aggressive immune response involving proinflammatory chemokines and cytokines. The blood vessels of the lungs become dilated and filled with leukocytes, causing destruction and necrosis of lung tissues.

How do vmRNA leave the nucleus?

Hitches a ride on the transportation that cellular pre-mRNA would originally take. Not affected by any difference, because influenza A transcriptase complex adds poly(A) tails onto 3' ends of viral miRNA. Cellular CPSF and PABII proteins are not required for processing the 3' ends of viral mRNA.

Neuraminidase

Homotetrameric integral membrane glycoprotein. N may also aid influenza viruses at the beginning of replication cycle by aiding transport through mucin layer of respiratory tract to attach to host epithelial cells.

Hemagglutinin

Homotrimeric integral membrane glycoprotein Influenza A virus Hemagglutinin attaches to sialic acid resides present on glycoproteins or glycolipids of the ciliated columnar epithelial cells lining the sinuses and airways. Composed of two polypeptides H1 and H2. Full-lengtgh H protein is regerred to as H0. H1 and H2 are linked by disulfide bonds between two cysteine amino acids. H1 portion of H0 contains sialic acid (binding site for attachment to host cell). H2 portion of H0 forms a membrane-spanning anchor or fusion peptide, directly involved in the fusion mechanism. H2 peptide interacts with the host membrane after the conformational change is induced by H0 in low pH.

1968 Hong Kong Influenza

Hong Kong early months of 1968, reaching the US by September. Deaths mostly in the elderly, killing 33,800 people in the US.

IgG antibodies protecting against influenza

IgG antibodies neutralize influenza A virus, providing protection in the lung.

Prevention of rotavirus

In 1998, the first human rotavirus vaccine called RotaShield was introduced. After 1 million doses were administered, development of intestinal intussesception led to withdrawal of the vaccine. Two new vaccines RotaRix and Rotateq are now available with no complications thus far.

Splicing segments 7 and 8

In both cases, RNA splicing events remove a large portion of the first OFT, leaving the AUG initiation codon in place. First alternative ORF is fused to the initiation codons of M1 and NS1 ORFs. Newly generated ORFs direct the synthesis of novel proteins M2, NS2, and M3. All the viral mRNA codes for nonstructural proteins, but M3 has not yet been detected in infected cells. Its role is unknown.

Evaluate the importance of reverse genetics in manufacturing influenza vaccines.

In order to recreate the influenza virus, the science team needed to use reverse genetics. Molecular basis of the H1N1 virulence was understood. Changing viruses so that 1 virus can't bind to either human or bird.

What living environment will we see the influenza A and B the worst?

In temperate or colder regions, influenza illness usually occurs during the winter In tropical regions, influenza occurs throughout the year.

Evolution in waterfowl vs. domesticated birds.

In wild waterfowl, evolution is very slow. Transmission between domesticated birds (chicken and turkey), evolution is very rapid.

Where does rotavirus infect?

Infects cells of villi of small intestine, multiply in enterocytes, and damage their transport system.

Clinical information

Infects young children Incubation period 1-3 days Transmitted through oral-fecal route and personal contact plus, nosocomial infections are common. Symptoms include watery diarrhea, vomiting, abdominal pain Severe loss of electrolytes and fluid can be fatal. Recovery 3-8 weeks. Viral excretion may last up to 2 months (individual infected is able to transmit).

Translational control mechanisms

Influenza A suppresses interferon response to viral infection in host cells. During replication, dsRNA intermediates are formed. Influenza A interferon suppression stops the dsDNA molecular from activating cellular kinase PKR.

12 steps of replication cycle of Influenza A virus

Influenza A virus hemagglutinin attaches to sialic acid host cell receptors. Virus enters the host cell by endocytosis. M2 ion channel of virus allows H+ ions to penetrate the virus. Low pH of endosome triggers irreversible conformational change in H, resulting in fusion of the viral and host endosomal membranes. As H+ ions enter through M2 ion channel, encapsidated genomic RNA (viral ribonucleoprotein complexes vRnPs) are dissociated from M1 matrix proteins and released into the cytoplasm where they travel through the nuclear pores int othe nucleus. In the nucleus, the viral polymerase initiates its mRNA synthesis by cleaving "snatching" 5' caps from host pre-mRNA. PB2 binds to 5' cap of host pre-mRNA, PA endonuclease cleaves host pre-mRNA 10-13 nucleotides downstream 5' cap. Replication/transcription of viral genome initiation. Viral mRNA are transported into the cytoplasm for translation into proteins. Surface proteins (H, N, and M2) are processed in the ER, glycosylated in the golgi apparatus, and transported to the host cell plasma membrane. NS1 suppresses host mRNA synthesis by inhibiting 3' end processing of host pre-mRNA. Consequently, host mRNA (including mRNA coding for interferon) are blocked from synthesis. vmRNA unaffected. Besides synthesizing vmRNA the viral transcriptase complex is involved in the replication of -vRNA through replicative intermediate +vcRNA. Final -vRNA containing transcriptase complex are encapsidated with M1, and NS2 is involved in their export through the nuclear pores to the site of virion assembly near the host cell plasma membrane. Newly assembled viruses exit by budding through the PM. Newly assembled influenza A viruses are released as N destroys sialic acid receptors on the surface of the host cell.

How is H activated?

Influenza A virus is not infectious unless the H protein is cleaved by cellular proteases. If H protein contains a furin cleavage site, it undergoes proteolytic cleavage by a cellular protease inside of the trans-golgi network. If no furin cleavage site, H0 precursor protein is cleaved into H1 and H2 by cellular proteases outside of the host cell, limiting their spread in hosts to tissues where the appropriate proteases are encountered. The H proteins are cleaved intracellularly by proteases, and have the capacity to infect various cell types and cause systemic infections, otherwise they're cleaved extracellularly by extracellular trypsin-like proteases (apathogenic).

What does receptor binding look like between influenza and epithelial cells?

Influenza targets (with H glycoproteins) sialic acid receptors on epithelial cells in the respiratory tract. Sialic acid receptors are bound to galactose through an alpha-2,6 linkage.

Standard naming for influenza A viruses

Influenza type/Species isolated from/Place of isolation/Strain of designation/Year of isolation/H#N# subtypes. If it's a human, you don't write the species isolated from portion.

Side effects of M2 inhibitors

Insomnia Lightheadedness Seizures Hallucinations CNS effects Kidney dysfunction GI discomfort

Elements of influenza that are observed

Intensity, severity, and frequency of symptoms.

Pathogenesis of Influenza A

Interferon induction 1 day. T-cell mediated immune response above baseline @~5 days. Causing rise in virus-specific antibodies and pathologic changes evident in the respiratory tract @8 days.

Why was the Spanish flu unique?

Killed 20-40 year old adults. Killed quickly Infected individuals suffered from hemorrhagic symptoms.

Lecture!

Lecture!

RotaRix RV1

Live attenuated human strain. provided as ly*** powder containing 10^6 particles/mL given 2 doses at 2 and 24 months.

Flu-mist

Live, attenuated influenza vaccine that was a nasal spray. Only approved for people between 2-49 years.

M2 ion channel integral membrane proteins

M2 is not present in influenza B virions. M2 ion channel proteins are inserted into the lipid bilayer membrane that surrounds the nucleocapsid of the virus (nucleoproteins + nucleic acids).

Pacific Flyway

Major north-south flyway for migratory birds in North America, extending from Alaska to Patagonia. Migratory birds travel in the spring and fall, following food sources to breeding grounds or overwintering sites. Most likely area of introduction for highly pathogenic influenza A viruses in the US and canada.

Gain of function experiments

Manipulation or mutation of influenza A viruses is a way for scientists to study and learn how a mutation works.

Who is affected by reoviridae?

Many transpitted by insects, except human Rotavirus. Genome is segmented. All have a double-layered capsid. Outter layer when removed, forms infectious subviral particles (ISVP) within which transcription of viral genes takes place. RNA replication is by conservative mode (RNA never leaves capsid), a single exception to more common semiconservative mode for dsnucleic acid genomes.

Antigenic drift

Mechanism for variation in viruses that involves the accumulation of mutations within the genes that code for antibody-binding sites... Result of mutations in H and or N gene that causes small changes in H and N glycoproteins continually over time of viral replication. New strains created, and can't be recognized by host immune system. Responsible for localized and/or seasonal influenza epidemics caused by A and B virus.

M1

Most abundant virion protein. Virus particle contains eight segments of -ssRNA ranging from 934 to 2,341.

Limitations

Must be taken 48 hours after onset of symptoms, or earlier. Some will be resistant to the drug if taken 36 hours or later, though. Peramivir is last ditch, strong IV dose effort used in emergency situations.

Limitations of M2 inhibitors

Must be taken within 48 hours of infection to be effective. Not effective against B viruses. M2 inhibitors are only 70-90% effective in preventing influenza A infection. Both discontinued because of drug resistance.

Nonstructural proteins

NS1 Only found in infected cells.

How does interferon suppression stop cellular kinase PKR production?

NS1 protein binds to dsRNA intermediate molecules of influenza A virus produced during replication, sequestering them. Activation of PKR is blocked. Influenza A virus activates a 58-kDa cellular protein that interacts with PKR directly, inhibiting PKR in infected cells.

Asymptomatic infection

Neonate and infants. Aged 6-24 months. Severe diarrhea leading to dehydration. One of the capsid proteins involved in pathogenesis (shortening villi, epithelial cells replaced by immature epithelial cells) Reduces glucose and Na+K+ transport systems, reducing intestinal absorption in general.

Reoviridae structure and replication.

Non-enveloped Icosahedral Double-layered capsid Inner core contains RNA polymerase, transcriptase, and regulatory proteins. dsRNA 10-12 segments, 18-24kb per segment, cytoplasmic replication, conservative mode... Genetic reassortment occurs between species within each genus.

Why is influenza more prevalent in the winter?

North America is temperate. November to March is influenza A time. Can happen throughout the year for tropical regions. Gathering of people indoors facilitating transmission of influenza A viruses. Aerosol spread of influenza A virus is dependent upon humidity and temp.

Treatment of rotavirus

Oral hydration therapy preferred over IV therapy

Taxonomy of viruses

Order to family to genera to species. Order is unassigned. Family: Orthomyxoviridae Genera: 5, Influenza A, B, and C, Isavirus, Thogotovirus. (respiratory illnesses). Species: only one for each genus Subtypes: 16H and 9N.

Viruses with segmented genomes

Orthomyxoviridae Bunyaviridae Arenaviridae Enveloped carrying 2-8 segments of -RNA. Orthomyxoviruses are only RNA that replicates in nucleus.

Influenza family

Orthomyxoviridae family of viruses. Contains Influenza A, B, and C.

Reye's syndrome symptoms

Persistent or recurrent vomiting Listlessness Personality changes (irritability or combativeness) Disorientation or confusion Deliirium Convolutions LoC No cure. Successful management depends on early Dx

Antigenic shift

Process by which two or more different strains (1 human, another of wildfowl or something) of a virus, or strains of two or more different viruses, combine to form a new subtype having a mixture of the surface antigens of the two or more original strains. Hybrid viruses contain major changes in H and N genes. Humans don't have immunity to it. Herd immunity does not exist. Result in high deaths for 2-3 years. Virus is able to reassort its genes, swapping RNA segments of the virus genome in co-infected host cells. New H#N# strains.

Cell culture microbiology testing for influenza virus

Propagating or isolation. Identify specific strains. Nasal swab, sputum samples, nasal washes, or combines nose and throat swab specimens. Special transport containers hold samples, labeled, and are transported to viral diagnostic lab quickly. Abx may be used to inactivate contamination by bacteria prior to inoculating the cell culture. Madin-Darby canine kidney cultures are used. Cytopathic effects are observed. Results won't be available for 2-10 days. Costly ($75). Requires skilled technicians and equipment for procedure. May use PCR, viral antigen detection through serology, or visualization of virions using electron microscopy, cytology, or histology.

Why can proteases of bacteria also found in the body be bad for humans infected with influenza A?

Proteases coinfecting bacteria may play a role in influenza A virus activation and cause an increase in the severity of the respiratory illness.

NS4

Protein that acts like an endotoxin

Matrix 1

Proteins that form a layer beneath the viral membrane, surrounding the 8 segments of viral genome.

How to identify influenza virus using microarray "Flu chips".

Quick way to ID a variety of respiratory pathogens within an hour or less. Can screen for flu A, B, and C, avian influenza viruses, SARS-CoV, RSV, and other airborne bioterrorist pathogens (including anthrax).

Nonstructural protein

RNA assortment, core binding, binds dsRNA, dispensable in cell culture.

Inner capsid proteins

RNA replicase, NTPase and RNA helicase, dsRNA binding proteins.

How can antigenic drift happen

Reassortment of H and gene in 3rd host 1968 H3N2 and 2009 virus Recycling of pre-existing strains (1971 H1N1) Gradual adaptation of avian virus to human host (1918 H1N1)

Mode of entry for reoviridae (2)

Receptor mediated endocytosis (Sialic acid is the receptor) Direct penetration into ER

Why was H1N1 deadly?

Replicated in absence of trypsin. Trypsin is a protease that cleaves influenza A hemagglutinin (H0 into H1/H2)... causing infectivity of the virus in cell cultures.

Initial stage of infection with Reoviridae

Requires proteolytic cleavage of the protein in the outermost capsid, followed by partial uncoating. mRNA synthesis in the partial capsid.

Where does the virus replicate in birds, and how does it come out?

Reservoirs are asymptomatic. Replicates in the GI tract. Comes out via fecal matter. Transmitted oral-fecal route.

Reoviridae

Respiratory Enteric Orphan virus. 12 genera; 4 infecting humans, 6 infecting fish, 1 infects non-human vertebrates and the last one infects fungi. Those infecting humans are: Orthoreovirus: mammalian Orbivirus-Bluetongue Rotavirus- Rotavirus A (humans) Coltivirus-Colorado tick fever virus

Cleavage and polyadenylation specificity factor (CPSF) and Poly(A)-binding protein II (PABII).

Responsible for processing the 3' end of cellular mRNA. Influenza A virus NS1 protein (most abundant) interacts with CPSF and PABII inhibiting their function. Cellular pre-mRNA is not cleaved, and contains very short 3' poly(A) tails that accumulate within the nucleus of the infected cell. pre-mRNA is trapped in the nucleus of infected cell, almost completely degraded. This retention facilitates removal of pre-mRNA 5' cap by PB2 protein of influenza A. Host mRNA is synthesized after infection and they do not survive long in the cell. Little or no cellular proteins will be synthesized in infected cells.

What is the primary source in which influenza can be shared?

Schools! Children bring the viruses home, and parents who contract influenza then disseminate the infection ot the workplace.

What's common between influenzas A, B, and C.

Segmented genomes composed of -ssRNA, and a host-derived envelope containing glyco-proteins critical for virus entry and exit of host cells.

How many proteins does each sequence of influenza viral -ssRNA?

Segments 1-6 code for only one viral protein. Segments 7 and 8 code for two.

Sero-prevalence studies

Serum samples collected from people in a different country prior to start of pandemic. Elderly had stronger antibody production than younger.

Why is influenza difficult to diagnose, what is an accurate diagnosis based on?

Several other infectious diseases that resemble it. Accurate diagnosis is based on the patient's history and PE in a clinical setting. Sometimes microbiology testing is needed.

Seasonal infection in elderly

Severe pneumonia, usually caused by Streptococcus aureus, pneumonia, or Haemophilus influenzae. Pre-existing chronic conditions like CAD can be exacerbated.

+vmRNA

Shorter than template genome segment Contains 3' poly(A) tail Contains 5' cap Synthesis is insensitive to inhibitors of protein synthesis

Reye's Syndrome Facts

Sometimes associated with influenza (or chickenpox) in children. Using aspirin or salicylate-containing medications to treat viral illnesses increases the risk of developing Reye's syndrome. Systematic infection, but causes acute intensive pressure in the brain, and massive accumulations of fat in the liver and other organs.

The 3 influenza pandemics

Spanish influenza 1918-1919 Asian influenza 1957-1958 Hong Kong influenza 1968-1969

2009 H1N1 Swine influenza

Started in North America. Late April, Mexico became epicenter. Rapidly identified via RT-PCR. 208 reported countries in 1 year. 7500-12000 this time of deaths in US. Less than annual seasonal deaths of Influenza A and B. Though, this number may be underestimated assuming deaths may have been due to comorbidities. 75% of cases were younger than 30 years old. 10-19 years was most affected. Less than 3% were over 65.

Retrospective study

Study that looks backward in time.

H1N1

Swine-Origin Influenza A virus Epicenter: 2009 Mexican Town of La Gloria, Veracruz. Mid-Feb. Triple reassortment between avian, human, and swine origin = H3N2. Contained genes: PBII-North American Avian PBI-Human H2N2 PA- North American Avian H1 - classical swine NP- classical swine N1- Eurasian avian-like swine NS- classical swine.

Uncoating of influenza A virus

Takes place in the cell endosome after viral membrane fuses with endosomal membrane. M2 transmembrane protein forms a transmembrane H+ ion channel in the viral envelope, and allows H+ ions to penetrate the virion. This weakens the interaction of the viral M1 matrix protein from the viral RNA, NP, and transcriptase and RNA-dependent RNA polymerase (RNP). RNP released into the cytoplasm and are exported to the nucleus.

List the M2 and N inhibitors of influenza viruses

Tamiflu (n) high dose and long duration. Peramivir (n) Zanamivir (n) Corticosteroids are steroid hormones that reduce inflammation. Have not shown benefit, and may be harmful. Cyanovirin-N (H) T-705 (ER n) siRNA (polymerase inhibitors) Interferon inducers Amantadine and Rimantidine block M2 ion channel function, resulting in incomplete release of viral RNP, ultimately interfering with uncoating.

Side effects of N inhibitors

Tamifly can cause nausea, vomiting, vertigo, and bronchitis. Relenza can cause nasal and throat irritation, headache, GI trouble, bronchitis, and cough especially in asthma sufferers or those with preexisting conditions like COPD.

What is the snatched host mRNA cap and associated nucleotides used for again? And what happens after that for the vRNA?

The cap and nucleotides are used as a primer to transcribe the 8 -ssRNA genome segments. The 6 newly transcribed vmRNA from segments 1-6 are exported to the cytoplasm and translated immediately into viral proteins by host translational machinery. Two of the primary viral transcripts from segments 7 and 8 are each spliced through exploitation of the host splice machinery into a least 2 different vmRNA.

Use of cilia in the respiratory tract

The cilia sweep mucus produced in goblet cells as well as mucus coming from deeper glands within the lungs and the particulate matter trapped in the mucus. The presence of cilia aids the removal of mucus materials from lower parts of the airway to the throat for disposal.

Influenza facts and symptoms.

The flu will begin 1-4 days after infection with sudden onset of headache, malaise x7 days, fever x3 days, extremity and back pain, dry cough x7 days, sore throat leading to husky or loss of voice, and myalgia. Many patients can pinpoint the hour they become sick. Tearing and burning in the eyes, and pain upon moving them. Red appearing. Congestion. Flush cheeks with the fever. Normal chest x-ray unless pneumonia infection. Flu resolves around 7 days. Feeling of listlessness can last a few more weeks.

What are the reasons influenza can go to unmanageable at alarming rates of cases?

The incubation period, also known as the time between infection and the first appearance of symptoms, is only 1-4 days. Copious members of infectious virus particles are shed in droplets discharged by sneezing or coughing. One droplet can contain 100,000-1,000,000 virus particles. The infectious dose of influenza A virus is 1,000-2,000 virus virions! Many symptomatic individuals do not stay at home. Instead, they continue their normal activities spreading the influenza virus to many contacts in the process. Individuals lack immunity to the strain of flu A or B virus circulating in the area at the time of the epidemic.

How much do influenza subtypes differ, and how many are there?

There are 18, and they have a 30% amino acid sequence homology.

Prevention of influenza

Vaccines (split or subunit) Vaccine normally trivalent consisting of 2 A and one B strains. Strains reviewed by WHO each year, giving a guideline of the combination. Vaccine given to high risk individuals. Amantadine used as prophylaxis before or after in emergencies.

Catarrhal fever

Very bad head cold involving copious mucus production.

Swine influenza

Very contagious. Fever, lethargy, weight loss in pigs. Hard to maintain hogs without influenza. No specific treatment. May administer antibiotics to sick sows to control or prevent secondary bacterial infections.

Subtyping kit in the US for swine.

VetMAX-Gold SIV Subtyping kit. Uses RT-PCR rapid and cost-effective. 20 minute completion. Automated, from sample prep to PCR reactions.

Cap snatching

Viral PB2 protein binds to the cap structures of host mRNAs located in the nucleus. The cap and 10-13 nucleotides from 5' end of host mRNA, is removed, and PB1 and PA of RdNP complex uses a short string of nucleotides as a primer to initiate transcription of vRNA by extending the primer.

How do viruses enter the epithelial cells?

Virions enter the cell within an endosomal vesicle by endocytosis. Inside the endosome, influenza A virus encounters a relatively low pH (dropping from 7 to 5). Lower pH change triggers fusion between the viral lipid membrane and the host cell endosomal membrane. H protein mediates low-pH dependent fusion. This is an irreversible conformational change in H.

How can pandemics of Waterfowl-Origin Influenza A viruses happen?

Waterfowl and shorebirds spread highly pathogenic influenza A viruses like H5N1 and H5N2 as they migrate. Extent of migratory movements can vary among and within species. Migratory groups of birds grouped together are called flyways. Birds can change habitats.

Why do pandemics of influenza occur?

When influenza A virus mutates dramatically, resulting in high morbidity and mortality.

mRNA synthesis and replication of virion RNA

v(-ssRNA) replication occurs in the nucleus of infected cells. Usually the RNA is replicated by RdRP in the cytoplasm. During synthesis, influenza A engages in cap snatching. Transcription of vmRNA terminated near the end of genome segment on the portion of the template that contains a run of uracil resides (17-22) from its 5' end. Uracil residues are copied into A residues in the mRNA, generating a poly(A) tail before mRNA dissociates from vRNA template. Synthesis of antigenome +RNA does not use a primer, and generates a complete complementary copy of the template RNA (vcRNA).