Virology Exam 3

MERS pathogenesis

o Discovered in Saudi Arabia in 2012 o 2491 infected, 858 deaths, 34.4% mortality rate

SARS-CoV-2 pathogenesis

o Discovered in late 2019 in Wuhan China o 73,258 infected (as of Feb 2020), 1,868 deaths, 2.5% mortality rate

DNA vaccines

o No DNA vaccine have ever been FDA approves but they have been studied for many years o Similar to RNA vaccine but just getting DNA

Rubella

togavirus

structure of the reovirus virion

· 3 shells, 6 structural proteins

Describe CMV pathogenesis, with a focus on the main risk factor for birth defects in humans;

· CMV: A herpesvirues · Causes infected cells to enlarge · Transmission: saliva, urine, semen, uterine, cervical, vaginal secretions, breast milk · Greatest risk factor for birth defects is if pregnant mother gets infected. Reactivation of virus from a latent stage is not efficiently transmitted to the fetus. · 1-2% of newborns are carried. · 15-20% of these show evidence of CNS damage · 1% of neonatal deaths are caused by CMV · 0.1-0.2% of all babies born have CMV-associated birth defects. · 80 to 95% of adults are already antibody positive in the US. · Congenital CMV o Splenomegaly, hepatomegaly, hepatitis, vascular involvement, anemia, petechial rash, respiratory infection, pneumonia o Microcephaly, hydrocephaly, intracerebral calcifications... o Some pattern of mental retardation or learning disability · No vaccine · Treatment: Ganciclovir

major risk factors for severe COVID-19 disease

· Cancer - radiation, immune suppression. · COPD (chronic obstructive pulmonary disease) · Immunocompromised state - AIDS, weakened immune system, organ transplant · Obesity - BMI over 30 or higher · Serious heart conditions - such as heart failure, coronary heart disease, or cardiomyopathies · Type 2 diabetes mellitus

5 reasons why there isn't a vaccine for the common cold

· Common cold caused by viruses and bacteria · Caused by many different viral agents, many of which are genetically unrealted · Large diversity of viral serotypes amongst the agents that do cause it · Mucosal adaptive immunity to "hit and run" viruses is generally not long-lasting Vaccine developers have no guarantee of making profit for preventing a nuisance disease

List the main birth defects associated with Congenital Rubella Syndrome;

· Deafness, heart disease, eye defects, hepatitis, bone inflammation (osteitis), mental retardation · Causes "blueberry muffin rash" · 1st trimester: 20% abort, high CRS · 2nd trimester: decreased CRS · 3rd trimester: almost no problems · Control: MMR vaccine highly effective at preventing birth defects

Diagnosis for rotavirus infection and history of the rotavirus vaccine

· Direct antigen test in fecal specimens (ELISAa rotazyme test), electron microscopy · Vaccine: Rotashield (1998), made with non-pathogenic monkey rotaviruses plus human rotavirus capsid proteins (recombinant, live attenuated). Caused intussusception of small intestine into large intestine but not a serious problem.

Major characteristics in the family reoviridae

· Genome: dsRNA (unique, much of lifestyle based on this), 10-12 segments, 16-27kb · Virion: non-enveloped, icosahedral, 2-3 capsid layers, 70-80nm · Transmission: fecal-oral for rotaviruses and mammalian reoviruses, tick bite for Colorado Tick Fever (CTF) · Natural host: humans and other mammals, also squirrels for CTF · Diseases: diarrhea, fever, gastroenteritis · Antiviral drugs: none · Vaccines: rotavirus vaccine exists. · 2 or 3 shelled capsid, disease range from gastroenteritis to CTF · Genome melts apart upon heating, resistant to S1 RNAse · Carries its own transcriptase in virion (dsRNA dependent RNA polymerase) · Reoviridae = viral pathogens of the gastrointestinal tract. Reo = Respiratory Enteric Orphan (no longer true)

Prevention of norovirus

· Hygiene, handwashing, fomite control, sewage handling, clean water, no vaccine

nomenclature of influenza strains

· Influenza virus type/host species (omitted for human types)/the geographical site/serial number/year of isolation/for type A: the subtype in brackets; H for hemagglutinin and N for neuraminidase · Ex: Influenza A/Moscow/10/99 (H3N2)

how do coronaviruses maintain a large genome?

· Mutation rate of RDRP usually limits that size of RNA but this RDRP has proofreading ability · Frequent recombination may allow for fixing mutations · Many coronaviruses encode RNA processing enzymes with 3 to 5 exonuclease activity - possible proofreading function

processes leading to antigenic shift

· Only influenza A exhibits this. It is the only virus that effectively infect multiple types of organisms. Influenza C also only has 1 type of HA/NA and thus cannot produce new combinations. · Swine are mixing vessel that have receptors for BOTH AVAIN and HUMAN viruses!!!

Describe the term "satellite virus" and explain how hepatitis D replication is aided by HBV.

· Satellite virus - a virus that is completely dependent upon another specific virus for replication. · HDV NEEDS HBV to replicate/infect · Uses proteins from HBV to replicate, uses HBV surface antigens for budding. · Will lead to more severe hepatitis, more chronic, more cirrhosis as compared to HBV alone

genome structure of bunyavirusess

· Spherical virions, enveloped, 2 glycoproteins, no matrix protein · Segmented minus RNA (3 segments) · 3 circular nucleocapsids · 5' caps pirated from cell cytoplasmic RNA's/ · Genetic re-assortment occurs but only between closely related viruses. · Always encode nucleocapsid, always encodes glycoproteins, always encode RDRP. In that order. Variation in sizes per segment. · Ambisense coding only used y some genera

List diseases caused by Zika virus, transmission mechanisms, and rate of detectable disease in humans;

· Transmitted by Aedes aegypti mosquitoes, sexual transmission also · Causes microcephaly - back of the brain doesn't develop. · Most common diseases: fever, rash, joint pain, conjunctivitis (lasts up to days or weeks) · Incubation period is up to two weeks, about 80% of people are asymptomatic. · No specific treatments, no vaccine Most people who get infected don't get sick - rate of detectable disease in humans

flu diagnostic techniques

· Virus isolation o Respiratory tract specimens o Rapid cultures o Madin Darby Canine Kidney cells o Standard culture · DFA on exfoliated NP cells - identifies type A, type B antigens

advantages of having a segmented genome

· ability to re-assort to make new viruses, ability to control gene expression from each segment independently (only make lots of proteins from particular genes)

most virulent influenza

A

3 main classes of anti-flu drugs target

M2 ion channel to uncoating NA inhibitor Block 5' snatching

unrelated vaccines

o There is evidence that when you get a vaccine for virus X it protects you from A, B, C, D, etc. o Not sure why - MO's could be more activated.

adenoviridae

really BIG knobs that stick out on the end!

Explain the priming strategy for transcription of Hantavirus mRNA;

· Cap snatching and prime-align for transcription of viral mRNAs, does not apply to production of vcRNAs or genomic RNAs. · Similar to flu but stolen from cytoplasm mRNA

astrovirus transmission

· fecal-oral route, person to person, in food and water.

replication of astroviruses relies on what?

a slippery stop codon

e

envelope

CMV

herpesvirus

inactivated vaccines

o Work in a similar fashion to protein vaccines o Hard to develop because we don't know the virus very well. o Not really any progression here.

s

spike

List 5 different respiratory-related diseases caused by adenoviruses in humans and be able to differentiate between viral and bacterial sore throat symptoms; and

· Acute febrile pharyngitis, acute respiratory disease, pharyngoconjunctival fever, pneumonia, pertussis-like syndrome

Explain how antigenic shift applies to vaccination strategies;

· Quick changes in the genome that are big time changes - recombination and re-assorting. · Both happen with influenza. · The viruses evolves in ways that other viruses cannot. Because of this, we have to update the flu virus every year.

What is the large group of viruses that's associated with gastroenteritis?

Norwalk

Particles of HBV

Spherical small particles Core particles/dane particles Filamentous

pathogenesis of Hep A

· 1.5 million cases per year in the world. Case fatality 0.1% in children, 2.1% in adults. o Symptoms: inapparent, jaundice, liver degradation, death o Pre-icteric: anorexia, malaise, nausea, diarrhea, abdominal discomfort, fever-chills, dark urine, light colored stools o Icteric: jaundice, release of liver enzymes o Virus sheds in blood, feces, urine o Very stable but susceptible to chlorine o Spread through FECAL-ORAL ROUTE! o Contaminated food, water, poor sanitation, food handlers, shellfish concentrate virus o Control: hygiene, vaccination

Difference between Hep A-E

· A and E are unique because most hepatitis viruses are blood transmission but they are fecal-oral transmission, no cancer, no lifelong infections · Hep A, B, C, D, and E are all from different virus families, yet they all cause the same disease. Example of convergent evolution. o A and B we have vaccines. o A and E are fecal-oral transmission, no lifelong infections, and no cancer. o HepB/C/D can all cause lifelong infections, can cause cancer, blood, sexual or vertical transmission.

explain the structure and gene sequence of the paramyxovirus genome, including the 2 purposes of intergenic signals

· All paramyxoviruses have intergenic signals - or between gene signals. · They all have an end gene which will be their first gene. Then they have the P/C gene that is necessary for polymerase to function and sometimes make bonus genes. · They generally have one protein that does the binding, and one protein the does fusion. · The purpose of having intergenic sequences is to establish a gradient. o Polymerase will start transcribing and when it reaches the end of the NP gene it reaches an intergenic signal. The intergenic signal has a bit of a confusing sequence. It is a series of U's that make a poly A tail. · This allows the mRNA to crease a poly A tail · When we reach the intergenic signal the polymerase can fall off. If it falls off it has to go back to the beginning and start again. The creates the gradient of genes and mRNA's. · There will be transcriptional polarity because the 3' end will see more transcription of mRNAs than the 5' end because the polymerase will fall off at intergenic signals, rarely making it to the L gene. · Make separate mRNA's - another purpose. · Polymerase stalls at intergenic signals. Puts on a poly A tail by transcribing U's. Then it can fall off, or keep going. If it keeps going it will spit out an mRNA for different proteins when it reaches the intergenic signals. Each time, there is about a 20% chance that polymerase will fall off. Makes it so that the L protein, which is the last protein transcribed, it transcribed the least.

pathogenesis of rotavirus gastroenteritis

· Decreased absorption of both water and nutrients · Incubation: 2 to 5 days · Fecal-oral transmission, virus readily survives drying out due to thick capsid shell · Outbreaks common in nurseries, day care centers and hospitals · Vomiting, diarrhea, dehydration. Metabolic acidosis · Lots of deaths in malnourished infants in the third world · Segment 10 of the genome encodes NSP4 which is a potent enterotoxin in and of itself. · Severe inflammation of small intestinal tract; virus prefers to replicate in cells at the tips of microvilli. o Creates a loss of surface area in the intestinal tract because it damages the microvilli

Explain the HBV replication cycle, with emphasis on the mode of genome replication;

· First step: viral genome reaches the nucleus and is repaired by host repair enzymes to make a regular old dsDNA circular genome called cccDNA o Removes protein from 5' end of (-) strand o Replaces single RNA nuleotide with DNA nucleotide on (+) strand o Fill in gaps to make fully dsDNA · Second step: RNA transcription o Host cell RNA polymerase II uses viral promoters to make a variety of mRNA transcripts o One of these transcripts is larger than the full length genome (pgRNA), blazes past the promoter where it started. o It will be longer than the genome because it needs to add a 3' poly A tail which is encoded after the C promoter where it starts o Also needs to preserve the ends of the genome · Third step: pgRNA reverse transcribed back into DNA to make the (-) DNA strand by the viral reverse transcriptase o Reverse transcriptase uses protein primer to make a small DNA copy using 5' end of pgRNA including direct repeat 1 - it is so small because it runs out of genome o Copied DNA releases 5 end and translocates to 3' end where it hybridizes to other DR1 o Reverse transcribe the rest of the genome to DNA, RNase H activity if RT degrades RNA as it goes. Small RNA fragment left undigested by RNase H acitivity. o This pgRNA is then used to make more viral genomes o How is it primed? With protein primer with a free -OH group à How protein gets on the (-) DNA strand. o This protein starts of the 5' end of the genome and has to be translocated to the 5' end of the genome once it has gotten to the end of its 5' strip that it copes first. o Protein = tyrosine o pgRNA is chewed up in this process of making (-) DNA strand, leaves one RNA nucleotide and the cap. · Fourth step: (-) DNA strand is a template for reverse transcriptase to make (+) strand, but (+) strand is left incomplete because the capsid is completed before the 2nd strand of DNA is fully polymerized. o DNA produces using this small RNA fragment as a primer, have to translocate again back to DR1 and then continue. o Cyclization of (-) strand; incomplete synthesis of (+) strand. o The cap that was on the pgRNA + the one nucleotide of RNA is translocated to serve as a primer for the (+) DNA strand. This is how the one nucleotide of RNA gets on the 5' end of the (+) DNA strand.

Symptoms of viruses that can cause gastrointestinal disease

· GI tract - surface area for absorbing nutrients and water is enormous. Infections that kill cells of microvilli cause diarrhea and malnutrition · Nausea and vomiting, abdominal cramps, diarrhea, headache, myalgia (muscle pain), low grade fever, dehydration (most serious) · Duration = 12-60 hours. Virus shed for 3 days. 10 particles cause infection.

Caliciviridae

· Genome: (+)ssRNA, about 7.5kb · Virion: non-enveloped, icosahedral, 30-38nm · Transmission: fecal-oral · Natural host: humans · Diseases: gastroenteritis · Vaccines: none · Antiviral drugs: none · Very stable, very difficult to decontaminate, one place of virion surface has a depression on it

Explain why HBV assumes a variety of morphological structures and how this is advantageous to the virus, and also be able to identify HBV virions in electron microscopic images;

· Hepa = liver topic, DNA = DNA genome · Hep B is only family member to infect humans · It creates a lifelong, chronic infection in children, a serious acute infection in adult (case fatality 1%) · Hepatitis: infection/inflammation of the liver · Blood-blood transfer, acute or chronic · Infection/cancer preventable by vaccine · A DNA virus that uses an RNA copy + reverse transcriptase to replicate the DNA genome · Forms 3 different particles: o Spherical small particles = decoys for the immune system, has the capacity to make these particles faster than actual infectious particles since they need a copied genome o The core particles/Dane particles = infectious, has core and envelope. o Filamentous - no core or genome, defective

Write about the pathogenesis, reservoirs, and control of Hantavirus pulmonary syndrome;

· Humans are unnatural hosts and get infected by exposure to rodents · Hantavirus pulmonary syndrome (HPS) in the US can be cause by Sin Nombre virus (SNV) · Not an arbovirus · Transmitted to human though rodent extreta by inhalation or direct contact with rodent urine or feces. · Virus is endemic in rodent population and causes little disease. · Infection occurs during harvest rimes and during winter when rodents invade houses, sheds, and garages and piles of firewood · Inhalation of virus takes it to the LRT · Virus invades lungs and causes a profound rapid pneumonia and pulmonary collapse · Edema, fluids influx, and debris fill the alveoli · Resulting in hypoxia, anoxia · Fever, deep muscle aches and severe shortness of breath · Occurs in all age groups but remarkably rapid in young, healthy adults. · Control: control rodent population, mask up when cleaning areas with possible rodent extreta, be careful and wear a mask when going caving, no good vaccines, not transmitted effectively from person to person

prevention, treatment, and vaccines for rotavirus

· Prevention: hygiene, handwashing, fomite control, sewage handling, clean water · Treatment: rehydration, electrolyte balance

pathogenesis of hep B

· Vertical transmission: about 20% chance that infected mother will pass to baby. · Sex · Parenteral incoluation · Transcplacental - congenital infections · Oral - not very common · 3 main diseases: o Hepatitis: inflammation of liver o Jaundice: destruction of liver tissue leads to abnormal liver function o Hepatocellular carcinoma: liver cancer o Over 1 million deaths worldwide per year · Incubation period: 40-180 days, a SLOW infection, not very cytocidal at all, innate immune system not very well alerted. 1-2 months before it is readily detectable. · Preicteric symptoms (before jaundice) o Anorexia, malaise, nausea, fever, chill, abdominal discomfort · Icteric symptoms (jaundice) o Dark urine. Light colored stools, release of liver enzymes, possible cirrhosis, liver cells killed by CTLs, hepatocellular carcinoma, death o Bilirubin is normally broken down in the liver but if the liver can't break it down, we will turn yellow. o Bilirubin produced from dying RBC's.

The covid controversy... lab created?

· Was it engineered by humans? o When we discover new viruses we commonly sequence their genomes and then submit those results to online databases o After SARS-1 epidemic in 2003, Dr. Shi Zhengli's lab started looking got bat coronaviruses · Is this really a new virus? o No one has found it in nature BUT w/ such a large genome and a 99% similarity between two viruses, that means they are still pretty different. o Shares highest overall genome sequence similarity to the bat coronavirus RaTG13 · There are quite a lot of differences between SARS-CoV-2 and closest known virus relative...essientially impossible that this virus was released from a lab unless it was unpublished, which is unlikely. · There are 931 synonymous substitutions (no diff in amino acid just diff in last nucleotide of codon) and 145 missense substitutions between SARS-CoV-2 and RaTG13

influenza A

· birds, mammals, waterfowl (not humans) o Co-infection of a single organism with multiple influenza A viruses can result in re-assortment and pandemics o Most severe disease in humans o Most important Orthomyxoviral pathogen of humans due to variation in HA/NA proteins and production of recombinants. o Principally a bird virus - all 15 subtypes found in birds. 5 subtypes found in humans (H1, H2, H3, H5, H9 - last two not as good at infecting humans)

4 unique aspects of the HBV genome

1) (+) sense DNA has the 1st 5' nucleotide as RNA, not DNA a. Serves as a primer for (+) DNA strand, RTase still sits on top of RNA 2) (-) sense has a protein attached on the 5' end that serves to prime a. Serves as a primer on the (-) DNA strand, builds DNA onto a protein. 3) (+) sense DNA is partial length of circle/virion a. Capsid proteins assemble before this strand is fully polymerized b. Capsid closes up around it before it is finished. 4) (-) sense DNA is greater than the full length of the circle a. End of protein primer base pairs with the other end of the genome which allows for production of the (+) strand but also makes (-) strand too large. b. pgRNA is too long and so our (-) strand is also going to be too long. · Messy genome à clean DNA genome (through cellular repair enzymes) à RNA genome à messy DNA genome to be packaged.

which type of influenza has the most diversity

A has tons of branching and diversity but we also have lots of diversity in B. C doesn't have as much.

types of influenza

A, B, C (NP and M Ags)

Xofulza

Blocking of 5' snatching

least virulent influenza

C

Advantages to using slippery stop codons

Cool trick for (+) RNA viruses because it allows them to produce different amounts of their various proteins. It is a waste of energy to make a billion copies of a polymerase just so you can produce a billion copies of a capsid protein. 100 copies of a polymerase can do tons or work for us.

MERS-CoV

Middle East Respiratory Syndrome Coronavirus o First reported infection in 2012, all reported infections too place in 9 countries around Arabian Peninsula o Human to human spread via close contact, but efficiency is low o Camels may be natural reservoir o The cirys resembles coronaviruses isolated from bats like SARS o Symptoms: severe acute respiratory illness including fever, cough, shortness of breath o Infect non-ciliated bronchial epithelial cells. o Evades immune responses, specifically INF o Case fatality rate: about 30-40% o People are still getting infected and dying as of 2017.

Generate a list of human viruses that cause birth defects

Rubella Zika CMV

n protein

bathrobe

Symmetrel

blocks M2 ion channel to uncoating. Essentially useless now because >99% of influenza viruses are resistant.

what would need to be done to see if someone has COVID-19?

chest x ray

Spherical small particles

decoys for the immune system, has the capacity to make these particles faster than actual infectious particles since they need a copied genome

Zika virus

flavivirus

pathogenesis of Hep C

flavivirus. Pathogenesis similar to HBV, even though it is a genetically unrelated virus.

coronaviridae

has a crown around it, native the large spikes and envelope, see the individual spike proteins

influenza C

humans and rarely pigs (common cold)

the core particles/dane particles

infectious, has core and envelope

major antigens of influenza

internal nucleocapsid (NP), hemagglutinin (HA/H), neuraminidase (NA/N), influenza C encodes HEE (hybrid of N and H), M protein (major structural proteins Ag od envelope).

Astrovirus

look like a star

m

membrane

myxo

mucus

COVID-19

name of the disease

SARS-CoV-2

name of the virus

filamentous

no core or genome, defective

orphan virus

no disease

Recombinant viral vectored vaccines

o All trying to use adenovirus to deliver genes into host o People have been using adenovirus to vaccinate for a while but nothing is approved. o Change the genome and eliminate most of adenovirus. Replace with Covid genes, like the spike gene. o Adenoviruses cause mild respitatory disease so it has to be attenuated. o Weakness: it naturally infects humans so if an individual has seen this specific adenovirus before, the immune system will neutralize the vaccine. o Some have tried with chimpanzee adenovirus.

SARS-CoV pathogenesis

o Discovered in China in 2003 o 8098 infected, 744 died, 9.6% mortality rate

pathogenesis of covid in different species

o Humans: respiratory tract, common colds (winter, early spring) § 15-25% of common colds have coronavirus etiology § Can infect lower respiratory tract (unlike rhinoviruses) § The Big 3: lethal pneumonia possible, mostly in the elderly or those with other health factors o Cattle, swine, turkeys § Gastroenteritis o Mice § Encephalitis, enteritis, rhinitis, hepatitis o Chickens § Infectious bronchitis

moderna mRNA

o No mRNA vaccine has ever been approved, this is a new idea o Want to use nucleic acids to hopefully create an adaptive immune response o Want to try and use mRNA because it would only have to reach the cytoplasm to work, not the nucleus like DNA. o Strengths: you can develop T cell responses in addition to antibody responses o Weaknesses: potential stability problems, tricky to get a new vaccine approach approved.

protein based vaccines

o Striking the spike protein in the lab and then injecting that protein into you o Immune response will be antibody based. o Virus has several different membrane proteins and so we could develop antibody against any of those as well.

vaccines that give T cell responses

o mRNA, DNA o Protein and inactivated à only Ab responses.

three reovirus groups that infect humans

orthoreoviruses, coltiviruses, rotaviruses

REOviridae

respiratory enteric orphan

reoviruses

rotavirus, has spokes, multiple layers of capsid

you're being tested for _________ when you get a "covid test"

sars-cov-2

What are we targeting in sars covid vaccines?

spike protein (antireceptor)

what are the three envelope proteins of covid?

spike, envelope, membrane, some also have an HA-esterase envelope protein possibly acquired from influenza C

Transmission of Noroviruses

· Stable in water and food by spread by fecal-oral route, survives for a long time in environment. Fomites, heat and pH stability. · Found in ice for soft drinks. · Raw clams and oysters. Uncooked shellfish and salads. · Found at sporting events. · Short term immunity after recovery, by 1 year patient can be re-infected. · 57% were food borne · 16% person to person · 3% contaminated water · 23% undetermined · Restaurants/catered meals: 36% · Nursing homes: 23% · Schools: 13% · Cruise ships/vacations: 10%

Calicivirus

· have a depression on surface or virion o Calici = chalice or cup, see depressions on surface of virions. o Also called Norwalk viruses o 30-38nm in diameter, icosahedral, 90 capsomers, one capsid protein, non-enveloped. o Resistant to heat (60 degrees) and stomach acid o 3 open reading frames on plus sense RNA o Noroviruses cause about 90% of viral gastroenteritis/50% of all gastroenteritis

disadvantages of having a segmented genome

· have to have a way to segregate genome pieces so that each virion gets 1 of each · Re-assorted virus is rarely viable/infectious · Most segments encode only 1 protein, there are 2 segments that encode 2 proteins

modifications to the reovirus virion

1) Proteolysis to produce ISVP can occur either outside cells or inside endosomes. 2) RDRP is packages inside the virion 3) Early genes encode RNA polymerase elements and core proteins (early/late phases not shown here) 4) (+) ssRNAs are packaged first into new cores, then RDRP converts ssRNA to dsRNA inside new cores. 5) Late genes encode outer capsid shell 6) Yield about 20,000 virions per cell; takes about 6 hours to complete. · Proteolysis within endosomes, modification of outer capsid (VP7), carried out by host cell trypsin (a protease). Creates ISVP form of virus. o Trypsin normally destroys virus particles; rotavirus has evolved to use this enzyme to promote virus replication. · ISVP is an intermediate, sub-viral particle. This form is able to exit the endosomes; region that allows for endosomal escape is exposed after the 1st step, loss of outer capsid. ISVP escapes endosome to cytoplasm which results in further rearrangement to produce viral core · Viral core = home of viral transcription · The genome is retained in the capsid because it is dsRNA, which is huge singal to cells to make INF and activate immune response. · No RNA replication to make dsRNA will occur until every segment is gathered and capsid assembly is nearly complete. · Uses a hit and run replication strategy and by genetic diversification through mutation. Virus replicate quickly and innate immunity largely controls virus. Poor adaptive immunity is typically developed which means that we can get re-infection by the same serotype.

SARS-CoV

o A new virus appeared starting an outbreak of severe respiratory disease. Started in Southern China. Spread to more places in Asia, then Canada, US and some other countries. o Transmission: human to human contact, close contact. Possibly fecal-oral. o Incubation period: 2-10 days then acute interstitial pneumonia, fever, non-productive cough, shortness of breath, hypoxia, alveolar damage, death, fever-chills, rigors, myalgia, diarrhea o Overall case fatality is about 10%. § <1% in patients under 24 § 6% in ages 25 to 44 § 15% in 45-64 § >50% in 65+ o Shedding: respiratory, fecal, urine o Reservoir: Chinese Horseshoe Bats o Virus was first seen by electron microscopy, genome then cloned and sequenced, called SARS-CoV o SARS-CoV and SARS-CoV2 transmitted readily from person to person even though humans are not natural hosts. Relatively uncommon for unnatural hosts. o These two viruses are 70% similar.

what is a slippery stop codon and how does it work?

o One way that viruses can pack more coding potential into a limited amount of space is to ignore stop codons part of the time. § Efficiency of ribosomal read-through is low, about 5%-20% of the time the 2nd proteins will also be translated/it misses the stop codon o A protein that is required in low abundance will be downstream of a stop codon § Virus encodes repeat sequences in RNA that make it easy for the ribosome to "get confused" and then to change frame, thus ignoring stop codon due to ribosomal frameshift (repeats such as AAAUUUA cause ribosomal slipping) · How does the slippery stop codon work? 1) Stop codon is read in frame, but is accidentally recognized as a regular codon (mis-matched tRNA, not a release factor) a. Results: second protein is in the same ORF as 1st proteins 2) Ribosomal frameshifting: ribosome shifts by -1 or +1 due to slippage a. Result: for any given gene, 2nd protein is in different ORF as 1st protein b. A nucleotide could be read twice by the ribosome, thus shifting the frame c. Ribosome will stop once it reaches a stop codon in this new reading frame For any given gene only ONE of these mechanisms is used.

why are nearly all GI tract viral pathogens non-enveloped?

they are tougher and can survive the low pH and digestive enzymes in the GI tract.

Describe the structure of the coronavirus virion and be able to identify virions (note the envelope and large spike proteins);

· (+)ssRNA, 27-31kb · Virion: helical, enveloped, 120-160nm · Transmission: aerosol, fecal-oral · Largest genome of any RNA virus - there is good evidence that RDRP may have proofreading ability

pathogenesis of Hep E

· (+)ssRNA, non-enveloped, natural pig host, no vaccine or antiviral drugs, FECAL-ORAL TRANSMISSION!Causes jaundice and hepatitis o 3 ORF's, 1 capsid protein, 5 nonstructural o Inc. period: 2 to 8 weeks o Damage occurs to hepatocytes, levels of ALT arise, symptoms similar to HepA. o Case fatality 0.5-3% but much higher (15-20% in pregnant women) o 10-40 yrs of age o Be careful of contaminated water. Cook food well, chlorinate water. No vaccines.

major features of the influenza virus

· (-)ssRNA, 6 to 8 segments · Virion: enveloped, helical, 80 to 120 nm · Transmission: aerosol, also fomites · Natural host: humans, waterfowl, pigs, birds, other mammals. · Diseases: respiratory disease and pneumonia · Vaccines: yes, but strains targeting vary annually · Antiviral drugs: yes, targeting NA activity, targeting cap-snatching, M2 ion channel (now obsolete due to ubiquitous viral resistance) · Influenza A killed a lot more people in the 1918 Spanish Flu pandemic than those killed in WWI. Segmented genomes results in regular re-assorted virus strains, causing pandemics. · Replicate in the nucleus: required to perform RNA splicing of some mRNAs and splicing machinery is in the nucleus.

major features and structure of the viruses in the bunyaviridae family

· (-)ssRNA, some ambisense coding, 3 segments · Virion: enveloped, helical · Transmission: arthropods, inhalation of rodent excreta · Natural host: mammal · Diseases: fever, encephalitis, Hemorrhagic fever, pneumonia · No vaccines · Antiviral drugs ribavirin reduces mortality for Hantavirus and Crimean-Congo · True worldwide distribution, human infections relatively rare, but can cause lethal hemorrhagic fever or pneumonia

Explain and be able to diagram how mRNAs are produced using the ambisense coding strategy (and all intermediates)

· Ambisense RNA's don't conform to regular (+) gene and (-) notation · Multiple genes can be encoded in a single RNA molecule, but they run in opposite direction relative to each other. Thus, you have to make 2 different kinds of mRNA running in opposite directions from a single RNA molecule template. o One mRNA produced from (-) sene genomes o Another produced from the (+) vcRNA molecule · S segment encodes in both direction · N protein made in typical fashion - make (+) mRNA from (-) template · NSs production: make mRNA from the vcRNA Summary · Allows virus to have true early and late gene expression - gives great control from one half of the genome from the other. o Half + and half (-) à figured out a different way to regulate the proteins that are produced · (-) ssRNA comes into cell as the genome · RDRP makes an mRNA that encodes the N protein. RDRP stops transcribing when it reaches stem-loop in middle of genome o Stem loop structure says STOP! · Ribosome translates N protein o N protein is made first - before we make antigenome we want to be able to cover ourselves up o Once we have enough N protein, we can make full copies of the genome. · Once enough N protein is made, RDRP now is able to transcribe all the way across (-) genomes and make (+) vcRNA · RDRP nor binds to 3' end of the vcRNA and makes another mRNA which encodes the NSs protein. Ribosome then translates NS's proteins. o Stops at stem loop · The mRNA that is produced is the only RNA with 5' cap and poly A tail · Use antigenome to make MORE genome · N protein will bind to stem-loop and make it so that it can be transcribed

Write about the steps of the replication cycle, with a focus on: a) the need for nuclear events, b) mechanisms/reasons for cap-snatching, and c) involvement of pH and proton pumps (viral and cellular) in the viral entry and egress processes;

· Attachment: not to a specific protein, but to a molecule attached to many extracellular proteins à sialic acid = receptor on cell for attachment of virus, attached to a protein on the outside of the cell. o HA binds to SA. o Neuraminidase will cleave the alpha2,6 linkage that attaches SA to the protein. Will prevent binding of soluble proteins. Also allows virus to escape from cell (coated with receptor). · HA binds to sialic acid and the cell endocytosis the virus in. Protons are then pumped into the endosome and that causes the virus envelope to fuse with the cellular lipid membrane of the endosome because of a conformational shift in HA. Then it goes into the nucleus and makes mRNA's, proteins and then the antigenome to make the genome. · Budding and release: (-) genomes are prodices within the nucleocapsid using (+) vcRNAs as templates. NO (+) vcRNA is packaged. · A) Nuclear event purpose o where mRNA's are made and cap snatching occurs. o Splicing is another reason why orthomyxoviruses replicate in the nucleus. § Some mRNA's are spliced to produce multiple protein products. § Both sliced and un-spliced mRNA serve as templates for translation \cap-snatching takes place before cellular mRNA's can be translated · B) Mechanisms for cap-snatching - mRNAs are primed by cap-snatching. Steals cap + some nucleotides. o Priming mRNA for production o Redirect gene expression from cellular to viral genes - interfere with INF production. Interferes with cell gene expression,. o No need for the virus to encode its own capping enzyme o Easy way to ensure that viral mRNA get usable 5' cap regardless of organism infected - humans are not a natural host so this ensures that it can effectively infect no matter what organism it infects. o Can cleave and steal caps. PB2 - binds to cellular capped pre-mRNAs. PA - removes the caps. o OB2 and PBA identify the structure and one of them cuts it off the cellular mRNA and it will then base pair a little bit with the viral mRNA. o Use 5' cap as a primer to make viruses own mRNA - builds off of the 3' end. · C) Why pH and protons? Proton pumps? o H+ into endosome promotes fusion of viral envelope with endosome lipid membrane a) Activates fusogenic activity of GA during entry. o H+ into virion for disassembly of nucleocapsid and polymerase activation H+ out of vesicle, prevents activation of fusion - M2 channel works opposite in this step. Prevents activation of HA during assembly/egress

astrovirus genome

· Genome (+)ssRNA, nonenveloped, icosahedral, fecal-oral transmission, natural human host, causes gastroenteritis, no vaccines, no anti-viral drugs, 5 to 6 pointed stars on virion is the origin of the virus family name. Treat the symptoms (rehydration) · 8 serotypes, 2 to 3 capsid proteins, resists 60˚C, 5 min, pH 3 (very stable!)

important features of the viruses in the paramyxoviridae family

· Genome: (-)ssRNA, 15-18kb · Virion: enveloped, helical · Transmission - aerosol, fomites, bodily fluids · Natural host: human, horses/bats (Hendra), cats/dogs/pigs (Nipah) · Diseases: respiratory disease, meningitis/encephalitis, fever/rash/immune suppression, measles, reproductive tract/sterility · Vaccines: yes, for mumps and measles · Antiviral drugs: an antibody is used to prevent RDV in at-risk hospital patients, also ribavirin vs. RSV · Broad spectrum of viruses and varying diseases · Myxo = mucus. Many use saliac acid as receptor and hence bind to mucus.

*****COVID Replication Cycle

· Incoming (+)RNA is translated - structural polyproteins are cleaved; ribosomal frameshifting occurs to produce polymerase · Virus encodes at least 5 RNA processing enzymes - uncommon for RNA viruses. Recent evidence shows that these can perform genome proofreading. · Nested set of subgenomic mRNA's - why do this since other mRNA's don't? · Nested set of transcripts - other (+)RNA viruses have 1-2 mRNA's maximum and here we have SIX, including genome!!! o How are subgenomic RNAs produce? 2 ways. 1) Primer directed synthesis from 3' end of (-)RNA - repeated sequences throughout genome allow polymerase to form an initial primer of about 60 nucleotides and then jump w/ primer to other sites w/ same sequence to make subgenomic transcripts 2) RNA transcription is primes at each site - short repeat sequences throughout genome may be sufficient to act as promoters. o True mechanism of priming is unknown but very high rates of recombination in coronavirus suggest evidence for #1.

Be able to describe how Flu strains are chosen for annual vaccines, how accurate the predictions are, and how the vaccines are generated;

· Influenza is so virulent because humans are an unnatural host. · The viruses used for making the flu vaccine are chosen each year based on information gathered over the previous year about the strains of the viruses that are circulating and how they are changing · Info is gathered by 122 national influenza centers in 94 countries - then analyze by WHO · Based on this info experts forecast which viruses are likely to circulate the following season · For recommendations being made in the Northern Hemisphere, the recommendations are made by WHO in early February · Each country then can use the recommendations made by WHO to assist with national decisions about what viruses to use in influenza vaccines for their country. · How they are generated: o 4 virus strains are produced independently then mixed together for injection · Accuracy o Getting the vaccine reduces getting the flu illness by 40-60% when vaccines are a good match. o Current flu vaccines are more effective against Flu A H1N11 and Flu B, and are less effective against H3N2 strains. H3N2 evolve faster in humans. o Even less effective flu vaccines can prevent severe illness I those that get infected.

neuraminidase

· NA cuts sialic acid. As the virus leaves the cell, there is SO much virus receptor on the surface of the cell that the HA on the progeny just made will stick to it if it isn't cut. NA cuts it and prevents HA on the new progeny from binding to the cell that has already been infected. HA does not recognize it once it has been cut. o Also removed soluble receptors in the mucus. They are called soluble receptors because sialic acid is just in the mucus and can neutralize HA so that it cannot infect any cells. NA cuts SA that is in the mucus to prevent this from happening. · Not all SA is equally bound to - there are different types of SA.

prevention and treatment of hep B

· Prevention: screen blood donors, vaccination · Viral hepatitis: liver inflammation, can lead to cirrhosis (tissue destruction), due to immune response to virus replication

treatments for gastroenteritis

· Rehydration, electrolyte balance (sodium, potassium, chloride, bicarbonate, glucose), no specific anti-viral, no vaccine available

rhinovirus pathogenesis

· Rhinoviruses are picornaviruses that cause the common cold more than anything else. · Respiratory spread - there are up to 20,000 droplets produced during a single sneeze. A few hundred per cough. Explains why these things are so contagious. · Nasal obstruction, edema (leakage of serum), rhinorrhea (runny nose), rhinitis (nose inflammation) · Pain: throat, nasal membranes, sinuses · Scratchy throat, sneezing, coughing, malaise, burning sensation upon inspiration of air · Sensation of saggy musculature · Possible lower respiratory tract infection, bronchitis and secondary pneumonia · No vaccines, no anti-viral drugs, hygiene prevents transmission, not a killer virus.

transmission and pathogenesis of parainfluenza and RSV

· Spread though URT route, aerosolized droplets. Aerosol, direct contact, fomites · Virus stable for hours in the environment · Seasonal community outbreaks · In adults mostly mild URT - replicates in mucosa - infections, causes inflammation in URTS, lower RT in infants. · In children, URT and severe LRT infections. · IN adults, common cold like infections, mild nasal congestion. · RSV - servere disease in infants, some children and the elderly. Spreads to LRTS in 25% to 40%. Rest of us = common cold symptoms. o CPE: large syncytia

symptoms of COVID-19

· Symptoms may appear 2-14 days after exposure to the virus. People with these symptoms may have COVID-19 · Fever (80%) or chills (63%) · Cough (84%) · Shortness of breath and difficulty breathing (57%) · Fatigue (62%) · Muscle or body aches (63%) · Headache (59%) · New loss of taste or smell (7-22%, less common is hospitalized) · Sore throat (14%) · Congestion or runny nose (21-51%) · Nausea or vomiting (13%) · Diarrhea (38%) · Effects on different organs - most people who have the virus it is found in heart cell 62% of the time · Effects on pregnancy - women who ae pregnant already have immune suppression so that they don't reject the baby. 0.16%) · Loss of smell and taste is also typical of other respiratory tract diseases.

why can the PVC gene complex encode so many proteins

· The PVC gene complex can encode so many protein because it has the ability to read in four different reading frames. · The P protein is made when the normal stop and start codon are used. Ribosome will come along and translate this mRNA and make the p protein. · Because of a weak Kozak sequence, there is another start codon that can be recognized by the ribosome. The stop codon ends much earlier, and this makes the C protein. It is read from a different reading frame. o A strong Kozak sequence would make it so that the P proteins start codon was ALWAYS recognized. This is not the case. · There is also the chance that the polymerase may reach the C nucleotide towards the end of the genome and when it reaches it, it can add +1G nucleotide. This would shift the reading frame from the P protein reading frame it was reading in, and create the V protein. · If the polymerase add +2G's instead of one, the reading frame is shifted yet again and this creates the W protein. · This all means that there will be THREE DIFFERENT mRNA's from the gene complex. · Only want this to be happening in the mRNA and NOT the antigenome. If these mutations happen in the antigenome then the genome would lose the ability to encode the P protein which is necessary for polymerase function. Don't want these G mutations inserted into the (+)vcRNA. · Multiple proteins are encoded in this process: o Multiple translation initiators from a single mRNA molecule: 2 AUG's or similar start codon sequences. o Frame shifting to produce multiple unique mRNA's from a single (-) RNA molecule: insertion of G's (can give +1, +2, or +4 frameshifts) · The Rule of Six o Even though +1 and +2 insertions are supposed to happen in mRNA, they can happen in the antigenome. o If you put in the extra G's there would be no way to fix it. You can never produce P protein once +1 G is inserted. o Virus has evolved a mechanism to figure if genomes or antigenomes have any +1, +2, or +4 insertions. We don't want this. o If you add one the virus will know because of the rule of six. Each copy of the N protein binds to 6 nucleotides specifically. If there is a frameshift due to extra nucleotides insertions the N protein cannot bind and this genome/antigenome will be disregarded. o The RNA polymerase efficiently replicates only viral genomes counting 6N = 0 nucleotides (exact multiples of six nucleotides). o The N protein interact with exactly 6 nucleotides (no more, no less) o The non-templated G's are only supposed to get incorporated into the mRNA, not the genome or antigenomes. This is a way for the virus to screen what is being put into the virion.

why do the impacts of seasonal flu and pandemic flu (like the 2009 swine flu) differ for humans?

· There are A BUNCH of different H1 genes that are all related. Different enough that there is not cross-immunity. · No swine flu outbreaks in humans for decades, so very little immunity in human populations. Occurrences like what happened in 2009 (re-assortment creating H1N1 swine flu) happen only every couple of decades and lead to pandemics. · It is hard to tell how many people got infected in 2009 since the disease was not sever and often subclinical · Nobody in the world has immunity to the pig portion of the virus unless you were is Eurasia taking care of pigs. · When these viruses recombined, nobody had immunity against it.

transmission mechanisms of sars-cov-2

· Through the air...inhalation o Transmission can occur through direct, indirect, or close contact with infected people through infected secretions such as saliva and respiratory droplets which are expelled when a person coughs, sneezes, talks or sings. o Respiratory droplets > 5-10 micrometers, aerosols < 5micrometers. Means that respiratory droplets fall out of the air faster, which is what we are trying to do with the six feet thing. It also means that other people have a better chance at getting infected because they are bigger. o Respiratory droplet transmission can occur when person is close in contact with infected peron or who is talking or singing · Through surfaces/fomites o Respiratory droplets can be expelled an contaminate surfaces and objects, crating fomites (contaminated surfaces). Virus can be found hours to days after. o Indirectly through touching surfaces in immediate environment with contaminated surface, followed by touching mouth, nose, or eyes. · Maybe through fecal, urine, or blood routes o Samples of virus found in urine and feces o Bloodborne, we are still not really sure. o Possibly breast milk

Characteristics of viruses in the coronavirus family

· Unique characteristics: o (+)ssRNA and helical à very abnormal for (+)ssRNA viruses o Incoming +RNA is translated, structural proteins are cleaved, ribosomal frameshifting occurs to produce polymerase o Virus encodes at least 5 RNA processing enzymes o Nested set of sub-genomic mRNA's o Up to 10% of coronavirus progeny are recombinants - due to hopping of polymerase to produce subgenomic RNAs may promote jumping from one strand to another to produce recombinants o SARS-CoV, MERS, SARS-CoV2 are emerging human pathogens with high mortality rates · Disease: mild to severe respiratory disease, also gastroenteritis in some · Natural host: humans, also bats and other mammals · Vaccines: none · Antivirals: none

*****Describe the common strategies used by all (-) RNA viruses for replication and generate a list of attributes shared by all (-) RNA viruses;

· Viruses in that have a (-)ssRNA genomes are more closely related to, for example, a (+)ssRNA virus · You have to make two different kinds of (+) o You start by making mRNAs from (-) genomes, all (+) mRNAs. o You then have to make full-length (+) RNA molecules (anti-genomes, vcRNA) as intermediates to make more (-) full-length genomes for packaging into new virions. · Basic features: o Incoming RNA is not a template for translation o Proteins packaged into virion. § P or NS: phosphoprotein needed for RNA replication § RNA-dependent RNA polymerase (L protein) § M: matrix protein (in all but Bunya/Arena) o ALL are enveloped, many spherical. Some have interesting morphology. Some can have more than one shape. o ALL have helical nucleocapsids. N protein associates with RNA to make nucleocapsid. § Makes all these viruses very flexible in size. § Several families have segmented genomes = rapid evolution § Genomes and anti-genomes ALWAYS remain associated with nucleocapsid during replication cycle. o After uncoating ALL use virion RDRP to make mRNAs (sub-genomic in size) § mRNA never as large as (-) genome § mRNA's NOT a template for making (-) genomes § separate (+) RNA used to replicate the genome (called vcRNA. Made later than mRNA in replication cycle. o Many genomes have inverted terminal repeats that may promote cyclization o Genes in 3' end of (-) genome expressed more heavily o Only rare polyprotein production followed by viral protease cleavage. o No 5' caps or poly A tails in (-) RNA. Why? Because they are translational signals. · General (-) RNA virus replication o (+) mRNA's released from nucleocapsid upon production o (+) vcRNAs are not translated and remain in nucleocapsid. Sole purpose is to make more (-) RNA o (+) mRNAs made from (-) RNAs are usually monocistronic (only gene encoded) § RDRP scans along (-) RNA and then starts transcribing when it finds an appropriate promoter; stops transcribing when it hits transcriptional terminator · Genome organization and genes themselves are similar for all (-) RNA viruses. All encode glycoproteins that help with binding to the cell. In some cases there are F proteins that promote fusion with the cell. General virus replication strategy (exception is orthomyoviruses!!!) (look at image on review slides)

Describe the major features and structure of the viruses in the family Adenoviridae;

· dsDNA · Virion: non-enveloped, icosahedral, long penton fibers · Natural host: found in many different organisms · Diseases: Many · Vaccines: used to be some from military personnel to prevent pneumonia, just a few virus types targeted · Antiviral drugs: non available · 68 types that infect humans discovered. Can cause cancer in unnatural hosts.

hepadnaviridae

· forms several different types of particles. The Dane particles, filamentous, spherical

influenza b

· humans, doesn't evolve as fast. o Recently, we've recognized that this virus causes more infections/disease than previously appreciated. Better targeted by vaccines now.

NA inhibitor

· prevent release of infected cells, prevents cleavage of sialic acid in mucus (HA unable to bind due to saturated binding sites)

vcRNA

· purpose for genome replication. Coated with nucleocapsid. Not capped or polyadenylated. Exact complements of viral genomic (-) RNA. Contain promoters for genome replication. Only (-) full-length copies have packaging signals.

mRNA

· purpose is to express viral genes. Not coated with a nucleocapsid, capped and polyadenylated, not an extract complement to genomic RNA. Shorter, lacking promoters for replication, lack packaging signals.

hemagglutinin

· responsible for cell binding and fusion. Anti-receptor on virus that binds to sialic acid receptor on cell. o Virus can sometimes bind to RBC because they have sialic acid on the surface. Virus does not want this because there is no machinery in RBC to replicate. But, the virus does cause agglutination of RBC and is one way that infection can be diagnosed.