VPBio 3554 Exam 4

Hepatitis

What is Hepatitis?•Hepatitis: Inflammation of the liver- Classic symptom is jaundice- Caused by a number of viruses and sometimes alcohol or prescription drugs•Hepatitis epidemics in history- War: Crowding and unsanitary conditions of military encampments created an ideal environment for hepatitis A viruses to be transmitted The History of Hepatitis Camp jaundice and outbreaks recorded:• Napoleonic Wars• U.S. Civil War: Camp jaundice• WWI: Trench warfare• WWII: Serum hepatitis (via vaccination) and infectious hepatitis (contaminated battle fields)• 8-9% of Veteran Affairs (VA) medical center patients are positive for hepatitis C antibodiesWhat is the cause of jaundice?Excess of bilirubin, which is formed when hemoglobin is broken down as part of the normal process of recycling old or damaged red blood cells. Normally, the bilirubin is processed by the liver and excreted into the digestive tract. Liver inflammation as caused by hepatitis reduces the organ's ability to move bilirubin into the bile ducts causing an excess of bilirubin.Liver inflammation is damaging the hepatocytes eventually causing cirrhosisLiver inflammation Viruses causing Hepatitis• Each named with a letter:• Hepatitis A virus (HAV; Picornaviridae, Hepatovirus), vaccine • Hepatitis B virus (HBV; Hepadnaviridae, Orthohepadnavirus), vaccine• Hepatitis C virus (HCV) (Flaviviridae; Flavivirus) treatable, chronic form is curable• Hepatitis Delta virus (HDV) treatable• Hepatitis E virus (HEV) treatableHAV, HBV, HCV - most common in USA Most people with Hepatitis A or Hepatitis B infection will recover on their own, with no lasting liver damage. Viruses causing HepatitisSay: non-enveloped Viruses That Cause Primary Hepatitis• Non-enveloped positive sense RNA virus, HAV is acid and bile resistant- Most outbreaks associated with contaminated food or water supplies• Shellfish may become contaminated with sewage and may concentrate and retain viruses- Major mode of transmission: Fecal-oral (hygiene!)- Average incubation period: 30 days- Adults experience signs and symptoms more often than childrenHepatitis A virus (HAV; Picornaviridae, Hepatovirus) "Oh, no........!" Hepatitis A virus (HAV)• Very stable, picornavirus (non-enveloped)• +ssRNA genome that is 7.5 kb in length• Viral genome and poly(A) tail at the 3′ terminus of the genome• 5′ end of the genome contains an IRES instead of m(7)G-cap for cap-independent translation of the viral mRNA• Polyprotein, which gets co-(post)translationally cleaved into functional viral proteins HAV Replication• HAV mRNA translation- +ssRNA viral genome acts directly as an mRNA for the synthesis of a large polyprotein- Polyprotein processed by a viral 3C protease into structural and nonstructural proteins of HAV• HAV genome replication and particle assembly- Viral RNA-dependent RNA polymerase encoded by 3D gene synthesizes -ssRNA intermediates used to create progeny genomic +ssRNAs- Newly assembled particles transported to surface of hepatocytes and exported Organization of the hepatitis A virus genome. Genes are listed within the genome, and the gene product names are listed above the genes. The genome is translated into a large polyprotein that is processed by the viral 3C protease into three distinct precursors that are processed by proteases to yield smaller precursors and mature viral proteins, etc.Secondary structure of the 5′ noncoding region of hepatitis A virus genome. The pseudoknots are involved in the recognition of the viral RNA-dependent RNA polymerase or replicase, whereas the IRES allows for cap-independent translation of the viral mRNA transcripts into a polyprotein product. HAV, a picornavirus HAV structure and replication cycleT = 1Non-enveloped virion made up of VP1, VP2, VP3. VP4 is situated internally. Prevalence of HAV HAV symptoms- Fatigue- Abdominal pain- Loss of appetite- Nausea and vomiting- Dark urine- Jaundice (occurs in 70-80% of individuals older than 14 years of age, less likely to occur in children)Course of disease and immune response toward HAV infection. Symptoms begin 1-2 weeks after infection. "ALT" represents levels of alanine aminotransferase activity detected in blood. ALT levels peak at 4 weeks postinfection. The IgM antibody response against HAV peaks 4 weeks after infection. HAV is shed in feces through weeks 1-5 after infection. Hepatitis B virus (HBV; Hepadnaviridae, Orthohepadnavirus)• Small ssDNA virus, partly dsDNA• Relatively rare in developed countries; vaccine is available• Blood-borne pathogen; Most infection occur through exposure to blood from unsafe injection practices, unsafe health care, unscreened blood transfusions, injection drug use and sexual practices that lead to exposure to blood.• For most people, HBV is a short-term illness. HBV has an 85 percent recovery rate, while 15 percent develop cirrhosis or cancer of the liver.• Endemic areas: Major model of spread is mother (carrier) to infant (90% of infants develop chronic infection)• Other high-risk groups:- IV drug users- Hemodialysis patients- Individuals with multiple sexual partners- Institutionalized patients- Healthcare workers Hepatitis B virus (HBV) • Average incubation period: 80 days• 30% of individuals have no signs or symptoms• If symptoms occur, they are similar to HAV with addition of joint pain• Chronic HBV infections occur in 5-10% of cases (when infected as adults)• Chronic infections lead to:- Cirrhosis of liver- Hepatocellular (liver) carcinoma cancer (HCC) Three types of HBV particles present during infection:1. Noninfectious Spherical 17-25 nm particle: Most abundant HBV particle in carriers2. Noninfectious filamentous particles: Up to 200 nm in length; less numerous3. Infectious Dane particle: 42 nm diameter; contains viral DNA polymerase (DT), protein kinase C, and heat shock 90 protein associated with viral genomeHBV genome• HBV is a hepadnavirus• Circular partially dsDNA• Full length strand is 3.2 kb- super-short!!!!• Shorter stand is 1.7 kb in length• Uses a replication strategy common to retrovirusesFeatures of Hepatitis B virus Three particles are observed during HBV infection: spheres, filaments, and Dane particles. Only the Dane particles are infectious. HBV envelope consists of S, M, L proteins. They can self-assemble to form non-infectious spheric SVPs or filaments. M not required for morphogenesis or infectivity of the virus . HBV core has icosahedral symmetry (34 nm). Hepatitis B virus particle structures Genomic organization of the hepatitis B virus There are 4 partially overlapping ORFs, which are translated into 7 proteins. The HBV DNA genome is transcribed by the cellular RNA polymerase II. It is under the control of four promoters: pre-S1, pre-S2, pre-C, and X (involved in cell cycle manipulation). HBV virions bind to surface receptors and are internalized. Viral core particles migrate to the hepatocyte nucleus, where their genomes are repaired to form a covalently closed circular dsDNA (cccDNA) that is the template for viral messenger RNA (mRNA) transcription. The viral mRNA that results is translated in the cytoplasm to produce the viral surface, core, polymerase, and X proteins. There, progeny viral capsids assemble, incorporating genomic viral RNA (RNA packaging). This RNA is reverse-transcribed into viral DNA. The resulting cores can either bud into the endoplasmic reticulum to be enveloped and exported from the cell or recycle their genomes into the nucleus for conversion to cccDNA. The small, peach-colored sphere inside the core particle is the viral DNA polymeraseHBV replication cycle Hepatitis B Replication • Episome (= minichromosome consisting of cccDNA)- Replicates independently of the host chromosome — HBV does not possess integrase activity- Acts as a template for viral pregenomic RNAs and genomic RNAs• HBV DNA codes for 4 partially overlapping ORFs, which are translated into 7 known proteins• Active viral reverse transcriptase converts pregenomic RNA into DNA inside of the Dane particlesHBV polymerase is a DNA polymerase that can utilize DNA and RNA templates and it also contains RT and RNAse H domains. Where to interfere in the HBV replication cycle? Prevalence of Hepatitis B Virus Anti-HBs: Ab against surface protein Anti-HBc: Ab against core protein Anti-HBe: Ab against extractable/soluble part of core antigen.Course of acute and chronic diseases and immune response toward HBV infection HBsAg can be detected very early in the acute course of infection and starts declining in serum to undetectable levels within 23-24 weeks post infection. The HbeAg is next and indicates the ability to infect others. The first HBV antibody produced is HBc IgM, and it may persist until 28 months post infection. Hence, detection of IgM represents an acute HBV infection. However, in the chronic infection phase, IgG becomes detectable and persists for a more extended period than IgM. During the recovery period, anti-HBs will not appear for a few weeks after HBsAg has been cleared. It is possible for both HBsAg and anti-HBs to be negative during recovery. This is called the window period in acute infection. Later, anti-HBs will be developed, and the immune system develops immunity as a result of an actual infection.The pathogenic events throughout HBV infection. Hepatitis C virus (HCV; Flaviviridae, Hepacivirus)• Positive sense RNA genome; enveloped particles.• The virus can cause both acute and chronic hepatitis, ranging in severity from a mild illness to a serious, lifelong illness including liver cirrhosis and cancer.• The hepatitis C virus is a bloodborne virus and most infection occur through exposure to blood from unsafe injection practices, unsafe health care, unscreened blood transfusions, injection drug use and sexual practices that lead to exposure to blood.• Globally, an estimated 58 million people have chronic hepatitis C virus infection, with about 1.5 million new infections occurring per year.• WHO estimated that in 2019, approximately 290 000 people died from hepatitis C, mostly from cirrhosis and hepatocellular carcinoma (primary liver cancer).• Antiviral medicines can cure more than 95% of persons with hepatitis C infection, but access to diagnosis and treatment is low.• There is currently no effective vaccine against Hepatitis C virus.• Hepatitis C is the most common blood borne virus in the USA. It is considered to be the most serious of the hepatitis viruses. Once exposed, the majority of people (60-85%) go on to develop chronic hepatitis C. Hepatitis C virus• Discovered in 1989, initially there was no cell culture system available to grow/study the virus in vitro. • Many infected people show few or no signs of disease for years and even decades ("silent epidemic")• HCV infection is common in the developed world• ~1.8% of U.S. population is infected with HCVTransmission Incubation period: 6-7 weeks Spread almost exclusively through blood contact Individuals who received blood products before 1992 are at risk for having contracted HCV since screening of donated blood for HCV did not begin until 1992 If signs or symptoms are present, they are similar to other hepatitis infections Between 55% and 85% of infected persons experience a chronic infection, resulting in chronic liver diseaseHepatitis C virus• Discovered in 1989, initially there was no cell culture system available to grow/study the virus in vitro. • Many infected people show few or no signs of disease for years and even decades ("silent epidemic")• HCV infection is common in the developed world• ~1.8% of U.S. population is infected with HCVTransmission Incubation period: 6-7 weeks Spread almost exclusively through blood contact Individuals who received blood products before 1992 are at risk for having contracted HCV since screening of donated blood for HCV did not begin until 1992 If signs or symptoms are present, they are similar to other hepatitis infections Between 55% and 85% of infected persons experience a chronic infection, resulting in chronic liver disease Hepatitis C virus genome organization RNA genome is 9.6 kb in length. Shorter than mosquito-borne flaviviruses (~11 kb). Cap-independent translation of viral proteins: IRES in 5'UTR. Viral RNA lacks a poly-A tail. Hepatitis C Virus life cycle The virus forms complexes with lipoproteins and circulates in the blood. HCV entry factors include scavenger receptor B1 (SCARB1), CD81, claudin 1 (CLDN1), occludin, epidermal growth factor receptor (EGFR), and Niemann-Pick C1-like protein 1 (NPC1L1). Cell entry occurs via receptor-mediated endocytosis (shown below). Subsequent to internalization, the viral envelope fuses with the endosomal membrane under acidic pH, and the viral genome is uncoated and released into the cytoplasm. Viral RNA is translated at the ER to produce the polyprotein, which is subsequently processed into mature structural and non-structural proteins. Viral non-structural proteins, in conjunction with host factors, form a membranous web where viral RNA replication occurs. HCV particle assembly most likely initiates near the ER and LD where core protein and viral RNA accumulate. Finally, HCV particles are secreted into the extracellular milieu via the secretory pathway. Apo, apolipoprotein; CD81, cluster of differentiation 81; ER, endoplasmic reticulum; HCV, hepatitis C virus; LD, lipid droplet; LVP, lipoviral particle.Wong, MT., Chen, SL. Emerging roles of interferon-stimulated genes in the innate immune response to hepatitis C virus infection. Cell Mol Immunol 13, 11-35 (2016). World-wide prevalence of HCV Geographic distribution of the HCV genotypes Genotype 1 is most prevalent in the United States, in comparison to countries in the Middle East and Africa, where genotypes 4 and 5 are the most dominant.HCV prevalence by selected groups in the United States Course of acute and chronic diseases and immune response toward HCV infection HCV antivirals Hepatitis Delta virus (HDV; Deltaviridae, Deltavirus)• Circular ssRNA genome• Requires presence of HBV to replicate; HDV particles make use of the envelope proteins produced by HBV. HBV is 42 nm in diameter• Symptoms of HDV are indistinguishable from HBV infection• Highest risk factor for HDV infection in the Eastern World is intravenous drug use- 39-90% of IV drug addicts test positive for HDV- HDV was discovered, in part, because of the detection of the delta antigen in hepatocyte nuclei- HDV is less dense and of somewhat smaller size, ∼39 nm in diameter. HDV contains not only the delta antigen but also an RNA genome- eight different "genotypes"HDV is a HBV satellite - it only occurs in the presence of HBV! Coinfection of HBV and HDV• HDV is a defective virus (discovered in 1977 as HBV antigen initially)- Requires the presence of a "helper" HBV for assembly- Associated with severe acute or chronic hepatitis- Probably uses the same cellular receptor as HBV- No treatment for HDV• HDV structure- Spherical enveloped particle- Internally contains -ssRNA genome and ~70 copies of delta antigen (large and small; L-HDAg and S-HDAg)• HDV genome- 1.7 kb length (similar to plant viral satellites and viroids), circular- Codes for 2 different types of the same protein (delta antigens), large and small.Coinfection of HBV and HDV• HDV is a defective virus (discovered in 1977 as HBV antigen initially)- Requires the presence of a "helper" HBV for assembly- Associated with severe acute or chronic hepatitis- Probably uses the same cellular receptor as HBV- No treatment for HDV• HDV structure- Spherical enveloped particle- Internally contains -ssRNA genome and ~70 copies of delta antigen (large and small; L-HDAg and S-HDAg)• HDV genome- 1.7 kb length (similar to plant viral satellites and viroids), circular- Codes for 2 different types of the same protein (delta antigens), large and small. Hepatitis D genome organizationThe RNA genome of HDV is single-stranded, very small (∼1700 nucleotides), and circular in conformation. There are two additional HDV RNAs in infected cells. One additional RNA (several-fold less abundant than the circular RNA genome) was the genome's exact complement, referred to as the "antigenome." Nucleotide sequence analysis indicated that the open reading frame (ORF) of the delta antigen was located on the antigenome.The other additional RNA ∼800 nt in length is 5' capped and 3′ polyadenylated appearing as a virus-derived mRNA. The per-cell abundances of genome, antigenome, and mRNA in infected liver tissues were estimated at ∼300,000, 60,000, and 600 copies, respectively.Hepatitis D genome organizationThe RNA genome of HDV is single-stranded, very small (∼1700 nucleotides), and circular in conformation. There are two additional HDV RNAs in infected cells. One additional RNA (several-fold less abundant than the circular RNA genome) was the genome's exact complement, referred to as the "antigenome." Nucleotide sequence analysis indicated that the open reading frame (ORF) of the delta antigen was located on the antigenome.The other additional RNA ∼800 nt in length is 5' capped and 3′ polyadenylated appearing as a virus-derived mRNA. The per-cell abundances of genome, antigenome, and mRNA in infected liver tissues were estimated at ∼300,000, 60,000, and 600 copies, respectively. Hepatitis D virus functional domains of the small and large forms of the delta antigen. RBD = RNA-binding domain; CCS = coiled-coil sequence; NLS = nuclear localization sequence; VAS = virus assembly signal. (b) Structure of hepatitis D virus RNAs.Rolling circle replication of viral RNA. As illustrated, both the genome and antigenome of HDV contain a ribozyme domain (hammerhead ribozyme Delta) to produce correctly structured 3' RNA ends. The small form of the antigen (195 aa) was shown to be essential for the accumulation of HDV RNA species in cells. The large form of the antigen (214 aa; extended at C-terminus) was first found to be a dominant negative inhibitor of replication supported by the small delta antigen. Later, it was shown to be essential for the assembly of HDV RNA into virus, presumably via interaction with HBV envelope proteins. The small and large forms are both highly basic proteins and share features, such as a nuclear localization signal and a region with a propensity to form α-helices. The unique carboxyl terminus of the large protein contains a cysteine which is essential for the ability of this protein to facilitate assembly using the envelope proteins of HBV.Hepatitis D virus small and large antigens Proposed model for hepatitis D virus replication cycle.Hepatitis D virus functional domains of the small and large forms of the delta antigen. RBD = RNA-binding domain; CCS = coiled-coil sequence; NLS = nuclear localization sequence; VAS = virus assembly signal. Hepatitis D structure and replication Global prevalence of Hepatitis Delta Virus Hepatitis E virus (HEV; Hepeviridae, Orthohepevirus)• Small positive sense RNA genome• Endemic the developing countries of Asia and Africa• Rare in industrialized nations• Sporadic cases have been reported in the United States, France, Italy, and the United Kingdom• Mode of transmission: Similar to HAV (fecal-oral route)• Person-to-person transmission rare• Incubation period: 40 days (average)• Epidemics often associated with fecally contaminated water supplies Hepatitis E virus (HEV) • Causes more severe illness than HAV• 1-3% mortality in general population• 15-25% mortality in pregnant women• Evidence HEV might be a zoonotic pathogen (from animals to people)The Orthohepevirus A genus is classified into hepatitis E virus (HEV) genotypes 1-8. Genotypes 1 and 2 are limited to human hosts and are transmitted via the fecal-oral route, primarily through contaminated water. Genotypes 3 and 4 have multiple hosts and can be transmitted to humans through the consumption of undercooked meats, including pork. Genotypes 5 and 6 are known to infect wild boar; however, it is unknown whether these genotypes can be transmitted to humans (although there have been reports of wild boar genotype 3 HEV transmission to humans). Genotypes 7 and 8 infect dromedary and Bactrian camels, respectively. There has been one case reported of genotype 7 HEV transmission to a liver transplant patient who consumed camel meat and milk.Hepatitis E virus (HEV) • Causes more severe illness than HAV• 1-3% mortality in general population• 15-25% mortality in pregnant women• Evidence HEV might be a zoonotic pathogen (from animals to people)The Orthohepevirus A genus is classified into hepatitis E virus (HEV) genotypes 1-8. Genotypes 1 and 2 are limited to human hosts and are transmitted via the fecal-oral route, primarily through contaminated water. Genotypes 3 and 4 have multiple hosts and can be transmitted to humans through the consumption of undercooked meats, including pork. Genotypes 5 and 6 are known to infect wild boar; however, it is unknown whether these genotypes can be transmitted to humans (although there have been reports of wild boar genotype 3 HEV transmission to humans). Genotypes 7 and 8 infect dromedary and Bactrian camels, respectively. There has been one case reported of genotype 7 HEV transmission to a liver transplant patient who consumed camel meat and milk. HEV• Fecal-oral transmission• HEV structure- +ssRNA genome- Clinically indistinguishable from HAV, but HEV particles much less stable- Viruses 32-34 nm in diameter, nonenveloped, and icosahedral shaped• HEV genome- Approx 7.2 kb in length- Short 5′ and 3′ noncoding regions- 3′ end contains a poly(A) tail- 3 ORFs• Overall knowledge of HEV replication is poor Negative contrast electron micrograph of human hepatitis E virus virions from a case stool collected in Nepal. (A) virion and (B) empty capsid. The bar represents 100 nm (photograph from M. Purdy).Genome organization of cutthroat trout virus and human hepatitis E virus. A short 5′ non-coding region is followed by ORF1, encoding non-structural proteins including the putative functional domains: MT, methytransferase; P, a putative papain-like cysteine protease; HUD, Hepeviridae unique domain, also called the Z domain; PP, a hypervariable polyproline region that is dispensable for virus infectivity; Macro, macro domain; Hel, helicase; and RdRP, RNA-dependent RNA polymerase. ORF2 encodes a capsid protein and is followed by a short 3′ NCR. ORF3 overlaps ORF2 in a different reading frame and encodes a small phosphoprotein with a multi-functional C-terminal region. The scale is in bases.Characteristic DescriptionTypical memberhepatitis E virus Burma (M73218), species Orthohepevirus A, genus Orthohepevirus,Virion Non-enveloped, 27-34 nm diameter with a single capsid proteinGenome 6.4-7.2 kb capped positive-sense monopartite RNA containing 3 open reading framesReplication Occurs in association with the host endoplasmic reticulum.Translation From genomic (ORF1) and subgenomic (ORF2 and ORF3) capped RNAHost range Mammals (Orthohepevirus A, C and D), birds (Orthohepevirus B) and trout (Piscihepevirus)Taxonomy two generaHepatitis E virus Step 1: Hepatitis E virus (HEV) is a quasi-enveloped virus, meaning it can exist in the non-enveloped state (HEV) or can be coated in a lipid-derived membrane (enveloped HEV (eHEV)). HEV and eHEV have distinct entry mechanisms. Little is known about entry mechanisms for HEV. For eHEV, the virus enters the cell through clathrin-dependent and dynamin-dependent, receptor-mediated endocytosis. A specific cell surface receptor mediating eHEV entry remains to be identified, Upon entering the cell, the envelope of eHEV undergoes lysosome-mediated lipid degradation and uncoats to expose the viral mRNA. The ORF1 polyprotein (pORF1) containing the RNA-dependent RNA polymerase (RdRp) is translated from the positive-sense (+) strand, and the RdRp then transcribes full-length negative-sense (-) RNA (step 2). The negative-sense RNA serves as a template for transcribing more full-length positive-sense RNA to be packaged into progeny virions, as well as a shorter subgenomic RNA (sgRNA) that contains ORF2 and ORF3. The capsid protein (pORF2) and protein ORF3 (pORF3), a viroporin essential for viral release, are translated from the sgRNA. pORF3 binds to tumor susceptibility gene 101 protein required for transport (ESCRT) pathway to bud from cell membranes (step 3). The lipid envelope of eHEV is thought to be derived from the trans-Golgi network, and viral particles contained in eHEV have been shown to be associated with pORF3. pORF3 has additionally been shown to exhibit viroporin activity, eHEV released from the apical membrane enters the bile duct, where the lipid envelope is thought to be degraded by detergents and proteases in the bile (step 4). This feature would explain why HEV in the feces is non-enveloped. On the other hand, eHEV released from the basal membrane of hepatocytes enters the serum in its quasi-enveloped form, where it is protected from neutralizing antibodies against pORF2 and pORF3 but is less efficient at infecting cells.Hepatitis E life cycle Global prevalence of hepatitis EGlobal prevalence of hepatitis E Course of HEV infection is similar to HAV infection except that symptoms are later and continue for a shorter duration than in HAV infections. Laboratory Diagnosis of Viral Hepatitis Infections• Serology (detection of IgM antibodies) used to diagnose HBV• Best way to prevent HBV is vaccination

HIV

•In 2019, there were approximately 1.2 million people living with HIV in the US•In 2019, there were 34,800 new infections in the US, which represented an 8% decrease since 2015•In 2019, an estimated 1 in 8 people living with HIV in the US did not know they had it•In 2019, about 66% of people living with HIV received some HIV care, 50% were retained in care, and 56% were virally suppressed•In 2016, an estimated 1.1 million people in the US were eligible for PrEP medicine based on their risk factors, but only 78,360 people filled prescriptions•MSM make up an estimated 2% of the US population, but accounted for 66% of new annual HIV infections in 2017•As of 2019, 56% of people living with HIV in the US contracted it through male-to-male sexual transmission•As of 2018, 1 in 6 MSM living with HIV was unaware of their status•Analysis of HIV diagnoses between 2010 and 2016 found that new infections among all MSM in the US remained stable, but they increased 65% among young Black American MSM aged 25 to 34 and 68% among Hispanic/Latino American MSM in that same age grouphttps://www.gileadhiv.com/landscape/state-of-epidemicHuman immunodeficiency virus (HIV) - Epidemic factsAnnual HIV infections in the US, 2015-2019New HIV diagnoses among MSM in the U.S. by race/ethnicity, 2019 AIDS found its way to the U.S. as early as 1960 but was first noticed after doctors discovered clusters of cancer (Kaposi's sarcoma) and Pneumocystis pneumonia in homosexual men in L.A., NY, and San Francisco in 1981.These diseases typically occur in severe immune-compromised people. How acquired immunodeficiency syndrome (AIDS) was discoveredPresident Ronald Reagan's administration may have dragged its feet in dealing with the crisis, while the gay community viewed early reports and public health measures with corresponding distrust, thus allowing the disease to infect hundreds of thousands more.Sexual transmission is the main route of HIV infection/transmission (genital tract, rectal mucosa). The disease was first termed GRID and therefore largely ignored. Furthermore, blood samples knowingly collected from symptomatic/infected people were further distributed fueling the epidemic . At the beginning of the epidemicRates of HIV Diagnoses in the US, 2017New HIV infections Number of people living with HIV in the United States, and the total cumulative number of deaths.38,281 new cases of HIV were diagnosed in the United States in 2017, a rate of 11.8 per 100,000 population.As of 2018, about 700,000 people have died of HIV/AIDS in the U.S. since the beginning of the HIV epidemic, and nearly 13,000 people with AIDS in the United States die each year.Male homosexuals are statistically the dominant group infected with HIV.HIV has been shown to have caused the 1969 death of Robert Rayford, a 16-year-old St. Louis male, who could have contracted it as early as 7 years old due to sexual abuse, suggesting that HIV had been present, at very low prevalence, in the U.S. since before the 1970s. There is no patient "zero". Origin of HIVRetroviridae; genus Lentivirus; Human immunodeficiency virus 1 (HIV-1), Human immunodeficiency virus 2 (HIV-2). HIV-1 is most similar to the chimpanzee infecting simian immunodeficiency virus (SIV-1). Both viruses share a common ancestor in other monkey species. Geographic origin of SIV-1/HIV-1 likely Kinshasa, Rep. Kongo. From there it moved to Haiti and from there to the U.S. Virus 'jumped' from chimpanzee to human. How? Likely happened during bush meet hunting. Earliest samples showing HIV-1 infection among people from Kongo region are from 1959. Clearly, HIV is of zoonotic origin.When did the virus approximately 'jump' from chimpanzees to human?Check sequence divergence between HIV-1 (earliest available sample) and SIV-1 and take average mutation rate into consideration: 4 × 10 −3 per base per cell, which is the highest reported mutation rate for any biological entity. Result: sometime in the 1920s is the likely date.HIV-2 viruses are related to viruses found in the sooty mangabey, a vulnerable West African primate. HIV-2 is less virulent and transmissible among humans than HIV-1. HEALTHCARE & PHARMA OCTOBER 30, 2007AIDS virus invaded U.S. from Haiti: studyBy Will DunhamWASHINGTON (Reuters) - The AIDS virus invaded the United States in about 1969 from Haiti, carried most likely by a single infected immigrant who set the stage for it to sweep the world in a tragic epidemic, scientists said on Monday.Michael Worobey, a University of Arizona evolutionary biologist, said the 1969 U.S. entry date is earlier than some experts had believed.The timeline laid out in the study led by Worobey indicates that HIV infections were occurring in the United States for roughly 12 years before AIDS was first recognized by scientists as a disease in 1981. Many people had died by that point."It is somehow chilling to know it was probably circulating for so long under our noses," Worobey said in a telephone interview.The researchers conducted a genetic analysis of stored blood samples from early AIDS patients to determine when the human immunodeficiency virus first entered the United States.They found that HIV was brought to Haiti by an infected person from central Africa in about 1966, which matches earlier estimates, and then came to the United States in about 1969.The researchers think an unknown single infected Haitian immigrant arrived in a large city like Miami or New York, and the virus circulated for years -- first in the U.S. population and then to other nations.It can take several years after infection for a person to develop AIDS, a disease that ravages the immune system. Sub-types of HIVThe HIV-1 group M viruses predominate and are responsible for the AIDS pandemic.Major group (Group M) and two or more minor groups, namely Group N, O, representing independent transmissions of SIV into humans. More than 90% of HIV/AIDS cases deriving from infection with HIV-1 group M. Zoonotic origin of HIV-1 M is SIV CPZ.N ("non-M, non-N"), extremely rare, discovered by a Franco-Cameroonian team in 1998, when they identified and isolated the HIV-1 variant strain, YBF380, from a Cameroonian woman who died of AIDS in 1995. When tested, the YBF380 variant reacted with an envelope antigen from SIVcpz rather than with those of Group M or Group O, indicating it was indeed a novel strain of HIV-1. As of 2015, < 20 Group N infections have been recorded.O ("Outlier") ~100,000 individuals located in West-Central Africa (Cameroon!) and is not usually seen outside of that area. Its zoonotic origin is SIVgor, which infects gorillas. Earlier test kits did not detect the O type. Green monkeysmaqaquechimpanzee The first identification of HIV-2 occurred in 1985 in Senegal by microbiologist Souleymane Mboup and his collaborators.The first case in the United States was in 1987. The first confirmed case of HIV-2 was a Portuguese man who was treated at the London Hospital for Tropical Diseases and later died in 1978. He was exposed to the disease in Guinea-Bissau where he lived between 1956 and 1966. As of 2010, there are 8 known HIV-2 groups (A to H). Of these, only groups A and B are pandemic. World-wide number of people infected with HIV and not receiving anti-retroviral drugs fell from 90% in 2006 to 63% in 2013. AIDS related deaths have fallen by 35% since 2005. In 2013, there were 1.5 million AIDS related deaths.HIV-2 Lente = latin for "slow"RetroviridaeAlpha-retrovirusBeta-retrovirusDelta-retrovirusEpsilon-retrovirusGamma-retrovirusLentivirus"slow" Avian leucosis virus,Avian sarcoma virus Human betaretrovirusBovine leukemia virus,Human lymphotropic virus walleye dermal sarcoma virus,salmon swim bladder sarcoma virus Feline leukemia virus Ovine progressive pneumonia virus OPPVEquine infectious anemia virus EIAV Caprine arthritis-encephalitis virus CAEV Feline immunodeficiency virus FIVBovine immunodeficiency virus BIVHuman immunodeficiency virus HIV (SIV) Long terminal repeats (LTR; HIV: 634 bp) are located at each end of the provirus as a direct repeat containing the U3, R, and U5 regions, functions as an eukaryotic transcription unit. The U3 region contains the viral promoter and enhancer elements. The R region includes the mRNA initiation site (+1) and ends at a polyadenylation termination site. The function of the U5 region is not well understood. HIV replication - what's specialHIV is a retro-transposon, which reverse transcribes its ss positive sense RNA genome into a dsDNA provirus, which gets integrated into the host genome. The virus per se is not oncogenic. In order to transcribe its RNA genome into DNA, the virus encodes a reverse transcriptase (RT), a unique feature. However, the RT is a very error-prone enzyme (no proofreading ability) , leading to very high mutation rates during transcription.HIV replication is driven by a molecular engine consisting of three viral enzymes: 1) reverse transcriptase (RT) containing, RNA-dependent DNA polymerase activity, ribonuclease H (RNase H), and DNA-dependent DNA polymerase activity.2) protease (PR)3) Integrase (IN). Integrase catalyzes the covalent insertion of the viral DNA produced by reverse transcription of the RNA into the chromosomes of infected cells.Each viral particle of a retrovirus contains two copies of the full-length viral genomic RNA, which is one of the characteristics of the retrovirus family. The two RNA molecules are both positive-sense and typically identical; furthermore, each RNA encodes the full complement of genetic information required for viral replication. The two strands of RNA are intricately entwined within the core of the mature infectious virus as a ribonuclear complex with the viral proteins, including nucleocapsid. Multiple steps in the biogenesis of the genomic full-length RNA are involved in achieving this location and dimeric state. Genome organization and expressionSU = surfaceTM = transmembranerev = regulator of viral gene expression in nucleustat = regulator of viral gene expression in nucleusNEF = accessory protein, promotes replication, infectivityMA = matrix protein p17CA = major capsid protein p24 gag proteins NC = nucleocapsid protein p7p6 NEFPR = protease p10RT= reverse transcriptase p66IN = integrase p32env proteinspol proteins(group specific antigen) HIV particle structure2 RNA genome molecules per virion! 1. HIV replication cycle, which begins when HIV fuses with the surface of the host cell. 2. A capsid containing the virus's genome and proteins then enters the cell. 3. The shell of the capsid disintegrates and the HIV protein called reverse transcriptase transcribes the viral RNA into DNA. 4. The viral DNA is transported across the nucleus, where the HIV protein integrase integrates the HIV DNA into the host's DNA. The host's normal transcription machinery transcribes HIV DNA into multiple copies of new HIV RNA. 5. Some of this RNA becomes the genome of a new virus, while the cell uses other copies of the RNA to make new HIV proteins. 6. The new viral RNA and HIV proteins move to the surface of the cell, where a new, immature HIV forms. 7. Finally, the virus is released from the cell, and the HIV protein called protease cleaves newly synthesized polyproteins to create a mature infectious virus.Source: NIH-NIAIDHIV infection cycle Late stages of HIV-1 replication cycleTo see if more details regarding the "maturation step" can be explained.Maturation, which leads to the formation of the conical capsid core, is triggered by proteolytic cleavage of the Gag and GagPol polyprotein precursors by the viral protease.Late stages of HIV-1 replication cycleTo see if more details regarding the "maturation step" can be explained.Maturation, which leads to the formation of the conical capsid core, is triggered by proteolytic cleavage of the Gag and GagPol polyprotein precursors by the viral protease. Host cell interactionsViral tropism:HIV can infect a variety of cells such as CD4+ helper T-cells and macrophages that express the CD4 molecule on their surface. HIV-1 entry to macrophages and T helper cells is mediated through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells and also with its chemokine coreceptors CCR5 (beta-chemokine) or CXCR4 (alpha-chemokine). Macrophage (M-tropic) strains of HIV-1, or non-syncitia-inducing strains (NSI) use CCR5 co-receptor for cell entry. T-cell tropic isolates (T-trophic), or syncitia-inducing (SI) strains replicate in primary CD4+ T-cells as well as in macrophages and use CXCR4 co-receptor for entry.Viruses that use only the CCR5 co-receptor are termed R5, those that only use CXCR4 are termed X4, and those that use both, X4R5. Viral pathogenesis in humansThere are four stages of HIV infection:1. Primary infection stage2. Clinically asymptomatic stage - provirus integration3. Symptomatic HIV infection - persistent stage begins4. Progression from HIV to AIDS.Innate immune responses Activated soon after an infection occurs but they are not virus-type specific. If the innate immune response is not capable of eliminating the virus or bacteria, or if these responses are evaded by the pathogen, the adaptive branch of the immune system kicks in. The adaptive immune responses, which include cellular immune responses (CD4+ and CD8+ T cells) and antibodies, are pathogen-specific and therefore take longer to become activated -- typically several days.Adaptive immune responseHIV prefer to infect activated memory CD4+ T cells that express CCR5 (co-receptor). Most of the T cells of this phenotype reside in the intestine and other mucosal sites.Viral pathogenesis in humansThere are four stages of HIV infection:1. Primary infection stage2. Clinically asymptomatic stage - provirus integration3. Symptomatic HIV infection - persistent stage begins4. Progression from HIV to AIDS.Innate immune responses Activated soon after an infection occurs but they are not virus-type specific. If the innate immune response is not capable of eliminating the virus or bacteria, or if these responses are evaded by the pathogen, the adaptive branch of the immune system kicks in. The adaptive immune responses, which include cellular immune responses (CD4+ and CD8+ T cells) and antibodies, are pathogen-specific and therefore take longer to become activated -- typically several days.Adaptive immune responseHIV prefer to infect activated memory CD4+ T cells that express CCR5 (co-receptor). Most of the T cells of this phenotype reside in the intestine and other mucosal sites. The course of HIV-1 infection defined by the level of viral replicationPlasma viraemia (top), and dynamic changes of the CD4+ T-lymphocyte compartments (bottom). Primary infection characterized by high plasma viraemia (red line, top), low CD4 cells (green line, bottom), and absence of HIV-1 specific antibodies (orange line, bottom). Viraemia drops as cytotoxic CD8+ T-lymphocytes (CTL) develop (blue line, bottom) and an individual viral-load set point is reached during chronic infection. Viral set points differ greatly among individuals (eg, red dotted line, top) and predict disease progression. Viral diversity increases through out the disease (closed circles, top). The risk of transmission is highest in the first weeks when viraemia peaks (closed circles, top). GALT=gut-associated lymphoid tissues.Following transmission of the virus, there is a period of ∼10 days, known as the eclipse phase, before viral RNA becomes detectable in the plasma.The course of HIV-1 infection defined by the level of viral replicationPlasma viraemia (top), and dynamic changes of the CD4+ T-lymphocyte compartments (bottom). Primary infection characterized by high plasma viraemia (red line, top), low CD4 cells (green line, bottom), and absence of HIV-1 specific antibodies (orange line, bottom). Viraemia drops as cytotoxic CD8+ T-lymphocytes (CTL) develop (blue line, bottom) and an individual viral-load set point is reached during chronic infection. Viral set points differ greatly among individuals (eg, red dotted line, top) and predict disease progression. Viral diversity increases through out the disease (closed circles, top). The risk of transmission is highest in the first weeks when viraemia peaks (closed circles, top). GALT=gut-associated lymphoid tissues.Following transmission of the virus, there is a period of ∼10 days, known as the eclipse phase, before viral RNA becomes detectable in the plasma. HIV control - vaccine efforts"Dream on....."In the four decades since the first cases of what would come to be known as AIDS were documented, scientists have made huge strides in HIV treatment, transforming what was once a death sentence to a manageable condition. What we still don't have is a vaccine that would train human immune systems to ward off the infection before it ever takes root.The quest to develop a preventive HIV vaccine was reinvigorated in 2009 when results from the large RV144 trial showed for the first time that an investigational vaccine regimen could confer a modest degree of protection against HIV infection. Moderna announced in August 2021 that it would soon launch a Phase 1 clinical trial for two new mRNA-based HIV vaccines, giving scientists fresh hope.In short, reasons generally given are: Lack of natural immunity to HIV Variability of HIV types Lack of correlates of protective immunity Lack of an animal model that reliably predicts vaccine efficacy in humans"The scale of mutations that HIV produces are beyond anything that's even in the same realm of what coronavirus does," Johnston said. "If you mapped out a genetic tree of all the different variants of HIV inside the body of one person, it's about as equivalent of all the genetic variations of all the influenza virus of all people around the world during one year." Control of HIV - anti-retroviral drugsAntiretroviral therapy (ART): generally includes the use of 3 HIV medicines from at lest 2 different drug classes. Drugs are FDA approved. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) block RTNon-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) block RTProtease Inhibitors (PIs) block polyprotein processingFusion Inhibitors block cell entry into CD4 T lympocyteCCR5 Antagonists block co-receptor - entry into CD4 T lympocyteIntegrase Strand Transfer Inhibitor (INSTIs) block provirus integration into genomeAttachment Inhibitors block entry into CD4 T lymphocytesCombinations blocking RT + protease inhibitors ART has made it possible for infected people to live a comparable live span to non-infected people. Successful ART prevents symptom development and viral transmission. What is "successful"? Virus is suppressed when there are <200 viral genome copies/ml blood. Possible side effects -short-term: headache, nausea, diarrhea, fatigue. Long-term (more rare): diabetes, high cholesterol, insomnia, kidney - liver damage. Control of HIV - anti-retroviral drugsAntiretroviral therapy (ART): generally includes the use of 3 HIV medicines from at lest 2 different drug classes. Drugs are FDA approved. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) block RTNon-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) block RTProtease Inhibitors (PIs) block polyprotein processingFusion Inhibitors block cell entry into CD4 T lympocyteCCR5 Antagonists block co-receptor - entry into CD4 T lympocyteIntegrase Strand Transfer Inhibitor (INSTIs) block provirus integration into genomeAttachment Inhibitors block entry into CD4 T lymphocytesCombinations blocking RT + protease inhibitors ART has made it possible for infected people to live a comparable live span to non-infected people. Successful ART prevents symptom development and viral transmission. What is "successful"? Virus is suppressed when there are <200 viral genome copies/ml blood. Possible side effects -short-term: headache, nausea, diarrhea, fatigue. Long-term (more rare): diabetes, high cholesterol, insomnia, kidney - liver damage. HIV infection cycle(again)Here also showing how/where antivirals are working.HIV infection cycle(again)Here also showing how/where antivirals are working. Selected observations, scientific developments, and treatment options pertinent to HIV-1/AIDSEstimates place the cross-species transmission events leading to the worldwide spread of HIV-1 to the early decades of the 20th century. Numbers circled by a hexagon identify the specific year of an event. PEPFAR=President's Emergency Plan for AIDS Relief.

Positive sense RNA viruses Arboviruses

Arbovirus overview(zoonotic parasites).blood-sucking arthropodsmosquitoes, flies, ticks.rodents, birds, primatesinvertebratehemorrhage; neurological (encephalitis); febrile illness; Class: Arachnida; Order: Parasitiformes; Family: Ixodidae (hard ticks).Class: Insecta; Order: Diptera. Arbovirus VectorsWhat is a vector? Current taxonomic status of selected arbovirusesCurrent Taxonomic ClassificationArbovirus Members Togaviridae Genus Alphavirus Chikungunya (M); Mayaro (M); O'Nyong-nyong (M); Ross River; Semliki Forest; Sindbis; Venezuelan, Eastern and Western equine encephalitis Flaviviridae Genus Flavivirus Dengue; Japanese encephalitis; Kunjin; West Nile; Murray Valley encephalitis; St. Louis encephalitis; yellow fever Omsk hemorrhagic fever (T); Powassan; tick-borne encephalitisOrder BunyaviralesFamily PeribunyaviridaeGenus OrthobunyavirusFamily NairoviridaeGenus NairovirusFamily PhenuiviridaeGenus PhlebovirusAkabene (midge); Cache Valley (M); La Crosse (M); Schmallenberg (midge); Tahyna (M) Crimean-Congo hemorrhagic fever (T); Nairobi sheep disease (T)Rift Valley fever (M); Toscana (sandfly); Heartland (T)ReoviridaeGenus OrbivirusGenus ColtivirusAfrican horse sickness (midge), bluetongue (midge), Colorado tick fever (T)Rhabdoviridae Genus Vesiculovirus Vesicular stomatitis (blackfly/sandfly) Bunyavirales10 families; >20 genera; >300 viruses species:u Arenaviridaeu Cruliviridae u Fimoviridaeu Hantaviridaeu MypoviridaeEither arboviruses or rodent-borne; spherical, enveloped RNA viruses. Nairoviridae Peribunyaviridae Phasmaviridae Phenuiviridae WupedeviridaeBunyavirales10 families; >20 genera; >300 viruses species:u Arenaviridaeu Cruliviridae u Fimoviridaeu Hantaviridaeu MypoviridaeEither arboviruses or rodent-borne; spherical, enveloped RNA viruses. Nairoviridae Peribunyaviridae Phasmaviridae Phenuiviridae Wupedeviridae RhabdoviridaeBovine ephemeral fever virus is mosquito transmitted; Vesicular stomatitis virus is transmitted by sandflies and/or black flies.nucleocapsidphospho Reoviridae (respiratory enteric orphan viruses)dsRNA viruses with segmented genomes (9-12 dsRNA segments, 10-14 proteins); wide host range (vertebrates, invertebrates, plants, fungi); no lipid envelope but multilayered protein shells instead.currently 97 species in this family, divided among 15 genera in two subfamiliesT=1T=13Spiked core capsidsSmooth core capsidsL segments encode for λ proteins, M segments encode for μ proteinsS segments encode for σ proteinsConsidered to be derived from bacteriophages Viral dsRNA segmentsSpiked core Smooth core Sedoreovirinae vs. Spinareovirinae Depending on viral genus, viruses have 9, 10, 11, or 12 linear dsRNA segments. Positive sense RNAs have a 5' cap but viral mRNAs lack poly-A tails. Purified viral dsRNA is considered to be non-infectious. Virion assembly Virion disassembly Rotaviruses first bind to target cell surface sialic acids via VP8* which lies at the tips of the virion spikes; they then enter the cell via receptor-mediated endocytosis. Uncoating of the triple-layered infectious particles (TLPs) occurs within early endosomes; loss of the outer capsid layer and release of the double-layered particle (DLP) into the cytosol activates the virion polymerase complex (VP1 and VP3) within the incoming virus, initiating the transcription of capped positive-sense RNA ((+) RNAs) from each of the 11 double-stranded RNA (dsRNA) genome segments. These mRNAs are translated and become associated with newly synthesized viral proteins to form RNase-sensitive subviral particles. (+) RNAs serve either as mRNAs for synthesis of viral proteins or as templates for synthesis of negative-sense RNA ((-) RNA) replicative intermediates. Non-structural protein 2 (NSP2) and NSP5 interact to form large intracytoplasmic inclusion bodies (viroplasms) that sequester components required for genome replication and virion assembly. Genome packaging involves the formation of "assortment complexes," wherein single copies of each dsRNA segment are bound together in the nascent virion core structure. This triggers more dsRNA synthesis by VP1. The intermediate capsid protein, VP6, then assembles onto the nascent core to form the DLP. Assembly of the outer capsid is not well understood; one model proposes that NSP4 recruits DLPs and outer capsid protein VP4 to the face of the endoplasmic reticulum (ER) membrane, from where DLP-VP4-NSP4 complexes bud into the ER. Subsequent removal of the ER membrane and NSP4 permits assembly of TLPs via VP7 crosslinking. Particles released from infected cells are exposed to trypsin-like proteases of the gastrointestinal tract, resulting in the specific cleavage of VP4 into VP5* and VP8*, yielding fully infectious virions.Rotavirus replicationRotaviruses first bind to target cell surface sialic acids via VP8* which lies at the tips of the virion spikes; they then enter the cell via receptor-mediated endocytosis. Uncoating of the triple-layered infectious particles (TLPs) occurs within early endosomes; loss of the outer capsid layer and release of the double-layered particle (DLP) into the cytosol activates the virion polymerase complex (VP1 and VP3) within the incoming virus, initiating the transcription of capped positive-sense RNA ((+) RNAs) from each of the 11 double-stranded RNA (dsRNA) genome segments. These mRNAs are translated and become associated with newly synthesized viral proteins to form RNase-sensitive subviral particles. (+) RNAs serve either as mRNAs for synthesis of viral proteins or as templates for synthesis of negative-sense RNA ((-) RNA) replicative intermediates. Non-structural protein 2 (NSP2) and NSP5 interact to form large intracytoplasmic inclusion bodies (viroplasms) that sequester components required for genome replication and virion assembly. Genome packaging involves the formation of "assortment complexes," wherein single copies of each dsRNA segment are bound together in the nascent virion core structure. This triggers more dsRNA synthesis by VP1. The intermediate capsid protein, VP6, then assembles onto the nascent core to form the DLP. Assembly of the outer capsid is not well understood; one model proposes that NSP4 recruits DLPs and outer capsid protein VP4 to the face of the endoplasmic reticulum (ER) membrane, from where DLP-VP4-NSP4 complexes bud into the ER. Subsequent removal of the ER membrane and NSP4 permits assembly of TLPs via VP7 crosslinking. Particles released from infected cells are exposed to trypsin-like proteases of the gastrointestinal tract, resulting in the specific cleavage of VP4 into VP5* and VP8*, yielding fully infectious virions.Rotavirus replication Natural transmission cycle of Colorado tick fever. Transfers of virus are shown with green arrows. Larval ticks feed on small mammals that can remain viremic for long periods of time and then transmit virus to other small mammals. Adult ticks, while not important for maintaining the virus in nature may then bite nonreservoir hosts such as deer or man. Adult ticks lay eggs to produce the next generation of larval ticks, but no transovarial transmission of virus occurs.In sheep, Blue tongue virus transmitted by biting midges causes an acute disease with high morbidity and mortality.High fever, excessive salivation, swelling of the face and tongue, and cyanosis of the tongue. fevers of an undifferentiated type with or without a maculopapular rash and usually benign; encephalitis, often with a high case fatality rate; hemorrhagic fevers, also frequently severe and fatal. These categories are somewhat arbitrary, and some arboviruses may be associated with more than one syndrome, eg, dengue.Diseases produced by the arboviruses may be divided into 3 clinical syndromes:

Coronaviruses

NidoviralesThese viruses infect vertebrates and invertebrates: mammals, birds, fish, arthropods. Single RNA molecule, 5'cap, polyA tail (polyadenylated).Nidus means Nest - referring to the nested set of 3' (coterminal) sgRNAs. Genomic organization and expression, and key domains of nidoviruses representing the 4 different families. The coding regions are partitioned into ORF1a (yellow), ORF1b (violet) and the 3′ORFs (blue). Black squares, ribosomal frameshifting sites. Within ORFs (white rectangles), colored patterns highlight domains identified in: all nidoviruses [TM2, TM3, 3CLpro, RdRp, and Zn-cluster binding domain fused with HEL1 (ZmHEL1) - light and dark blue], large nidoviruses (ExoN, OMT - orange), certain clades (NMT, NendoU - red; ronivirus-specific domain (RsD) - light green; arterivirus-specific domain (AsD) - dark green). Genomic organizations are shown for Beluga whale coronavirus SW1 (corona), gill-associated virus (roni), Nam Dinh virus (mesoni), and porcine respiratory and reproductive syndrome virus (arteri). • There are dozens of described coronaviruses, many of them discovered in bats.• Only 7 coronaviruses are known to affect humans (HCoV). • 4 human coronaviruses only cause mild cold- or flu-like symptoms: 229E, NL63, OC43, HKU1. They contribute to 15-30% of common colds in human adults. • The 3 other human coronaviruses pose more severe risks: SARS CoV, MERS, SARS CoV-2. • Until 2003, only 2 HCoV were used for research: 229E and OC43. The other 5 viruses were discovered thereafter.Coronaviruses - OverviewAll seven types of human coronaviruses cause upper respiratory infections. Symptoms resemble those of the common cold or flu and may include• nasal congestion• sore throat• cough• headaches• feverCoronaviruses such as SARS-CoV-2 can cause complications in the lower respiratory tract, such as pneumonia. These complications are often among: • infants• older adults• people with other illnesses or weakened immune systems Order: Nidovirales ("nest" for nested set of 3' co-terminal sgRNAs)Families: Coronaviridae, Arteriviridae, Mesoniviridae, RoniviridaeGenera: Alpha bat reservoir species: HCoV-229E (aminopeptidase N receptor) HCoV-NL63 (ACE-2 receptor)Beta bat reservoir A lineage: species: HCoV-OC43, HCoV-HKU1 (sialic acid receptors), possess HE B lineage: SARS, SARS-CoV-2 (ACE-2 receptor) do not possess HE (?) C lineage: MERS (ACE-2 receptor), no HE(Gamma bird reservoir infectious bronchitis virus of chickens)(Delta bird reservoir found in several species of songbirds)Taxonomic overview of human coronaviruses The 4 common HCoVsHCoV-229E (named after a student specimen coded 229E) was isolated in 1966 using standard tissue culture.HCoV-OC43 (Organ Culture 43) was later recovered (1967) using tracheal organ culture and found to be serologically distinct from HCoV-229E. HCoV-NL63 (NetherLand 63) was isolated from the aspirate of a 7-month-old infant with bronchiolitis in 2004. HCoV-HKU1 (Hong Kong University 1) was isolated from a Hong Kong patient with pneumonia in 2005.The open reading frame 1a (ORF1a) and ORF1b are represented as shortened navy blue-boxes. The genes encoding structural proteins spike (S), envelope (E), membrane (M), nucleocapsid (N), and hemagglutinin-esterase (HE) are shown as orange boxes. The genes encoding accessory proteins are shown as dark gray boxes.HCoV-229E isolated at geographically distinct locations shows little genetic variability, whereas for HCoV-OC43 the opposite is true.As for HCoV-NL63, it shares homology with HCoV-229E and phylogenetic analyses suggest that HCoV-NL63 and HCoV-229E diverged approximately 1000 years ago.aabb Severe Coronaviruses 1. Severe Acute Respiratory Syndrome (SARS): 2002-2003; 8096 cases; 774 deaths; CFR: 9.6% Emerged in China (Foshan, Guangdong) in late 2002Spread rapidly in healthcare setting among workers, patients, and visitorsSuper-spreading in 29 countries or regions:• Droplet transmission• Longer incubation period than other viral diseases; people traveled without symptoms2. Middle East Respiratory Syndrome- Coronavirus (MERS-CoV): 2012-present; 2553 cases; 876 deaths; CFR: 34.3%Exact origin unknown (Jordan); Dromedary camels studied as a possible animal reservoir 3. Severe Acute Respiratory Syndrome-2 (SARS-CoV-2): Dec. 2019- present; Emerged in China, most likely at the Wuhan Seafood MarketRaTG13 (horse-shoe bat virus sample) and SARS-CoV-2 share the highest homology regarding their overall genomic sequence (96.2%), SARS-CoV-2 exhibits the highest sequence similarity (97.4%) to pangolin-CoV in terms of the receptor-binding domain. Thus, horse-shoe bats or pangolins could be the animal reservoirs for the virus, from which transmission to humans occurred. Currently in the U.S.: ~80 million cases reported; 965,336 deaths; CFR: 1.2% Worldwide: 460 million cases; 6.05 million deaths; CFR: 1.3%3. Severe Acute Respiratory Syndrome-2 (SARS-CoV-2): Dec. 2019- present; Emerged in China, most likely at the Wuhan Seafood MarketRaTG13 (horse-shoe bat virus sample) and SARS-CoV-2 share the highest homology regarding their overall genomic sequence (96.2%), SARS-CoV-2 exhibits the highest sequence similarity (97.4%) to pangolin-CoV in terms of the receptor-binding domain. Thus, horse-shoe bats or pangolins could be the animal reservoirs for the virus, from which transmission to humans occurred. Currently in the U.S.: ~80 million cases reported; 965,336 deaths; CFR: 1.2% Worldwide: 460 million cases; 6.05 million deaths; CFR: 1.3% Genome organization of the Betacoronavirus mouse hepatitis virusSubgenomic RNAs encoding structural / accessory proteins; 5' 70 nt leader sequence acts as primer Replicase polyproteins pp1a and pp1b = 22 kb in lenght; expression of pp1b requires ribosomal frameshift. Proteolytic processing leads to formation of 16 mature replicase proteins.In addition, 7 ORFs (encoding structural proteins) expressed from nested subgenomic RNAs.nsp1-112a to 7 are expressed via sgRNAsnsp12-16 5'UTR 3'UTRORF1aORF1bS(pike)3a/bE Mp67a/b8 9 10N12 13 nsp16nsp15nsp14SARS-CoV-2 genome organization Replicase complex - ORF1a and ORF1b which code for 16 non-structural proteins (named nsps 1-16) important for virus replication. 3' end of the genome (~10 kb) codes for the structural and accessory genes of the virus, The four structural genes of the virus include: the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The spike is a ~150 kD trimeric, heavily glycosylated protein for receptor binding (ACE2). S is cleaved by a host protease into a surface (S1) domain that interacts with ACE2 and a transmembrane domain (S2) that allows virus fusion with the host cell membrane. E is a small (~8-12 kD) protein that is highly divergent between viral strains, but with a common structure. Similar to the HIV-1 Vpu protein, E is important for virus assembly and release of virus particles. It also has an ion channel activity important for viral pathogenesis. M (~25-30 kDa in size) is the most abundant protein of the virus important for virion shape. Finally, N is the largest structural protein of the virus which interacts with the genomic RNA, giving it its classical "helical" structure that is characteristic of coronaviruses. nsp1-11Ribosomal frameshiftnsp2 nsp3 nsp4 5 6 7 8 9 101 11nsp12-1627 proteins total Coronavirus proof-read replicase and mutation ratesN-terminal exoribonuclease (ExoN) encoded by nsp14. nsp10 acts as a co-factor for nsp14. The exonuclease (ExoN) domain that proofreads the nascent RNA strand and excises the misincorporated nucleotides. Low genomic mutation rate (estimated): 3.7x10-6 nt-1 cycle-1 for SARS-CoV2 with the Spike gene accumulating mutations at a mean rate 16×10-6 nt-1 per infection cycle across backgrounds, five-fold higher than the genomic average. IFAV: 7.1×10−6 − 4.5×10−5 nt-1 substitutions per nucleotide per cell infection cycle.Polio: 3.0x10-4 nt-1 substitutions per nucleotide per cell infection cycleHIV: 4.0×10−3 nt-1 substitutions per nucleotide per cell infection cycle. Atomic resolution structure of viral replicase consisting of multiple proteinsCoronavirus proof-read replicase and mutation ratesN-terminal exoribonuclease (ExoN) encoded by nsp14. nsp10 acts as a co-factor for nsp14. The exonuclease (ExoN) domain that proofreads the nascent RNA strand and excises the misincorporated nucleotides. Low genomic mutation rate (estimated): 3.7x10-6 nt-1 cycle-1 for SARS-CoV2 with the Spike gene accumulating mutations at a mean rate 16×10-6 nt-1 per infection cycle across backgrounds, five-fold higher than the genomic average. IFAV: 7.1×10−6 − 4.5×10−5 nt-1 substitutions per nucleotide per cell infection cycle.Polio: 3.0x10-4 nt-1 substitutions per nucleotide per cell infection cycleHIV: 4.0×10−3 nt-1 substitutions per nucleotide per cell infection cycle. Atomic resolution structure of viral replicase consisting of multiple proteins Kumar S, Nyodu R, Maurya VK, Saxena SK. Morphology,. Coronavirus Disease 2019 (COVID-19). 2020;23-31. Published 2020 Apr 30. doi:10.1007/978-981-15-4814-7_3Virions: 70-90 nm diameterSARS-Cov-2 Virion Architectureno Hemagglutinin Esterase;M and E are needed for morphogenesis. • Spike (S): 180 kDa• Membrane glycoprotein (M): 23 kDa• Envelope (small membrane protein) E: ~11 kDa• (Hemagglutinin esterase (HE): 65 kDa) • Nucleocapsid protein (N): 50 kDa surrounding viral RNA genome.S (1200 aa) is composed of 2 subunits, S1 and S2. To enter a host cell, the SARS-CoV-2 spike protein interacts with the cellular surface protein angiotensin-converting enzyme 2 (ACE2), while being cleaved by cellular proteases to activate its membrane-fusion capacity. The genomic RNA (gRNA), which is capped on its 5′ end (red circle) and polyadenylated ((A)n) on its 3′ end, is released from the viral particle and translated into two replicase polyproteins, pp1a and pp1ab. Proteases embedded in viral non-structural protein 3 (nsp3) and nsp5 cleave pp1a and pp1ab into 16 non-structural proteins that assemble into replication-transcription complexes (RTCs). Viral RNA synthesis occurs within double-membrane vesicles that are part of virus-induced membranous replication organelles. The RTCs produce new gRNAs and a set of subgenomic mRNAs (sg-mRNAs) that include open reading frames (ORFs) 2-9b, which encode the structural spike, membrane, envelope and nucleocapsid proteins, and also a number of accessory proteins. Newly made gRNAs can be translated to yield additional non-structural proteins, serve as a template for further RNA synthesis or be packaged into new virions. SARS-CoV-2 assembly starts with the coating of gRNAs with nucleocapsid proteins, generating nucleocapsid structures that bud into the endoplasmic reticulum-Golgi compartment (ERGIC), thereby acquiring a lipid bilayer containing the viral spike, membrane and envelope proteins. SARS-CoV-2 Replication CycleMalone, B., Urakova, N., Snijder, E.J. et al. Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat Rev Mol Cell Biol 23, 21-39 (2022). To enter a host cell, the SARS-CoV-2 spike protein interacts with the cellular surface protein angiotensin-converting enzyme 2 (ACE2), while being cleaved by cellular proteases to activate its membrane-fusion capacity. The genomic RNA (gRNA), which is capped on its 5′ end (red circle) and polyadenylated ((A)n) on its 3′ end, is released from the viral particle and translated into two replicase polyproteins, pp1a and pp1ab. Proteases embedded in viral non-structural protein 3 (nsp3) and nsp5 cleave pp1a and pp1ab into 16 non-structural proteins that assemble into replication-transcription complexes (RTCs). Viral RNA synthesis occurs within double-membrane vesicles that are part of virus-induced membranous replication organelles. The RTCs produce new gRNAs and a set of subgenomic mRNAs (sg-mRNAs) that include open reading frames (ORFs) 2-9b, which encode the structural spike, membrane, envelope and nucleocapsid proteins, and also a number of accessory proteins. Newly made gRNAs can be translated to yield additional non-structural proteins, serve as a template for further RNA synthesis or be packaged into new virions. SARS-CoV-2 assembly starts with the coating of gRNAs with nucleocapsid proteins, generating nucleocapsid structures that bud into the endoplasmic reticulum-Golgi compartment (ERGIC), thereby acquiring a lipid bilayer containing the viral spike, membrane and envelope proteins. SARS-CoV-2 Replication CycleMalone, B., Urakova, N., Snijder, E.J. et al. Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat Rev Mol Cell Biol 23, 21-39 (2022). SARS Cov-2 surveillance through CDC and other Public Health Institutions •Mutation: A mutation refers to a single change in a virus' genome (genetic code). Mutations happen frequently, but only sometimes change the characteristics of the virus.•Lineage: A lineage is a group of closely related viruses with a common ancestor. SARS-CoV-2 has many lineages; all cause COVID-19.•Variant: A variant is a viral genome (genetic code) that may contain one or more mutations. In some cases, a group of variants with similar genetic changes, such as a lineage or group of lineages, may be designated by public health organizations as a Variant of Concern (VOC) or a Variant of Interest (VOI) due to shared attributes and characteristics that may require public health action.CDC definitions:December 2021: Omnicron declared as Variant of concern. Why? Major routes how SARS-CoV-2 is spreading• Detection of cases attributed to Omicron in multiple countries, including among those without travel history.• Transmission and replacement of the Delta variant in South Africa.• The number and locations of substitutions in the spike protein.• Available data for other variants with fewer substitutions in the spike protein that indicate a reduction in neutralization by sera from vaccinated or convalescent individuals.• Available data for other variants with fewer substitutions in the spike protein that indicate reduced susceptibility to certain monoclonal antibody treatments. Overview over mutants/variants and their phylogenic relationship (2020)Nextstrain phylogenetic tree of local cases of SARS-CoV-2 and the most similar reference sequences in the GISAID database. Phylogenetic analysis suggests that S1, S10, S12, and S19 are similar to reference sequences that are predominately European. S21 and S23 are A.1 lineage viruses similar to reference sequences from the USA. The other samples are composed of B.1 and B.1 derived lineages and share genomes with reference sequences described predominantly in the USA. S_m refers to the sample sequenced on the MinION and S_i refers to the sample sequenced on the Ion Torrent next generation sequencing platforms; r_ refers to reference genome.https://nextstrain.org/ncov/gisaid/global SARS CoV-2 (December 2020 onwards)Variants being monitoredAlpha variant (B.1.1.7 and Q lineages), Dec. 2020Beta variant (B.1.351 and descendent lineages), Dec. 2020Gamma (P.1 and descendent lineages), Dec 2020Epsilon (B.1.427 and B.1.429), March 2021Eta (B.1.525), Feb. 2021Iota (B.1.526), Feb. 2021 Kappa (B.1.617.1), May 2021Mu (B.1.621, B.1.621.1) Sept. 2021Zeta (P.2), Feb. 2021Variants of concern: Beta, GammaDelta variant (B.1.617.2 and AY lineages), June 2021Omnicron variant (B.1.1.529 and BA lineages), Nov. 2021Delta - 15 spike protein aa substitutions: T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681R, D950N Omnicron - 33 spike protein aa substitutions: A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, R346KDeescalating based on European Centre for Disease Prevention and Control SARS CoV-2 (December 2020 onwards)Variants being monitoredAlpha variant (B.1.1.7 and Q lineages), Dec. 2020Beta variant (B.1.351 and descendent lineages), Dec. 2020Gamma (P.1 and descendent lineages), Dec 2020Epsilon (B.1.427 and B.1.429), March 2021Eta (B.1.525), Feb. 2021Iota (B.1.526), Feb. 2021 Kappa (B.1.617.1), May 2021Mu (B.1.621, B.1.621.1) Sept. 2021Zeta (P.2), Feb. 2021Variants of concern: Beta, GammaDelta variant (B.1.617.2 and AY lineages), June 2021Omnicron variant (B.1.1.529 and BA lineages), Nov. 2021Delta - 15 spike protein aa substitutions: T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681R, D950N Omnicron - 33 spike protein aa substitutions: A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, R346KDeescalating based on European Centre for Disease Prevention and Control Schematic representation of SARS-CoV-2 genome organization, the major amino acid substitutions, and stability of amino acid changes. Stabilizing mutations are colored green, destabilizing mutations are colored red, and mutations that neither stabilize nor destabilize are colored yellow.SARS-CoV-2 MutationsSpikereplicasenucleocapsid Using wastewater monitoring for SARS-Cov-2 surveillance in real timeWastewater surveillance is a much cheaper and an efficient means of tracking infectious agents in communities.Detection of SARS-CoV-2 RNA is currently the most sensitive and quantitative means of monitoring viral presence in the wastewater. Virus particles present in the wastewater protect the viral genome from decay by environmental factors. Relative Abundance of SARS-CoV-2 Sequences Observed in Fenton, MO sewershed from February to March. Results from sequencing of spike amplicons of the NTD, RBD and S1S2 junction regions are shown. All amplicons show a population shift from the reference with D614G to B.1.1.7 sequences with the appearance of P.1 sequences at the last time point. Gregory, D.A.; Wieberg, C.G.; Wenzel, J.; Lin, C.-H.; Johnson, M.C. Monitoring SARS-CoV-2 Populations in Wastewater by Amplicon Sequencing and Using the Novel Program SAM Refiner. Viruses 2021, 13, 1647. https://doi.org/10.3390/v13081647 The ongoing COVID-19 pandemic has resulted in numerous accounts of different transmission routes between humans. Droplet transmission (>5 μm) is the most pronounced and heavily implicated mode of transmission reported during the pandemic. Direct contact spread from one infected individual to a second, naïve person has also been considered a driver of human-to-human transmission, especially in households with close interactions between family members. Both airborne and fecal-oral human-to-human transmission events were reported in the precursor SARS-CoV epidemic but have yet to be observed in the current crises. Solid arrows show confirmed viral transfer from one infected person to another, with a declining gradient in arrow width denoting the relative contributions of each transmission route. Dashed lines show the plausibility of transmission types that have yet to be confirmed. SARS-CoV-2 symbol in 'infected patient' indicates where RNA/infectious virus has been detected.SARS-CoV-2 Transmission Routes SARS-CoV-2 is genetically similar to SARS-CoV-1, but characteristics of SARS-CoV-2—e.g., structural differences in its surface proteins and viral load kinetics—may help explain its enhanced rate of transmission In the respiratory tract, peak SARS-CoV-2 load is observed at the time of symptom onset or in the first week of illness, with subsequent decline thereafter, indicating the highest infectiousness potential just before or within the first five days of symptom onset Reverse transcription polymerase chain reaction (RT-PCR) tests can detect viral SARS-CoV-2 RNA in the upper respiratory tract for a mean of 17 days; however, detection of viral RNA does not necessarily equate to infectiousness, and viral culture from PCR positive upper respiratory tract samples has been rarely positive beyond nine days of illness Symptomatic and pre-symptomatic transmission (1-2 days before symptom onset), is likely to play a greater role in the spread of SARS-CoV-2 than asymptomatic transmission. A wide range of virus-neutralizing antibodies have been reported, and emerging evidence suggests that these may correlate with severity of illness but wane over time.SARS-CoV-2 Symptomatology: the COVID19 disease SARS-CoV-2 is genetically similar to SARS-CoV-1, but characteristics of SARS-CoV-2—e.g., structural differences in its surface proteins and viral load kinetics—may help explain its enhanced rate of transmission In the respiratory tract, peak SARS-CoV-2 load is observed at the time of symptom onset or in the first week of illness, with subsequent decline thereafter, indicating the highest infectiousness potential just before or within the first five days of symptom onset Reverse transcription polymerase chain reaction (RT-PCR) tests can detect viral SARS-CoV-2 RNA in the upper respiratory tract for a mean of 17 days; however, detection of viral RNA does not necessarily equate to infectiousness, and viral culture from PCR positive upper respiratory tract samples has been rarely positive beyond nine days of illness Symptomatic and pre-symptomatic transmission (1-2 days before symptom onset), is likely to play a greater role in the spread of SARS-CoV-2 than asymptomatic transmission. A wide range of virus-neutralizing antibodies have been reported, and emerging evidence suggests that these may correlate with severity of illness but wane over time.SARS-CoV-2 Symptomatology: the COVID19 disease COVID-19 manifestations in humans have been described to incorporate multiple body systems with varying degrees of onset and severity. Both the upper respiratory tract and lower respiratory tract manifestations are often the most noticeable. Red-highlighted signs/symptoms tend to be over-represented in severe patients, but common symptoms are also present in more advanced COVID-19. A virus symbol denotes where a live virus and/or viral RNA has been isolated. Abbreviation: ARDS: acute respiratory distress syndrome.Clinical Symptoms of COVID-19COVID-19 manifestations in humans have been described to incorporate multiple body systems with varying degrees of onset and severity. Both the upper respiratory tract and lower respiratory tract manifestations are often the most noticeable. Red-highlighted signs/symptoms tend to be over-represented in severe patients, but common symptoms are also present in more advanced COVID-19. A virus symbol denotes where a live virus and/or viral RNA has been isolated. Abbreviation: ARDS: acute respiratory distress syndrome.Clinical Symptoms of COVID-19 Following inhalation of SARS-CoV-2 into the respiratory tract, the virus traverses deep into the lower lung, where it infects a range of cells. Upon entry, SARS-CoV-2 is likely detected by cytosolic innate immune sensors, as well as endosomal toll-like receptors (TLRs) that signal downstream to produce type-I/III interferons (IFNs) and proinflammatory mediators. The high concentration of inflammatory cytokines/chemokines amplifies the destructive tissue damage via endothelial dysfunction and vasodilation, allowing the recruitment of immune cells, in this case, macrophages and neutrophils. Vascular leakage and compromised barrier function promote endotheliitis and lung edema, limiting gas exchange that then facilitates a hypoxic environment, leading to respiratory/organ failure. The inflammatory milieu induces endothelial cells to upregulate leukocyte adhesion molecules, thereby promoting the accumulation of immune cells that may also contribute to the rapid progression of respiratory failure. Hyperinflammation in the lung further to irreversible lung damage. Recent evidence suggests that systemic inflammation induces long-term sequela in heart tissues. Abbreviations: BALF, bronchoalveolar lavage fluid; IRF3, interferon regulatory factor 3; NF-κB, nuclear factor-κB; RIG-I, retinoic acid-inducible gene I; STAT1/2, signal transducer and activator of transcription 1/2; STING, Stimulator of interferon genes. Harrison AG, Lin T, Wang P. Mechanisms of SARS-CoV-2 Transmission and Pathogenesis. Trends Immunol. 2020;41(12):1100-1115. SARS-CoV-2 Pathogenicity Construct of two types of RNA vaccines: (A) A typical conventional mRNA construct with Cap, untranslated regions (UTRs), antigen of interest, and poly-A tail; (B) self-amplifying mRNA or Replicons construct with the sequences of non-structural proteins (NSPs) derived from another virus (e.g. Alphavirus) introduced between the 5ʹ-UTR and the antigen of interest.Borah et al., 2021; Perspectives on RNA Vaccine Candidates for COVID-19. Frontiers, Mol bioscience.RNA vaccine developmentConstruct of two types of RNA vaccines: (A) A typical conventional mRNA construct with Cap, untranslated regions (UTRs), antigen of interest, and poly-A tail; (B) self-amplifying mRNA or Replicons construct with the sequences of non-structural proteins (NSPs) derived from another virus (e.g. Alphavirus) introduced between the 5ʹ-UTR and the antigen of interest.Borah et al., 2021; Perspectives on RNA Vaccine Candidates for COVID-19. Frontiers, Mol bioscience.RNA vaccine development (1) In this illustration, both conventional mRNA and SAM are shown to be formulated in lipid-derived nanoparticles (LNPs) to provide better stability; (2) The LNP formulated mRNA enters the cell through membrane-derived endocytosis processes; (3) The mRNA content shows endosomal escape to reach the cytosol; (4) In case of conventional type, the escaped mRNAs are immediately translated by the ribosomes to generate the protein of interest (Top), while SAM constructs undergoes translation to produce the replicase complex to exhibit self-amplification of the encoded mRNA, followed by translation of the antigen of interest to express the desired protein (Bottom); (5) Then the expressed proteins undergo subsequent post-translational modification to appear as trans-membrane, intracellular or secreted proteins; (6) The expressed proteins are then broken down to peptides by the proteasome, and the peptide formed are presented to the immune system by the major histocompatibility complex (MHC).Mechanism of antigen expression by the conventional mRNA and self-amplifying mRNA (SAM) vaccines (1) In this illustration, both conventional mRNA and SAM are shown to be formulated in lipid-derived nanoparticles (LNPs) to provide better stability; (2) The LNP formulated mRNA enters the cell through membrane-derived endocytosis processes; (3) The mRNA content shows endosomal escape to reach the cytosol; (4) In case of conventional type, the escaped mRNAs are immediately translated by the ribosomes to generate the protein of interest (Top), while SAM constructs undergoes translation to produce the replicase complex to exhibit self-amplification of the encoded mRNA, followed by translation of the antigen of interest to express the desired protein (Bottom); (5) Then the expressed proteins undergo subsequent post-translational modification to appear as trans-membrane, intracellular or secreted proteins; (6) The expressed proteins are then broken down to peptides by the proteasome, and the peptide formed are presented to the immune system by the major histocompatibility complex (MHC).Mechanism of antigen expression by the conventional mRNA and self-amplifying mRNA (SAM) vaccines The SARS-CoV-2 situation in the USA - what happens next? Human societies anywhere are unable so far to stamp out the virus. Best measures involve 1) mass vaccination, 2) monitoring/tacing, 3) masking, 4) social distancing, 5) hygiene. People are not able to rigorously/consequently adhere to these measures over prolonged periods ("Corona-fatigue"). What sets SARS-CoV-2 apart from SARS (2003) and massively complicates the situation:There seem to be a lot of asymptomatic carriers of the virus who are also shedding/transmitting the virus. Vaccines do not provide full protection against infection. Following infection and recovery, people can be re-infected. Thus, there is not much selection pressure imposed onto the virus with plenty of susceptible hosts around everywhere. Tracing and detection of the virus is insufficient. Based on these circumstances, and if no nation-wide mass vaccination campaigns will be implemented/enforced, the virus will prevail among human populations for a prolonged period of time, perhaps even permanently. Is the pandemic turning into a permademic???

Flaviviruses

Overview• Taxonomy• Genome organization and replication strategy• Cell entry / exit• Structure of flavivirus virions: mature/immature forms• Host interactions and Pathogenesis • Flavivirus life cycle / transmission• Flavivirusmosquito interactions• Epidemiology• Control • Bibliography Aroa virusCacipacore virusDengue virus groupDengue virusJapanese encephalitis virus groupJapanese encephalitis virusKoutango virusMurray Valley encephalitis virusSt. Louis encephalitis virusUsutu virusWest Nile virusKedougou virusKokobera virus groupModoc virus groupMontana myotis leukoencephalitis virusNtaya virus groupRio Bravo virus groupRio Bravo virusSpondweni virus groupZika virustick-borne encephalitis virus groupKyasanur forest disease virusLangat virusLouping ill virusOmsk hemorrhagic fever virusPhnom Penh bat virusPowassan virusRoyal Farm virusTick-borne encephalitis virusYellow fever virus groupYellow fever virusunclassified FlavivirusAedes flavivirusCell fusing agent virusCulex flavivirusKamiti River virus...............................Taxonomy of flavivirusesTaxonomy of flaviviruses>70 assigned virus species within genus FlavivirusAmong the Flaviviridae only viruses of the genus Flavivirus are arthropodborne (=arboviruses) Hepacivirusgenotypes 1-6Flaviviridae - 3 genera:FlavivirusPestivirusAntelope pestivirusBorder disease virusBovine viral diarrhoea virus 1Bovine viral diarrhoea virus 2Bungowannah pestivirusClassical swine fever virusGiraffe pestivirusHobi-like pestivirusTunisian sheep virus Taxonomy of flavivirusesTaxonomy of flavivirusesViruses of the genus Flavivirus can be grouped into mosquito-transmitted viruses(i) the neurotropic viruses (encephalitis) often associated with Culex species and bird reservoirs (ii) the non-neurotropic viruses, associated with haemorrhagic disease in humans, associated with Aedes mosquitoes and primate hoststick-transmitted viruses (neurotropic and nonneurotropic viruses)unclassified viruses (include viruses with unknown vectors and also 'insectonly' flaviviruses) Origin of flaviviruses: old world Originally classified as Group B arboviruses as opposed to Group A arboviruses (=alphaviruses) Vector or Host SerogroupLouping IllTBE (Hypr)TBE (Neu)Langat BatPowassanAPOIRio BravoJEVMurray V. Kunjin West Nile St. Louis E. Dengue 4 Dengue 2 Dengue 1 Dengue 3 YFV CFAV tick rodent/ batmosquitoCulexAedestickborne encephalitis Rio Bravo Japanese encephalitis dengue YFV CFAV mosquito only= distance of 0.13Phylogenetic relationship of flaviviruses based on NS3 gene Phylogenetic relationship of flaviviruses based on NS3 gene Vector or Host SerogroupLouping IllTBE (Hypr)TBE (Neu)Langat BatPowassanAPOIRio BravoJEVMurray V. Kunjin West Nile St. Louis E. Dengue 4 Dengue 2 Dengue 1 Dengue 3 YFV CFAV tick rodent/ batmosquitoCulexAedestickborne encephalitis Rio Bravo Japanese encephalitis dengue YFV CFAV mosquito only= distance of 0.13Phylogenetic relationship of flaviviruses based on NS3 gene Phylogenetic relationship of flaviviruses based on NS3 gene Genome organization and replication strategyGenome organization and replication strategystructural non-structural 3'viral genome ~11,000 nt5'UTR ~100 nt 3'UTR ~400-700 C prM E NS1 NS2a NS2b NS3 NS4a NS4b NS5NH2 COOHtranslationpolyprotein precursor ~3400 aapolyprotein processing 10 mature proteins C prM E NS1 NS2a NS2b NS3 NS4a NS4b NS5nucleocapsid E protein chaperone binding and fusion replication, pathogenesis, immunoevasion assembly, replication NS3 serine protease co-factor serine protease, helicase, replication, RNA triphosphatase replication, assembly, induction of membrane rearrangements assembly methyltransferase, guanylyltransferase , RdRp Umareddy, I. (2007). Virology journal 4, 91. Viral Zone: Flavivirus. (2011). Expert Protein Analysis System. http://expasy.org/viralzone/all_by_species/24.html&gtPredicted layout of polyprotein prior to cleavage by the various listed proteases. Black arrows show viral protease cleavage at the indicated sites and the blue/red arrows show host protease cleavage sites.Protein produced directly from template RNA strand, which was encapsidated (no antisense strand replication required for protein production.Genome organization and replication strategyGenome organization and replication strategy Flaviviruses are internalized by receptor-mediated endocytosis and trafficked to early endosomes, where the acidic environment induces fusion between the virus and the host membrane resulting in genome release.Translation of viral RNA is followed by processing of the resulting polyprotein by host and virus-encoded proteins. Following translation, a replication complex is assembled and associated to virus-induced membranes where viral replication takes place. The replication complex starts to transcribe the RNA (+) template into RNA (−), which then serves as template for new RNA (+) synthesis. Progeny RNA (+) strands can either initiate a new translation cycle or be assembled into virions. Packaging occurs on the surface of the ER, followed by budding of the structural proteins and newly synthesized RNA into the lumen of the ER. The resultant immature virions are transported to the trans-Golgi where furin-mediated cleavage of prM to M generates mature infectious particles that are released by exocytosis. However, not all flaviviruses exit the cell as complete packages. Many are released without a nucleocapsid or without proper cleavage of the prM proteins. Flavivirus replication cycle Flavivirus replication cycle Perera, R., Khalig, M., Kuhn, R.J. (2008). Antiviral Res, 80(1), 11-22. Mature virion enters cell: 180 copies of E are arranged in 30 rafts, each containing 3 parallel E-E homodimers (T=3 symmetry) In low pH endosome, 60 EEE homotrimers (prM is cleaved [=M]) are formed, which tilt upwards to expose fusion peptide of E domain II. FP inserts into target membrane, initiates fusion. Fusion with endosome membrane leads to uncoating of RNA.Replication cycle begins; de novo synthesized virions possessing 60 trimeric spikes each consisting of 3 heterodimers of prM and E. prM is covering fusion peptide of E to avoid premature fusion when passing through low pH TGN.During channeling through TGN (before cell exit), virion maturation occurs: furin cleavage of prM: the ME heterodimers dissociate, 30 rafts, each containing 3 parallel E-E homodimers are formed. As a consequence, virion diameter shrinks by ~10 nm.Conformational changes in the membrane proteins during the replication cycle Conformational changes in the membrane proteins during the replication cycle Class II fusion system: fusogenic state is generated by proteolytic cleavage of the accessory protein and not of the fusion protein itself.Mature virion enters cell: 180 copies of E are arranged in 30 rafts, each containing 3 parallel E-E homodimers (T=3 symmetry) In low pH endosome, 60 EEE homotrimers (prM is cleaved [=M]) are formed, which tilt upwards to expose fusion peptide of E domain II. FP inserts into target membrane, initiates fusion. Fusion with endosome membrane leads to uncoating of RNA.Replication cycle begins; de novo synthesized virions possessing 60 trimeric spikes each consisting of 3 heterodimers of prM and E. prM is covering fusion peptide of E to avoid premature fusion when passing through low pH TGN.During channeling through TGN (before cell exit), virion maturation occurs: furin cleavage of prM: the ME heterodimers dissociate, 30 rafts, each containing 3 parallel E-E homodimers are formed. As a consequence, virion diameter shrinks by ~10 nm.Conformational changes in the membrane proteins during the replication cycle Conformational changes in the membrane proteins during the replication cycle Class II fusion system: fusogenic state is generated by proteolytic cleavage of the accessory protein and not of the fusion protein itself. Stiasny K., Heinz, F.X. (2006); J. Gen. Virol. 87, 2755-2766.Flavivirus mature / immature virionsFlavivirus mature / immature virionsStiasny K., Heinz, F.X. (2006); J. Gen. Virol. 87, 2755-2766.Flavivirus mature / immature virionsFlavivirus mature / immature virions http://www.dnatube.com/video/12225/lifecycleofdenguevirusGreat movie!Great movie!http://www.dnatube.com/video/12225/lifecycleofdenguevirusGreat movie!Great movie! Dengue virus interference with IFN-α/β signaling. DENV non-structural proteins impede IFN-α/β signaling in virally infected cells. NS2A, NS4A, NS4B, NS5.Flavivirus-host interactionsFlavivirus-host interactionsType I Interferon Signal Transduction Pathwayvirus infectiontype 1 interferons (IFN-1)bind to IFN-α receptors activation of JAK-STAT pathwaySTAT1/STAT2/IRF-9 complex to nucleustranscription of interferon-stimulated (antiviral genes)Dengue virus interference with IFN-α/β signaling. DENV non-structural proteins impede IFN-α/β signaling in virally infected cells. NS2A, NS4A, NS4B, NS5.Flavivirus-host interactionsFlavivirus-host interactionsType I Interferon Signal Transduction Pathwayvirus infectiontype 1 interferons (IFN-1)bind to IFN-α receptors activation of JAK-STAT pathwaySTAT1/STAT2/IRF-9 complex to nucleustranscription of interferon-stimulated (antiviral genes) Pathogenesis of YFV Pathogenesis of YFV Infection of dendritic cells Lymph nodes liver Infection of Kupffer cells (=liver specific macrophages) lining blood vessels in liver. Death of Kupffer cells damages surrounding hepatocytes. Liver damage prevents the bile precursor bilirubin from being converted to biliverdin, leading to the most visible symptom of yellow fever; jaundice. Late stage symptoms: flulike symptoms, jointpain, encephalitis, and hemorrhagic disease. Case fatality rate: 1030%. Pathogenesis of YFV Pathogenesis of YFV Infection of dendritic cells Lymph nodes liver Infection of Kupffer cells (=liver specific macrophages) lining blood vessels in liver. Death of Kupffer cells damages surrounding hepatocytes. Liver damage prevents the bile precursor bilirubin from being converted to biliverdin, leading to the most visible symptom of yellow fever; jaundice. Late stage symptoms: flulike symptoms, jointpain, encephalitis, and hemorrhagic disease. Case fatality rate: 1030%. Pathogenesis of DENVPathogenesis of DENV3 organ systems play an important role in the pathogenesis of DHF/DSS: immune system liverendothelial cell (EC) linings of blood vesselsDENV can also infect skin cells, spleen, bone marrow, lung, and brain.EC from the pulmonary and abdominal territories react specifically to DENV, resulting in the selective vascular leakage syndrome characteristic of DHF/DSS.Selective apoptosis of microvascular EC in pulmonary and intestinal tissues has been detected in fatal cases of DHF/DSS . NS1 is suspected to be directly involved in this process.Disease comes in 3 variants: DF; DHF; DSS Phenomenon of antibody enhancement: subsequent infections with different DENV serotypes increase disease symptoms (from DF to DHV). Certain DENV strains typically cause DF; others DHF Model of antibody-dependent enhancement of dengue infectionAntibody (Ab)dependent enhancement of infection occurs when preexisting antibodies present in the body from a primary (first) dengue virus (DENV) infection bind to an infecting DENV particle during a subsequent infection with a different dengue serotype. The antibodies from the primary infection cannot neutralize the virus. Instead, the Ab-virus complex attaches to receptors called Fcγ receptors (FcγR) on circulating monocytes. The antibodies help the virus infect monocytes more efficiently. The outcome is an increase in the overall replication of the virus and a higher risk of severe dengue.Antibody enhancementModel of antibody-dependent enhancement of dengue infectionAntibody (Ab)dependent enhancement of infection occurs when preexisting antibodies present in the body from a primary (first) dengue virus (DENV) infection bind to an infecting DENV particle during a subsequent infection with a different dengue serotype. The antibodies from the primary infection cannot neutralize the virus. Instead, the Ab-virus complex attaches to receptors called Fcγ receptors (FcγR) on circulating monocytes. The antibodies help the virus infect monocytes more efficiently. The outcome is an increase in the overall replication of the virus and a higher risk of severe dengue.Antibody enhancement Flavivirus life cycle / transmissionFlavivirus life cycle / transmissionurban dengue virus West Nile virus dead end hostsMosquito transmissionFlavivirus life cycle / transmissionFlavivirus life cycle / transmissionurban dengue virus West Nile virus dead end hostsMosquito transmission Flavivirus life cycle / transmissionFlavivirus life cycle / transmissionAlso common: transovarial / veneral transmission among mosquitoes!Tick transmissisonMansfield, K.L., Johnson, N., Phipps, L.P., Stephenson, J.R., Fooks, A.R., Solomon, T. (2009); J. Gen .Virol. 90 (8), 17811794.tick transmissionFlavivirus life cycle / transmissionFlavivirus life cycle / transmissionAlso common: transovarial / veneral transmission among mosquitoes!Tick transmissisonMansfield, K.L., Johnson, N., Phipps, L.P., Stephenson, J.R., Fooks, A.R., Solomon, T. (2009); J. Gen .Virol. 90 (8), 17811794.tick transmission Flavivirus-mosquito interactionsFlavivirus-mosquito interactionsFlaviviruses do not cause any pathology in their arthropod vectors!Mosquito vector needs to get systemically infected with a flavivirus to be able to transmit the virus to a new host. The EIP for flaviviruses is ~10-14 days.'Old' mosquitoes, which have taken multiple bloodmeals are the most dangerous ones in terms of flavivirus transmission.In an endemic region, typically only a small proportion of females (<10%) within a mosquito population is infected and ready to transmit the virus.The mosquito midgut is the major organ that determines vector competence. It is the first tissue that gets infected.To allow transmission, the mosquito salivary glands need to get infected.Flavivirus-mosquito interactionsFlavivirus-mosquito interactionsFlaviviruses do not cause any pathology in their arthropod vectors!Mosquito vector needs to get systemically infected with a flavivirus to be able to transmit the virus to a new host. The EIP for flaviviruses is ~10-14 days.'Old' mosquitoes, which have taken multiple bloodmeals are the most dangerous ones in terms of flavivirus transmission.In an endemic region, typically only a small proportion of females (<10%) within a mosquito population is infected and ready to transmit the virus.The mosquito midgut is the major organ that determines vector competence. It is the first tissue that gets infected.To allow transmission, the mosquito salivary glands need to get infected. Infection pattern of DENV2 in Aedes aegypti Days post-infection3 7 10-11 14 21Virus Secondary tissues: fat body, hemocytes, nerve tissue, salivary glandsInfection pattern of DENV2 in Aedes aegypti Days post-infection3 7 10-11 14 21Virus Secondary tissues: fat body, hemocytes, nerve tissue, salivary glands Factors that affect productive infection of a mosquito with a flavivirusFactors that affect productive infection of a mosquito with a flavivirus • virus titer in viremic bloodmeal: [dead end hosts]• innate immune pathways: RNA interference, Toll/Imd, JAKSTAT• receptor recognition: mutations in viral structural genes enable / abolish midgut infection.RNAi is the major antiviral immune pathway in the mosquitoFlaviviruses are triggers and targets of the RNAi mechanism.DENV2 siRNAs in midguts of Ae. albopictus at 10 days pbm. EpidemiologyEpidemiology• Flavivirus epidemiology and vector ecology are tightly linked! • Vector mosquitoes are globally spread around due to trade and traffic. • (Flavivirus)infected people also travel a lot internationally.Examples: Introduction of Asian DENV strains into Central and South America; 2014: ZIKV arrives in Brazil.; since then - active spread throughout the Americas. Aedes albopictus in Europe, North America (Missouri!)Introduction of Ae. japonicus into the US (NY) in 1998; first report in Missouri in 2006. United States: all relevant arbovirus vectors are present! .... and many more.Ae. albopictus Ae. aegypti Ae. Japonicus Culex tarsalis Cumulative number of cases of yellow fever reported to the World Health Organization for the years 1992 through 2004, by country. It is suspected that cases in Africa may be underreported by a factor of 10 or more. Immunization coverage in Africa has remained low and the disease has continued to spread. Major epidemics (>250 cases) have occurred in Liberia, Burundi, and Peru in 1995, Guinea in 2000, Burkina Faso in 2002, and the Democratic Republic of Congo in 2004. Note that while Nigeria had 19,891 cases between 1980 and 1991, since 1994 there have been only 12 cases.Epidemiology of YFVEpidemiology of YFV Epidemiology of ZIKVEpidemiology of ZIKVThis map includes countries and territories where mosquitoborne transmission of Zika virus has been reported, including countries with current travel notices issued for Zika. Shading does not necessarily indicate that mosquito-borne transmission is occurring throughout the entire country or territory. India 25,856Nepal 1283Bangladesh 5Sri Lanka 1965Thailand 836Malaysia 104Singapore 10Bali 5Philippines 174Hong Kong 4Vietnam9745Taiwan 127Japan 122S. Korea 6P.R. China 122,955reported cases 19861990none11201212002012000>2000no data, but endemism suspectedRange and reported cases of Japanese encephalitis, 19861990. Adapted from MMWR (1993) Vol. 42,RR1, p. 2. Since this detailed report, the first human cases were reported in Papua New Guinea in 1997, there were two fatal cases on islands in the Torres Strait in 1995, and the virus was detected in mainland Australia (the Cape York Peninsula) in 1998Epidemiology of JEVEpidemiology of JEV Distribution of WNV in the western hemisphere in 2005Distribution of WNV in the western hemisphere in 2005First incident: NY in 1999; the virus strain originated from Israel.Distribution of WNV in the western hemisphere in 2005Distribution of WNV in the western hemisphere in 2005First incident: NY in 1999; the virus strain originated from Israel. WNV activity in 2012/2015 (U.S.)WNV activity in 2012/2015 (U.S.)human disease cases neuroinvasive disease incidencesCDC webpage: http://www.cdc.gov/ncidod/dvbid/westnile/clinicians/epi.htm20152012 201220152019WNV activity in 2012/2015 (U.S.)WNV activity in 2012/2015 (U.S.)human disease cases neuroinvasive disease incidencesCDC webpage: http://www.cdc.gov/ncidod/dvbid/westnile/clinicians/epi.htm20152012 201220152019 Control strategies Control strategies Vaccines: Vaccines are available for TBEV, YFV and JEV. Major effort is undertaken to develop a DENV vaccine.YFV: vaccine consists of a live, but attenuated, strain of the yellow fever virus called 17D; JEV: Three secondgeneration vaccines: SA14142, IC51 and ChimeriVaxJE. • The liveattenuated SA14142 strain was introduced in China in 1988. It is administered to 20 million Chinese children each year.• A purified, formalininactivated, wholevirus vaccine known as IC51 was licensed for use in the United States, Australia, and Europe in 2009. It is based on a SA14142 strain and cultivated in Vero cells. • Another vaccine, a liveattenuated yellow feverJapanese encephalitis chimeric vaccine known as ChimeriVaxJE was licensed for use in Australia in August 2010.TBEV: Based on purified, inactivated tickborne encephalitis virus.Antiviral drugs: under development for DENV and WNV. The idea is to inhibit methyltransferase or guanylyltransferase of NS5. Vector controlVector control•Insecticide treated bednets / window curtains;•Residual spraying•Removal of potential oviposition containers / larvicide •SIT

Picornaviridae

Picornaviruses are among the most diverse (more than 200 serotypes) and "oldest" known viruses (temple record from Egypti ca. 1400 BC).FMDV was the first animal virus to be recognized (Loeffler & Frosch,1898)Poliomyelitis virus was first recognized as a viral disease by Landsteiner and Popper, 1909; successful virus isolation in the 1930s.Name: 'Pico' (Greek: 'very small') Picornavirales - Eight families including PicornaviridaeThe family Picornaviridae comprises small non-enveloped viruses with RNA genomes of 6.7 to 10.1 kb and contains >60 genera and several hundred species. Most of the known picornaviruses infect mammals and birds. Many picornaviruses are important human and veterinary pathogens and may cause diseases of the central nervous system, heart, liver, skin, gastrointestinal tract or upper respiratory tract. Most picornaviruses are transmitted by the fecal-oral or respiratory routes. Typical member: poliovirus 1 (Mahoney), species Enterovirus C, genus EnterovirusVirion Non-enveloped, 30-32 nm virions comprising 60 protomers, icosahedral (T=3).Genome 6.7-10.1 kb of positive-sense, non-segmented RNA with a poly(A) tailReplication RNA synthesis occurs in reorganized cytoplasmic replication organelles containing non-structural proteins derived from the 2BC-P3 region of the encoded polyprotein; RNA structures at the 5′ and 3′ ends of the genome direct initiation of RNA synthesis and VPg encoded by 3B serves as the primer for synthesis of both RNA strandsTranslation Directly from genomic RNA containing an internal ribosome entry site (IRES)Host range Mammals, birds, reptiles, amphibians and bony fishesTaxonomy Member of the order Picornavirales; >60 genera containing several 100 speciesCharacteristics of the family Picornaviridae ccXXXX Figure 1 Genome organization of representative members of the order Picornavirales. Each RNA is shown with the ORF(s) represented with the boxes. Circles depict VPg molecules covalently attached at the 5′ end of the RNAs. Black circles represent VPg confirmed experimentally and open circles represent putative VPgs. Poly(A) tails are represented at the 3′ end of the RNAs [A(n)]. Conserved motifs for the helicase (orange diamonds), 3C or 3C-like proteinase (yellow diamonds), RNA-dependent RNA polymerase (red diamonds) and the domains for coat protein(s) (blue rectangles) are shown. ICTV Report, 2009. Picornavirus genome organization and replication cycle Translational initiation on the genomic RNA, in a cap-independent manner directed by one or two IRESs. A polyprotein is synthesized that is co- and post-translationally cleaved to capsid proteins (derived from the P1 region of the genome and non-structural proteins (from the P2 and P3 regions) by cognate viral proteinases. These are 3C proteinase (3Cpro) encoded by all picornaviruses, 2Apro of viruses in the genus Enterovirus (both are chymotrypsin-like cysteine proteinases), and papain-like Lpro of viruses in the genera Aphthovirus. Replication of viral RNA occurs in complexes associated with cytoplasmic membranes that contain most of the functional proteins and their precursors. VPg serves as the primer for both positive- and negative-strand RNA synthesis. A diagrammatic representation of the enterovirus genome is shown. The 11 mature polypeptides are shown, together with the three main cleavage intermediates. The main biological functions are included for each polypeptide. UTR, untranslated region; IRES, internal ribosome entry site; VPg, viral protein genome-linked.Lin, JY., Chen, TC., Weng, KF. et al. Viral and host proteins involved in picornavirus life cycle. J Biomed Sci 16, 103 (2009). Special features:VPg: A viral protein covalently linked to 5' end of their genomes instead of 7-methylguanosine cap like cellular mRNAs. Viral RNA polymerase uses VPg as a primer for both positive- and negative-strand RNA synthesis. A VPg primer mechanism is used by picornaviruses (entero- aphtho-, and others), as well as poty-, como-, calici-viruses and the picornavirus-like (coronavirus, notavirus, etc.) supergroup of RNA viruses.IRES: Internal ribosome entry site (IRES) elements were discovered in picornaviruses. These elements are cis-acting RNA sequences that adopt diverse three-dimensional structures and allow/enable 5' cap-independent translation of its viral RNA in the host cell.Secondary RNA structure of an IRESStructural features of type I, II, III, and IV picornavirus IRES elements. RNA secondary structures of four IRES types based on M-fold software. (A). Enteroviruses (PV, CVB3 and EV71) and rhinoviruses contain type I IRES elements. (B). Aphthoviruses (FMDV) and cardiovirus (EMCV and TMEV) contain type II IRES elements. (C). HAV contains type III IRES element. (D). porcine reschovirus serotype 1 (PTV-1) contains type IV IRES element. Specific viruses: Hepatitis A, Coxsackie, Food and Mouth, Entero, Polio, Rhino Architecture of PicornavirusesPicornaviruses: non-enveloped viruses 22-30 nm in diameter 4 capsid proteins (VP1-VP4) per capsid subunit T=1 icosahedral symmetry: 60 subunits/particleSpecific viruses: Hepatitis A, Coxsackie, Food and Mouth, Entero, Polio, Rhino Architecture of PicornavirusesPicornaviruses: non-enveloped viruses 22-30 nm in diameter 4 capsid proteins (VP1-VP4) per capsid subunit T=1 icosahedral symmetry: 60 subunits/particle Picornavirus receptor interactionsMany picornaviruses have a deep cleft formed by around each of the 12 vertices of icosahedrons. The outer surface of the capsid is composed of regions of VP1, VP2, and VP3. Around each of the vertices is a canyon lined with the C termini of VP1 and VP3. The interior surface of the capsid is composed of VP4 and the N termini of VP1. (A, B) Picornavirus uses different receptors to enter the cell, some implicated in the signaling internalization (A), meanwhile others can act as carriers that transport the viral particle to meet the primary receptor (B). (C, D) This infection event can be impeded by the action of specific neutralizing antibodies that can destabilize the viral particle (C) or opsonize or stabilize the particle to impair receptor binding or conformational changes required for infection (D). (E) Once the virus enters the cell, the viral RNA delivery mechanism is triggered, and the viral genome (black wavy line) is released into the cytoplasm. (F) Upon removal of VPg (magenta oval), the genome starts the IRES-driven translation leading to the production of the viral polyprotein. (G) The proteolytic cascade produces all viral proteins, structural and non-structural. (H) Some proteins act by hijacking the host cellular systems such as the nuclear pore, the cell translation machinery, and the apoptotic systems and initiate the remodeling of the internal cell membranes. (I) The structural proteins assemble into the capsid intermediates, the protomer and the pentamer, and also procapsids (L). (J) The formed replication complex assembled from non-structural proteins and modified internal membranes firing the picornaviral genome replication by the 3D polymerase via RNA complementary (red wavy lines) and using VPg as a primer. (K) The new progeny genomes including eventual mutations (yellow stars). (M) Mature virions assemble from pentamers that surround and package the new viral genomes. Viral particles escape from the cell by cell lysis or budding within membranes that can protect the viral progeny (P). (N) Some progeny virus with mutations in their capsids (yellow star) may escape from to the action of specific NAbs. (O) Empty capsids can act as molecular decoys for Abs to protect the infecting particles from neutralization.Picornavirus replication cycle Picornavirus cell entry receptorsCAR = Coxsackie-adenovirus receptor; DAF, decay-accelerating factor; ICAM-1, intracellular adhesion molecule type 1; LDLR, low density lipoprotein receptor; PVR, poliovirus receptor. Hepatitis A virus (HAV)Hepatitis A is a liver infection caused by HAV, which is found in the stool and blood of people who are infected. Hepatitis A is very contagious. It is spread when someone unknowingly ingests the virus through close personal contact with an infected person or through eating contaminated food or drink. Symptoms of hepatitis A can last up to 2 months and include fatigue, nausea, stomach pain, and jaundice. Most people with hepatitis A do not have long-lasting illness. The best way to prevent hepatitis A is to get vaccinated.Contact with infected personHepatitis A can be spread from close, personal contact with an infected person, such as through certain types of sexual contact, caring for someone who is ill, or using drugs with others. Hepatitis A is very contagious, and people can even spread the virus before they feel sick.Eating contaminated food or drinkContamination of food with the hepatitis A virus can happen at any point: growing, harvesting, processing, handling, and even after cooking. Contamination of food and water happens more often in countries where hepatitis A is common. Although uncommon, foodborne outbreaks have occurred in the United States from people eating contaminated fresh and frozen imported food products.A single shot of the hepatitis A vaccine can help prevent hepatitis A if given within 2 weeks of exposure.Virus can stay infectious outside the cell for months! 2016: The Hawaii Department of Health (HDOH) is continuing to investigate a cluster of hepatitis A infections in the state.On August 15, 2016, HDOH identified raw scallops served at Genki Sushi restaurants on Oahu and Kauai as a likely source of the ongoing outbreak. The product of concern is Sea Port Bay Scallops (Wild Harvest, Raw Frozen) that originated in the Philippines (states "Product of the Philippines" on the box), distributed by Koha Oriental Foods and True World Foods.Recent HAV Outbreaks in the U.S. 2016: Multistate outbreak of hepatitis A linked to frozen strawberries (Final Update)Epidemiologic and traceback evidence indicate frozen strawberries imported from Egypt were the likely source of this outbreak. In interviews, nearly all ill people interviewed reported drinking smoothies containing strawberries at Tropical Smoothie Café locations prior to August 8 in a limited geographical area, including Maryland, North Carolina, Virginia, and West Virginia, but there have been a small number of cases outside of that geographic area with no Tropical Smoothie Café exposure.As of December 13, 2016: 143 people with hepatitis A have been reported from nine states: Arkansas (1), California (1), Maryland (12), New York (5), North Carolina (4), Oregon (1), Virginia (109), West Virginia (7), and Wisconsin (3). 129 of these cases reported eating a smoothie from Tropical Smoothie Café. Polio (poliomyelitis) virus [PV] = Enterovirus C [EV-C])Family: Picornaviridae; Genus: Enterovirus; Species: Enterovirus C; Serotypes: PV-1, PV-2, PV-3• Poliovirus was first isolated in 1909 by Karl Landsteiner and Erwin Popper. • The structure of the virus was first elucidated in 1958 using x-ray diffraction by a team at Birbeck College led by Rosalind Franklin, showing the polio virus to have icosahedral symmetry.• In 1981, the poliovirus genome was published by two different teams of researchers: by Vincent Racaniello and David Baltimore at M.I.T. and by Naomi Kitamura and Eckard Wimmer at Stony Brooks University.• The three-dimensional structure of poliovirus was determined in 1985 by James Hogle at Scripps Research Institute using X-ray crystallography.• Poliovirus is one of the most well-characterized viruses and has become a useful model system for understanding the biology of RNA viruses.Possible origin from a Coxsackie A ancestor with change of receptor preference from CAM receptor to IgG-like.High RNA mutation rate: substitution rate of 3.0 x 10−4 nt substitutions/site/year.Polio (poliomyelitis) virus [PV] = Enterovirus C [EV-C])Family: Picornaviridae; Genus: Enterovirus; Species: Enterovirus C; Serotypes: PV-1, PV-2, PV-3• Poliovirus was first isolated in 1909 by Karl Landsteiner and Erwin Popper. • The structure of the virus was first elucidated in 1958 using x-ray diffraction by a team at Birbeck College led by Rosalind Franklin, showing the polio virus to have icosahedral symmetry.• In 1981, the poliovirus genome was published by two different teams of researchers: by Vincent Racaniello and David Baltimore at M.I.T. and by Naomi Kitamura and Eckard Wimmer at Stony Brooks University.• The three-dimensional structure of poliovirus was determined in 1985 by James Hogle at Scripps Research Institute using X-ray crystallography.• Poliovirus is one of the most well-characterized viruses and has become a useful model system for understanding the biology of RNA viruses.Possible origin from a Coxsackie A ancestor with change of receptor preference from CAM receptor to IgG-like.High RNA mutation rate: substitution rate of 3.0 x 10−4 nt substitutions/site/year. • Infection occurs via the fecal-oral route, meaning that one ingests the virus and viral replication occurs in the alimentary tract. Virus is shed in the feces of infected individuals. • In 95% of cases only a primary viremia occurs, and the poliovirus infection is asymptomatic. • In about 25% of cases, flu-like symptoms occur (sore throat, fever, tiredness, nausea, headache, stomach pain) associated with a secondary viremia. • Paralytic poliomyelitis occurs in <1% of poliovirus infections. Paralytic disease occurs when the virus enters the CNS and replicates in motor neurons within the spinal cord, brain stem, or motor cortex, resulting in the selective destruction of motor neurons leading to temporary or permanent paralysis. • Between 2 and 10% of people who have paralysis from poliovirus infection die, because the virus affects the muscles that help them breathe ("iron lungs").Polio PathogenesisIt lives in an infected person's throat and intestines. It enters the body through the mouth and spreads through: Contact with the feces of an infected person. Droplets from a sneeze or cough of an infected person (less common). The virus can live in an infected person's feces for many weeks. It can contaminate food and water in unsanitary conditions. No Lockdowns: The Terrifying Polio Pandemic of 1949-52In 1916, U.S. faced one of the worst polio epidemics of the 20th century, killing 6000 people, paralyzing 27000. 1945: many polio outbreaks resulted in ~20,000 cases/year until 1948. Children avoided swimming pools and public water fountains, fearing that it was transmitted through water. The CDC reports that "travel and commerce between affected cities were sometimes restricted. Public health officials imposed quarantines on homes and towns where polio cases were diagnosed." In 1952, the polio epidemic reached a peak in U.S.: almost 58,000 reported cases and more than 3,000 deaths.1955: nationwide vaccination campaign using Salks inactivated (dead) vaccine. Seasonal Polio outbreaks: San Angelo, TX: cases came up in May (despite shutdown inside city), disappeared in August. Some towns and cities across the United States tried to prevent the spread of polio by closing swimming pools, libraries, and movie theaters. The Stop Transmission of Polio (STOP) program, through the Centers for Disease Control and Prevention (CDC) and in collaboration with the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), trains volunteer international public health professionals and deploys them to countries around the world. Polio eradication campaignPrevention & Treatment: Inactivated poliovirus vaccine (IPV) given as an injection in the leg or arm, depending on the patient's age. Only IPV has been used in the United States since 2000.Oral poliovirus vaccine (OPV) developed by Sabin is still used throughout much of the world.PV-1 is the most common form encountered in nature, but all three forms are extremely infectious. As of March 2020, wild PV-1 is highly localized to regions in Pakistan and Afghanistan. Certification of the eradication of indigenous transmission occurred in September 2015 for wild PV-2, after last being detected in 1999, and in October 2019 for wild PV-3, after last being detected in 2012.Jonas Salk in his laboratory vaccinating a girl with his polio vaccine in 1956. The Cutter incidence:In 1954, Jonas Salk came up with Polio dead vaccine - formaldehyde inactivated virus (mixture of the 3 serotypes grown in Vero cells)In April 1955 more than 200 000 children in five Western and mid-Western USA states received a Salk polio vaccine in which the process of inactivating the live virus proved to be defective. Within days there were reports of paralysis and within a month the first mass vaccination programme against polio had to be abandoned. Subsequent investigations revealed that the Salk-type vaccine, manufactured by the California-based family firm of Cutter Laboratories, had caused 40 000 cases of polio, leaving 200 children with varying degrees of paralysis and killing 10. The Cutter incident led to the replacement of Salk's formaldehyde-treated vaccine with Sabin's attenuated strain. Though Sabin's vaccine had the advantages of being administered orally and of fostering wider `contact immunity', it could also be re-activated by passage through the gut, resulting in occasional cases of polio (still causing paralysis in six to eight children every year in the 1980s and 1990s, when a modified Salk vaccine was re-introduced). Ironically, the Cutter incident—by creating the perception among scientists and the public that Salk's vaccine was dangerous —led in part to the development of a polio vaccine that was more dangerous'.Albert SabinOPV is an attenuated vaccine, produced by the passage of the virus through nonhuman cells at a subphysiological temperature, which produces spontaneous mutations in the viral genome. Oral polio vaccines were developed by several groups, one of which was led by Albert Sabin.Fifty-seven nucleotide substitutions distinguish the attenuated Sabin 1 strain from its virulent parent (the Mahoney serotype), two nucleotide substitutions attenuate the Sabin 2 strain, and 10 substitutions are involved in attenuating the Sabin 3 strain. A patient in an iron lung being checked by a nurse, July 30, 1938. A woman in Kansas City is one of the remaining people in the U.S. to require an iron lung.It started in 1956 with the most intense headache she had ever felt. Days later, Mona Randolph was unable to breathe without the help of an iron lung.Aged 20 at the time, she was told to be too old for the vaccine prioritized for children, the Kansas City Star reported. At first, she noticed her senses were overwhelmed with everyday sights and sounds. Then she gradually grew weaker. More than six decades later, Randolph is believed to be one of only three people in the U.S. still using an iron lung to deal with the ravages of the polio virus on her body.An iron lung, known in medical terms as a negative pressure ventilator, is a piece of equipment that helps a person breathe. The machinery features pressurized cylinders which create a vacuum to push oxygen into a person's lungs.A patient in an iron lung being checked by a nurse, July 30, 1938. A woman in Kansas City is one of the remaining people in the U.S. to require an iron lung.It started in 1956 with the most intense headache she had ever felt. Days later, Mona Randolph was unable to breathe without the help of an iron lung.Aged 20 at the time, she was told to be too old for the vaccine prioritized for children, the Kansas City Star reported. At first, she noticed her senses were overwhelmed with everyday sights and sounds. Then she gradually grew weaker. More than six decades later, Randolph is believed to be one of only three people in the U.S. still using an iron lung to deal with the ravages of the polio virus on her body.An iron lung, known in medical terms as a negative pressure ventilator, is a piece of equipment that helps a person breathe. The machinery features pressurized cylinders which create a vacuum to push oxygen into a person's lungs. For centuries, protection from polio was passed down through the generations. Mothers who had survived polio infection themselves passed on immunity to their babies in the womb and through breast milk.There are two stages to the polio infection. In the first mild stage the infection stays in the digestive system and throat and doesn't reach the central nervous system. Most babies with maternal immunity are able to fight off the disease at this stage with only mild flu-like symptoms. At the same time, exposure to the first stage gives them their own long-term immunity.But the unforeseen consequence of better hygiene and sanitation at the end of the 1800s was that babies in clean surroundings stopped encountering the infection while they still had maternal immunity.So they failed to develop their own long-term immunity and were not protected when they encountered the disease later in life. And exposure to polio in late childhood or as an adult, was more likely to develop to the second, more aggressive stage of the disease.What happened with polio in the U.S. ? There are 81 non-polio and 3 polio enteroviruses (A-L) that can cause disease in humans. Of the 81 non-polio types, there are 22 Coxsackie A viruses, 6 Coxsackie B viruses, 28 echoviruses, and 25 other enterovirusesEnteric = transmission through the intestineThe most common sign of enterovirus is a common cold. More intense symptoms of enterovirus include hypoxia, aseptic meningitis, conjunctivitis, hand, foot and mouth disease, and paralysis.Rhinovirus A, B, C are also within the genus Enterovirus. Rhinovirus is the most common virus responsible for giving humans the common cold. It is transmitted from person to person by nasal fluid of an infected person getting into the respiratory tract of another person. The virus attaches to cell membranes inside a person's nasal cavity and begins to reproduce and travel down the respiratory tract. Foot and Mouth Disease VirusA picornavirus of the genus Aphthovirus, family Picornaviridae, which causes foot-and- mouth disease of cattle, swine, sheep, goats, and wild ruminants; it has wide distribution throughout Africa and Asia, causing serious economic losses; the virus is spread by contamination of the animal environment with infected saliva and excreta.Sometimes fatal viral disease that affects cloven-hoofed animals, including domestic and wild bovids. The virus causes a high fever lasting two to six days, followed by blisters inside the mouth and on the feet that may rupture and cause lameness.Seven major serotypes: O (most common), A, C, SAT-1, SAT-2, SAT-3, and Asia-1.Foot and Mouth Disease VirusA picornavirus of the genus Aphthovirus, family Picornaviridae, which causes foot-and- mouth disease of cattle, swine, sheep, goats, and wild ruminants; it has wide distribution throughout Africa and Asia, causing serious economic losses; the virus is spread by contamination of the animal environment with infected saliva and excreta.Sometimes fatal viral disease that affects cloven-hoofed animals, including domestic and wild bovids. The virus causes a high fever lasting two to six days, followed by blisters inside the mouth and on the feet that may rupture and cause lameness.Seven major serotypes: O (most common), A, C, SAT-1, SAT-2, SAT-3, and Asia-1. One of the most contagious animal diseases, with important economic losses Low mortality rate in adult animals, but often high mortality in young due to myocarditis Cattle are usually the main host, although some strains appear to be specifically adapted to domestic pigs or sheep and goats All wild cloven-hoofed animals are also susceptible, including deer, antelope, wild pigs, elephant, giraffe, and camelidsTransmission Direct contact between infected and susceptible animals Direct contact of susceptible animals with contaminated inanimate objects (hands, footwear, clothing, vehicles, etc.) Consumption (primarily by pigs) of untreated contaminated meat products (swill feeding). Ingestion of contaminated milk (by calves) Artificial insemination with contaminated semen Inhalation of infectious aerosols. Airborne, especially temperate zones (up to 60 km overland and 300 km by sea) Humans can harbor FMDV in their respiratory tract for 24-48 hours, leading to the common practice of 3-5 days of personal quarantine for personnel exposed in research facilities. Foot and Mouth Disease Virus FMDV research at the Plum Island Animal Disease CenterSince 1954, the DHS S&T Office of National Laboratories (ONL) Plum Island Animal Disease Center (PIADC) has served as the nation's premier defense against accidental or intentional introduction of transboundary animal diseases (a.k.a. foreign animal diseases) including foot-and-mouth disease (FMD) and African Swine Fever (ASF). PIADC is the only laboratory in the nation that can work on live FMD virus (FMDV). The lab and its staff of nearly 400 employees provide a host of high-impact, indispensable preparedness and response capabilities, including vaccine R&D, diagnostics, training, and bioforensics among others.In 2012, PIADC scientists developed the first, licensed FMD vaccine that does not require live FMDV in the manufacturing process. This means that for the first time, these next-generation FMD vaccines can be safely manufactured on the U.S. mainland. PIADC's groundbreaking innovation promotes enhanced biosecurity, efficiency, and rapid response capability. PIADC scientists also collaborate with animal health industry partners through Cooperative Research and Development Agreements to help support the transition of transboundary animal disease countermeasure product candidates.

Alpha viruses

Togaviridae1 Genus: Alphavirus (mosquito-borne, 31 species; typical virus - Sindbis virus)Alphaviruses are mosquito-borne virusesMembers of the genus Alphavirus can be classified antigenically into six (seven) complexes. The alphaviruses have classically been described as either Old World or New World viruses, depending on their geographic distribution, Old World viruses can often cause fever, rash, and arthritic symptoms and diseases. New World viruses may succumb to encephalitis. In humans and other mammals, alphavirus infection is acute and in many cases characterized by high-titer viremia, rash, fever and encephalitis until the death of the infected host or clearance of the virus by the immune system. Old WorldNew WorldPhylogenicity of Alphaviruses Alphavirus structureThere are 30 alphaviruses, 13 of them can infect humans.Virions are spherical, 60-70 nm in diameter, icosahedral nucleocapsid enclosed in a lipid-protein envelope. Alphavirus RNA is a single 42S strand of approximately 4x106 daltons Virion RNA is positive sense: it can function intracellularly as mRNA, and the RNA alone has been shown experimentally to be infectious. The single capsid protein (C protein) Two envelope viral glycoproteins (E1 and E2) of molecular weights of 48,000 to 52,000 daltons. A small third protein (E3) of molecular weight 10,000 to 12,000 daltons remains virion-associated in Semliki Forest virus but is dispatched as a soluble protein in most other alphaviruses. Alphavirus structureThere are 30 alphaviruses, 13 of them can infect humans.Virions are spherical, 60-70 nm in diameter, icosahedral nucleocapsid enclosed in a lipid-protein envelope. Alphavirus RNA is a single 42S strand of approximately 4x106 daltons Virion RNA is positive sense: it can function intracellularly as mRNA, and the RNA alone has been shown experimentally to be infectious. The single capsid protein (C protein) Two envelope viral glycoproteins (E1 and E2) of molecular weights of 48,000 to 52,000 daltons. A small third protein (E3) of molecular weight 10,000 to 12,000 daltons remains virion-associated in Semliki Forest virus but is dispatched as a soluble protein in most other alphaviruses. (A) Surface-shaded view of Sindbis virus at 9 Å resolution and viewed down at the icosahedral twofold axis. The flower-like trimeric spikes are seen in blue, and small portions of the lipid bilayer are seen in green. (B) A central cross-section of the virus particle showing the organization of the particle with the glycoproteins (blue), skirt region of the envelope (turquoise), the lipid bilayer (green) penetrated by the transmembrane helices of glycoproteins, ordered protease domain of the capsid protein (yellow), disordered protein-RNA region (orange) and RNA region (red). (C) Radial section at 208 Å of the 9 Å structure showing the nucleocapsid core viewed down at the icosahedral twofold axis. (D) Structure of the Semliki Forest virus envelope protein E1 in the monomeric conformation (protein data bank identification The domains (DI, DII and DIII) are colored according to the scheme DI: red; DII: orange; and DIII: blue. (E) Structure of the ordered C-terminal domain of the Sindbis virus capsid protein.Alphavirus structure(A) Surface-shaded view of Sindbis virus at 9 Å resolution and viewed down at the icosahedral twofold axis. The flower-like trimeric spikes are seen in blue, and small portions of the lipid bilayer are seen in green. (B) A central cross-section of the virus particle showing the organization of the particle with the glycoproteins (blue), skirt region of the envelope (turquoise), the lipid bilayer (green) penetrated by the transmembrane helices of glycoproteins, ordered protease domain of the capsid protein (yellow), disordered protein-RNA region (orange) and RNA region (red). (C) Radial section at 208 Å of the 9 Å structure showing the nucleocapsid core viewed down at the icosahedral twofold axis. (D) Structure of the Semliki Forest virus envelope protein E1 in the monomeric conformation (protein data bank identification The domains (DI, DII and DIII) are colored according to the scheme DI: red; DII: orange; and DIII: blue. (E) Structure of the ordered C-terminal domain of the Sindbis virus capsid protein.Alphavirus structure Genome organization/expression of viral proteinsnsP1- methylation and capping of viral RNAnsP2- helicase and proteasensP3- converts RNA replicase to plus strand replicasensP4- replicaseC- capsidE1, E2, (E3)- Envelope peplomer• Following translation of the non-structural viral proteins, the positive sense genome is transcribed to produce a negative sense copy. • From this negative sense copy, a full length positive sense genome is produced for packaging, and a sub-genomic 3' mRNA fragment encoding the structural proteins is made.• The polyprotein is autolytic for the C protein, which is cleaved while the protein is in the cytosol. • The remaining envelope proteins insert into the endoplasmic reticulum, where they are cleaved by host-cell signalase. • Following glycosylation the envelope proteins are released back into the cytosol, where they bind to the cellular membrane. The lifecycle starts with the attachment of a virion to the cellular receptor (top left), after which receptor-mediated endocytosis, fusion of the viral envelope, disassembly of the core and release of the genomic RNA occur. The replication proteins are then translated and processed (bottom left). These replication proteins enable the replication of the input genomic RNA and translation of the subgenomic mRNA into structural proteins (bottom center). Glycoproteins are translocated across the ER, processed and transported through the Golgi to the plasma membrane (right). Cytoplasmic assembly of genomic RNA and capsid produces the nucleocapsid core, which associates with processed glycoproteins at the plasma membrane, resulting in budding (top right). Scale varies. ER: Endoplasmic reticulum; nsP: Nonstructural protein; PM: Plasma membrane.Joyce Jose, Jonathan E Snyder, and Richard J Kuhn, A structural and functional perspective of alphavirus replication and assembly. Future Microbiol. 2009 Sep; 4: 837-856Alphavirus replication cycle Recombinant alphaviruses as transducing systemsDuplication of subgenomic promoter sequence 5' of C or 3' of E1.non-structural structural5' 3'sgRNA1sgRNA2Recombinant alphaviruses as transducing systemsDuplication of subgenomic promoter sequence 5' of C or 3' of E1.non-structural structural5' 3'sgRNA1sgRNA2 Medically important alphavirusesNew WorldVirus Clinical Syndrome Vector Host Distribution Eastern equine encephalitis (EEEV) Encephalitis, 30% case fatality rateCuliseta melanura, Culex spp.Birds Americas Western equine encephalitis (WEEV) Encephalitis, 5-15% case fatality rateCulex tarsalis,Culiseta spp.Birds North America???Venezuelan equine encephalitis (VEEV) Mayaro virus (MAYV)Febrile illness, encephalitis (VEEV)Febrile illness, rash, arthralgiaCulex taenopius, Aedes taeniorhynchusHemagogus spp., Ae. albopictus, Ae. aegyptiRodents, horsesNew World primates, rodents, opossumsCentral-, South AmericaCaribbean,Central-, South AmericaMedically important alphavirusesNew WorldVirus Clinical Syndrome Vector Host Distribution Eastern equine encephalitis (EEEV) Encephalitis, 30% case fatality rateCuliseta melanura, Culex spp.Birds Americas Western equine encephalitis (WEEV) Encephalitis, 5-15% case fatality rateCulex tarsalis,Culiseta spp.Birds North America???Venezuelan equine encephalitis (VEEV) Mayaro virus (MAYV)Febrile illness, encephalitis (VEEV)Febrile illness, rash, arthralgiaCulex taenopius, Aedes taeniorhynchusHemagogus spp., Ae. albopictus, Ae. aegyptiRodents, horsesNew World primates, rodents, opossumsCentral-, South AmericaCaribbean,Central-, South America Virus Clinical Syndrome Vector Host Distribution Chikungunya (CHIKV) worldwide most important alphavirus Febrile illness, rash, arthralgiaAe. aegypti, Ae. albopictusPrimates, humansAfrica, India, Southeast Asia, Southern Europe, South-, Central America, CaribbeanO'nyong-nyong (ONNV) Febrile illness, rash, arthralgiaAnopheles ssp. Primates AfricaSindbis (SINV) Febrile illness, rash, arthralgiaCulex spp.,Ae. aegypti,Ae. taeniorhynchus Birds Northern Europe, Africa, Asia, AustraliaSemliki Forest (SFV) Febrile illness, rare encephalitisAedes spp. Birds AfricaMedically important alphaviruses - Old World One hallmark of alphavirus infection in the vertebrate cell is the ability to shutdown host transcription and translation processes without affecting viral protein and nucleic acid synthesis. Importantly, these shutdowns result in a decrease in IFN-α/β production in the host cell, and the ability of the innate immune system to attenuate the infection is diminished. These functions appear to map, in part, to nsP2 in Old World viruses, and to the CP in New World viruses.The shutoff of host-cell protein synthesis in both groups may be mediated by phosphorylation of the host-cell translation initiation factor eIF2α, which is likely the result of detection of viral dsRNA by the host protein kinase R. Translation of the viral subgenomic RNA does not require host eIF2α. In concert with shutdown of host macromolecular synthesis, the development of cellular cytopathic effect typically arises. Alphavirus infection of the vertebrate cell usually leads to the induction of the apoptotic pathway. This pathway has proven important for the development of neuronal pathogenesis in infected organismsAlphavirus virulence factors/pathogenesis Alphavirus transmission cycles(urban) chikungunya virus Mosquito transmissisonSindbis virusdead end hosts CHIKV - mosquito interactionsCHIKV - mosquito interactions Alphavirus epidemiologyEnzootic disease cycles between mosquitoes and wild animal reservoirs:New World alphaviruses (EEEV, WEEV, VEEV) causing encephalitis EEEV: 4 lineages, lineage I found in U.S. Cycles between birds & mosquitoes; accidental hosts in epizootic disease cycle: humans, horses, ostriches, pheasants. Virus is fatal in horses; case fatality rate in humans: ~30%. Recurrent epidemic outbreaks.WEEV: recombinant between EEEV and 'Old World' Sindbis virus; 3 epizootic and 5 enzootic lineages; only epizootic strains are neurovirulent. Cycles between birds or jackrabbits & mosquitoes; accidental hosts in epizootic disease cycle: humans, horses. WEEV is found west of the Mississippi. Virus occurs as sporadic cases. VEEV: 6 subtypes (I-VI); subtype I has seven variants of which A/B and C are epizootic. no cases in USA since 1971, but USA has the sylvatic hosts & the vectors. Cyles between cotton rats & mosquitoes; has to "jump" to horse (=amplifying host), then becomes dangerous for humans (=accidental host).CHIKV: ('Old World' alphavirus) The urban disease cycle between humans & mosquitoes is relevant during epidemic outbreaks. Humans are amplifying hosts, producing high viremia. EEEV in U.S.WEEV in U.S.Alphavirus epidemiologyEnzootic disease cycles between mosquitoes and wild animal reservoirs:New World alphaviruses (EEEV, WEEV, VEEV) causing encephalitis EEEV: 4 lineages, lineage I found in U.S. Cycles between birds & mosquitoes; accidental hosts in epizootic disease cycle: humans, horses, ostriches, pheasants. Virus is fatal in horses; case fatality rate in humans: ~30%. Recurrent epidemic outbreaks.WEEV: recombinant between EEEV and 'Old World' Sindbis virus; 3 epizootic and 5 enzootic lineages; only epizootic strains are neurovirulent. Cycles between birds or jackrabbits & mosquitoes; accidental hosts in epizootic disease cycle: humans, horses. WEEV is found west of the Mississippi. Virus occurs as sporadic cases. VEEV: 6 subtypes (I-VI); subtype I has seven variants of which A/B and C are epizootic. no cases in USA since 1971, but USA has the sylvatic hosts & the vectors. Cyles between cotton rats & mosquitoes; has to "jump" to horse (=amplifying host), then becomes dangerous for humans (=accidental host).CHIKV: ('Old World' alphavirus) The urban disease cycle between humans & mosquitoes is relevant during epidemic outbreaks. Humans are amplifying hosts, producing high viremia. EEEV in U.S.WEEV in U.S. Alphavirus controlu control of larvae and adult mosquitoes, sometimes by using ultra-low-volume aerial spray techniques; in some areas, insecticide resistance (for example, resistant Culex tarsalis) is a major limitation to effective control. u Inactivated vaccines are used to protect laboratory workers from EEEV, WEEV, and VEEV. An effective live attenuated VEEV vaccine has been employed extensively in equines as an epidemic control measure, and a similar vaccine is used to protect laboratory workers. u A live attenuated CHIKV vaccine has proven safe and immunogenic in investigational human trials. Not licensed so far.Alphavirus controlu control of larvae and adult mosquitoes, sometimes by using ultra-low-volume aerial spray techniques; in some areas, insecticide resistance (for example, resistant Culex tarsalis) is a major limitation to effective control. u Inactivated vaccines are used to protect laboratory workers from EEEV, WEEV, and VEEV. An effective live attenuated VEEV vaccine has been employed extensively in equines as an epidemic control measure, and a similar vaccine