Additional Virology notes

angiotensin-converting enzyme (ACE2 receptor)

- present in many cell types and tissues including the lungs, heart, blood vessels, kidneys, liver, and gastrointestinal tract
- ACE2 is present in epithelium in the nose, mouth and lungs.
>> In the lungs, ACE2 is highly abundant on type 2 pneumocytes, an important cell type present in chambers within the lung called alveoli, where oxygen is absorbed and waste carbon dioxide is released.
- Type 1 pneumocytes responsible for the gas (oxygen and carbon dioxide) exchange that takes place in the alveoli
- Type 2 pneumocytes produce surfactant and promotes elastic properties of the lungs

Arboviruses

a heterogeneous group of RNA viruses - mainly in the families of:
- Togaviridae (aka Alpha) (Chikungunya, Equine Enchephalitis Virus): + ssRNA
>>enveloped, positive-sense, non-segmented, single-stranded RNA viruses
- Flaviviridae (Dengue, Zika, West Nile, Yellow Fever)
>>enveloped viruses +ssRNA genome of approximately 11 kb in length are transmitted to vertebrate hosts by arthropod vectors in nature

Bats

- Bats are known to carry many different strains of viruses but do not get sick from them
- 500 known CoV in bats (estimated reservoir is 5000)
- live a long time, about 30 yrs
- Tolerate a high concentration of viruses- especially Coronavirus
>> Shed in saliva, feces and aerosol
- Immune system tolerates and sustains high concentration of virus
- A lot of contact with humans
- Bats are the only flying mammal
- Increased levels of interferons and heat shock proteins in bats leads to handling flight stress
>> Decrease in immune signaling mechanisms leads to immune tolerance without excessive inflammation while still repressing viral replication
>> Leads to a somewhat "symbiotic" relationship with the virus

Corona receptors

-Different coronaviruses use a variety of receptors for viral attachment and entry.
- various coronaviruses from four major genera:
>alpha-, beta-, gamma-, and delta-coronavirus
>>distinct receptors on the surface of host cell mediates the viral entry of the specific coronavirus.
- Coronaviruses: PRCV, TGEV, PEDV, MERS-CoV, MHV, BCoV, IBV, PdCV.
- Host cell receptors: APN, CEACAM1, DPP4, ACE2, ASGPR and the spike protein

Coronavirus emergence from zoonotic reservoirs

-Emergence of coronaviruses into human populations has occurred by spillover from bat reservoir hosts into intermediate hosts.
- The intermediate hosts during the 2003 SARS-CoV epidemic included civets and other small carnivore species located in wet animal markets.
- MERS-CoV has been identified in dromedary camels, and is particularly associated with active infection of juvenile camels.
- Novel emerging CoVs may occur in the future via infection from bat populations into other intermediate animal hosts.
- More severe disease in SARS and MERS cases resulted in patients that were over the age of 65 or had comorbidities such as obesity, heart disease, diabetes, renal disease, or hypertension.

Dual infection

- Hepatitis B virus/Hepatitis C virus (HBV/HCV) dual infection is common, especially in highly endemic areas and among individuals with a high risk for parenteral infections including injection drug users (IDUs) and patients on hemodialysis
- The incidence of HBV/HCV dual infection is approximately 2-10% in anti-HCV positive and 5-20% in HBsAg individuals
- Increasing evidences indicate a greater likelihood of the advancement of chronic hepatitis to cirrhosis and hepatocellular carcinoma (HCC) in patients with HBV/HCV dual infection and the increased difficulty for therapy relative to HBV or HCV single infections
- Competition between both viruses with virus that infected first dominating
- However, impaired antibody response to both HepB and HepC

E5/E6/E7 roles in oncogenesis

- The integrated E6/E7 early genes are overexpressed in cancer cells
- The E6/E7 proteins inactivate the products of tumor suppressor gene products p53 and Rb
>>E6- promotes degradation of p53
>>E7- E7 inhibits tumor suppressing function of Rb
>> E5- inhibits apoptosis
- The integrated E6/E7 early genes are overexpressed in cancer cells
- The E6/E7 proteins inactivate the products of tumor suppressor gene products p53 and pRb
- DNA is found in nearly all cervical cancers (Typically HPV-16, -18, -45, -3)
- upon integration, there is a major overexpression of HPV E6, whihc means it facilitates a bind between P53 and E6-AP (promotes cellular proliferation) P53 is a really important tumor suppressor.
>> absence of P53 means there is the chance of tumor development
- E7 binds to a region of the Rb protein
>>Rb is to bind E2F-family transcription factors
>>Rb is cell-cycle regulatory protein/prevent entry into S phase/cell-cycle arrest
- E2F is a cell cycle transcription factor
- E7 disrupts the interaction between Rb and E2F
- Resulting in the release of E2F factors in their transcriptionally active forms

Early epicenter of COVID-19

- reports confirm that most of the earliest human cases centered around the Huanan Seafood Wholesale Market
>>Live SARS-CoV-2-susceptible mammals were sold at the market in late 2019
>>Within the market, SARS-CoV-2-positive environmental samples were spatially associated with vendors selling live mammals
- Spatial analyses within the market show that SARS-CoV-2-positive environmental samples, including cages, carts, and freezers, were associated with activities concentrated in the southwest corner of the market.
>>This is the same section where vendors were selling live mammals, including raccoon dogs, hog badgers, and red foxes, immediately before the COVID-19 pandemic.
- Multiple positive samples were taken from one stall known to have sold live mammals, and the water drain proximal to this stall, as well as other sewerages and a nearby wildlife stall on the southwest side of the market, tested positive for SARS-CoV-2 (24).
>> These findings suggest that infected animals were present at the Huanan market at the beginning of the COVID-19 pandemic

EBV genetic aberrations

- DNA of BL tumors contain genetic aberrations of chromosomes 8 and either 14, 22, or 2
- c-myc (transcription factor promoting cell division) gene moves near the immunoglobulin heavy chain or light chain gene
>>Abnormal expression of the c-myc gene and increased tumorigenicity of the cells
- BL affects the jaw and spreads to the soft tissues and the parotid glands

Emerging virus

- A new recently identified virus to humans not observed before
- Usually zoonotic
- many times new viral infections are caused by viruses that crossed species barrier
- examples: HIV, Zika, SARS, MERS-CoV, West Nile, SARS-CoV2

Environmental factors of emerging diseases

- Many emerging infectious disease outbreaks occur after environmental disruptions (hurricanes, floods, droughts, etc.)
- Global climate changes are expected to increase
>>Broadens the range of vector borne diseases and expands mosquito range
- 1999, WNV outbreak, NYC:
>>Correlated with a dry spring and summer that decreased the mosquito predators and breeding sites. Mosquitoes thrived in drainpipes and sewer puddles that attracted birds in search of water. Brought the vector and the reservoir together. Mosquitoes fed on WNV-infected birds

Epstein-Barr virus (EBV)

•Herpes virus 4
•Causes mononucleosis
•Associated with Burkitt's Lymphoma
-In those with weakened immune system (chronic malaria or AIDS)
-Solid tumor of B lymphocytes
-In the U.S., about 1,200 people are diagnosed each year, and about 59% of patients are over age 40.
- Burkitt lymphoma is especially likely to develop in people infected with HIV, the virus that causes AIDS

evasion fo the innate immune response

- After viral genome entry into the host cells during infection, viral genome ssRNA as well as dsRNA intermediate found in virus life cycle is sensed by innate immune sensors, RIG-I/MDA5 in cytoplasm or Toll-like receptors TLR3/7/8 in endosome.
- Response generated from these sensors initiates a downstream signaling cascade leading to IFN-β gene expression.
-RIG-I/MDA5-dependent signaling involves a mitochondrial adaptor MAVS, whereas TLR signals through TRIF/MyD88. Both pathways involve common TRAF adaptor to activate transcription factors.
- The SARS-CoV-2 encoded proteins shown in yellow box are known to intervene the host innate immune signaling at various action points as evasion mechanisms to sustain viral replication and propagation.
>>One key strategy is to effectively suppress the activation of TNF receptor-associated factors (TRAF) 3 and 6, thereby limiting activation of the transcription factors NFκB and IRF3 and 7. This leads to severely dampened early pro-inflammatory response mediated by type I interferons (IFN) and pro-inflammatory effector cytokines IL-1, IL-6, and TNF-α.
- Furthermore, novel SARS-CoV-2 inhibits activation of STAT transcription factors (ISRE) in response to type I IFN receptor activation, which further limits antiviral response mechanisms.
- Altogether, this prohibits virus containment through activation of antiviral programs and the recruitment of immune cells.

Flyways

- Flyways traverse large paths, north to south, breeding grounds and food source, giving them a great transportation of potential influenza virus and for it to circulate regionally.
- Even though they appear to be regional, they are actually global. Potential of risk for transfer globally.

HBV and HCV

- HBV and HCV causes chronic hepatitis that can progress to cirrhosis and liver cancer (HCC)
- HBV and HCV infect the hepatocytes of the liver
>>HCV also infects lymphocytes
- HBV DNA integrated into host (not HCV RNA)
- Rate of progression to cancer much higher with HCV
- HBV vaccine was the first vaccine to prevent a cancer
- HCV has no vaccine but effective treatment

HBV DNA integration

- Integration of HBV DNA into the host hepatocyte genome is key to HCC
- HBV does not have integrase nor is integration part of life cycle
- Unlike retroviruses, genomic integration has no role in HBV replication and does not produce integrase enzymatic activity protein, meaning that the integrative process is likely mediated by cellular topoisomerase I activity
- Whole-genome sequencing indicates that the integration of HBV DNA into the host hepatocyte genome can be detected in 80-90% of cancer cells and ~30% of liver tissue adjacent to the tumor
- Integration appears prior to the occurrence of HCC
- A common effect, increasing with rising numbers of integrations, seems to be increased genomic instability, and a correlation was observed between the number of chromosomal aberrations and the status of tumor suppressor genes
- Comparing HBV‐associated HCC to other aetiologies shows a significantly more frequently altered p53 pathway (47%)

HBV Hbx

- Hepatitis B Hbx contributes to Hepatocellular Cancer
- absolute requirement for in-vivo HBV replication
•154 amino acids
•Numerous actions centered around viral and Cellular proliferation (stimulate transcription)

HBV life cycle

- HBV life cycle, including virion entry, nucleocapsid uncoating, nuclear import, cccDNA formation, viral RNA transcription, viral protein translation, capsid formation, viral replication, virion assembly and secretion.
- Mechanisms of main causing factors in HBV life cycle leading to HCC development and progression.
>> cccDNA, dslDNA, ER, HBeAg, HBsAg, HBV, HBx, LHB, MHB, NTCP, pgRNA, SHB, rcDNA

HCC mechanisms

- The are three major molecular mechanisms of hepatocarcinogenesis caused by HBV infection:
(1) First, the expression of viral proteins, particularly hepatitis B virus X protein (HBx), promotes cell proliferation and viability.
(2) Second, the integration of HBV DNA into the host genome alters the expression and function of endogenous genes and induces chromosomal instability.
(3) genetic damage accumulates as a result of inflammation and ongoing hepatocyte division to replace cells killed by virus-specific T cells.

HCV

- Icosahedron-shaped, enveloped, +ssRNA flavivirus
- Similar to picornaviruses (poliovirus), except HCV particles are enveloped
- Infects B lymphocytes and hepatocytes
- No evidence of sexual transmission for HepC
- Does not contain an obvious v-onc (like Hep B) or integrate
- Mechanism of oncogenesis?? Not entirely clear (genomic instability)
- Multiple avenues to Hepatocellular carcinoma (HCC) from HCV

HCV and oncogenesis

- Chronic HCV and associated liver cirrhosis represent major risk factors for HCC development.
>> Hepatocarcinogenesis is a multistep process that may last for years; it involves progressive accumulation of different genetic alterations which lead to malignant transformation.
>> Malignant transformation of hepatocytes occurs through increased liver cell turnover, induced by chronic liver injury and regeneration, in the context of inflammation and oxidative stress.
- HCV proteins may directly up-regulate mitogenic pathways, block cell death and induce ROS production. Moreover, HCV triggers persistent inflammation with accumulation of liver-infiltrating lymphocytes and production of several cytokines, such as LTα and LTβ, which are tightly linked to HCC development.
- Chronic inflammation exacerbates ROS production, which is considered a main source of genetic mutations. ROS are also associated with TGF-β pathway induction, leading to hepatic stellate cell activation and fibrogenesis.
- TGF-β, together with TLR4, plays an important role in the epithelial-mesenchymal transition. HCV dysregulates host lipid metabolism, causing liver fat accumulation which in many patients is associated with HCC.
- HCV is also able to induce angiogenic and metastatic pathways. Polymorphisms, mainly in DEPDC5 and MICA genes, have been recently shown to increase the risk of developing HCC.
- HCC, hepatocellular carcinoma; HCV, hepatitis C virus; ROS, reactive oxygen species; TGF, transforming growth factor.

Hendra Virus

- Hendra virus: through flying fruit bats, it can infect a horse which can infect other horses and humans.
- Highly fatal
- Humans tend to be dead end host

High Risk Types 6, 11, 16, 18, 31, 33, 45, 52, and 58

•90% OF CERVICAL CANCER CASES
•90% OF ANAL CANCER CASES IN MALES AND FEMALES*
•90% OF VULVAR CANCER CASES*
•85% OF VAGINAL CANCER CASES*
•90% OF GENITAL WARTS CASES IN MALES AND FEMALES

How do bats handle viruses?

- Bats show an excellent balance between enhanced host defence responses and immune tolerance through several mechanisms.
- Examples of enhanced host defences include: constitutive expression of IFNs and interferon-stimulated genes (ISGs), increased expression of heat-shock proteins (HSPs), a higher base level expression of the efflux pump ABCB1 and enhanced autophagy.
- On the other hand, dampened STING and suppressed inflammasome pathways—such as dampened NLRP3, loss of PYHIN and downstream IL-1β—contribute to immune tolerance in bats.

HPV pathogenesis

- HPV infect stratifying basal epithelial cells
>> *CANNOT infect cells of dermis because they are not metabolically active*
- HPV attach and gain entry into basal epithelial cells through break in skin
- HPV binds to heparin sulfate proteoglycans (HSPGs)
- Species specific (animal HPV's cannot infect human)
- Genome replication, nucleocapsid and virion formation occurs in nucleus of epithelial cells
- Most cause benign tumors
- *Lifecycle occurs entirely in the nucleus*

HPV structure

•Small (52-55 nm in diameter)
•Naked
•Icosahedral-shaped
Circular dsDNA genome (~8000 bp in length)

HPV vaccines

- 3 HPV vaccines available (no, only one):
>Virus-like particles (no genome) empty capsids
>Made from yeast
>Gardasil®9, Gardasil®, Cervarix®
- The bivalent HPV-16 and HPV-18 vaccine was made by expressing the HPV-16 and HPV-18 L1 genes in Saccharomyces cerevisiae (yeast) cells.
Steps 1-3: The L1 genes from the HPV-16 and HPV-18 genomes were inserted into separate yeast DNA vectors.
Step 4: The recombinant yeast vectors were transformed into yeast cells.
Step 5: The L1 mRNA was transcribed in the yeast nucleus and exported to the cytoplasm where the L1 protein was made in high concentrations.
Step 6: The L1 proteins assembled into empty capsids or virus-like particles (VLPs).
Step 7: Large quantities of yeast expressing the VLPs were grown.
Step 8: The yeast expressing the VLPs was pelleted by centrifugation and the VLPs were harvested and purified to create a vaccine.
Step 9: The VLPs resemble the HPV viral particle but do not contain the DNA genome. VLPs cannot infect human cells and cause papillomas or cancer. The bivalent vaccine contained both HPV-16 and HPV-18 VLPs. The vaccine was injected intramuscularly into the arm.

HTLV

- The virus is transmitted from mother to child, between sexual partners, by needle sharing and in contaminated blood products.
•HTLV-1 is a complex retrovirus. So, in addition to the standard repertoire of structural proteins and enzymes shared by all retroviridae (gag, pol, pro and env), the 3'region of the HTLV-1 genome (pX region) also encodes a number of accessory genes: tax, rex, p12, p21, p13, p30 and HBZ.
•Disease typically occurs decades after initial infection and affects less than 10% of carriers. Total number of HTLV-1 carriers globally is estimated to be between 5-10 million.
•Only a minority of HTLV-1-infected individuals develop disease. Depending on ethnicity and gender, approximately 2-3% of infected individuals develop ATL

Human Coronavirus history

- Among the seven known human coronaviruses:
SARS-CoV, MERS-CoV, and SARS-CoV-2 have evolved as severe pathogenic forms infecting the human respiratory tract.
- 2521 cases and 866 deaths were reported for the MERS-CoV that emerged during 2012. (34% mortality, epidemic ongoing)
•Recently ~ 637M cases and 6.6 M deaths (data as 11/7/2022) were reported from SARS-CoV-2 infection (around 2%, pending and variable, very age dependent).

Human exposure to zoonotic disease

- Zoonoses: Infectious diseases transmissible from animals to humans or from humans to animals
- "Species" Jump
- Close proximity of wildlife to humans may contribute to viruses crossing the species barrier
-Monkeypox, U.S. exotic pet trading (prairie dogs)
-Open-Markets: SARS and Coronavirus by "horseshoe bat"
-HIV

Human factors contributing to new and reemerging Viral infections

- Human demographics drive viral disease emergence more than virus evolution or ecological factors (sex/gender, race/ethnicity, mobility/travel, occupation, etc.)
- Increasing human populations lead to urbanization
>>Higher population densities favor the spread of viral diseases
-Crowding
-Sanitation
-Contamination of drinking water
-Healthcare facilities
- Air travel allows infected travelers to reach any part of the world in less than 24 hours
-the spread of viral diseases to new areas can happen at any time
- Virus have no borders

Human Papillomaviruses (HPV)

- HPV infections are most common among sexually active adults and adolescents
>>Entry through minor break in skin
- Two HPV types (16 and 18) cause 70% of cervical cancers and precancerous cervical lesions.
- HPV with cancers of the anus, vulva, vagina and penis
- HPV implicated in cancers of oral cavity, oropharynx, and throat
- Risk of being infected at least once in a lifetime among both men and women is 50%
- About 120 papillomavirus types identified: Approximately 40 can infect genital tract
>>Low-risk types cause warts or papillomas (e.g., genital warts) such as HPV 6 and 11, cause about 90% of genital warts, which rarely develop into cancer
>>Intermediate-risk types found in precancerous lesions
>>High-risk types can cause cancers of the cervix, vulva, vagina, anus, and penis (e.g., types 16 and 18)
>>Significant latent period in causing cancer (4-20 years, approximately)

Human retroviruses

•5 that have been identified
-HIV-1, HIV-2, HTLV-1, HTLV-2, and Human Foamy
•Human T-cell leukemia virus type 1(HTLV-1)
-Can induce acute T-cell leukemia (ATL)
-Most people infected are asymptomatic carriers
•HTLV-2
•HTLV-1 causative agent of ATL

Hypothesis on SARS-CoV-2 spread

- If the virus had been human-made, that would show in its genome
- If you were going to create a coronavirus that can be transmitted by humans, you would almost certainly start with the first SARS virus.
- SARS-CoV-2 is like nothing we have seen before. It really is highly unlikely that someone created it; it is not put together from pieces we know about.
- SARS-CoV-2 is closely related to other beta coronaviruses such as RaTG13, a bat virus that the Wuhan Institute of Virology has been working on.
>>But it only shares 96% of its genome sequence with RaTG13, which makes them roughly as similar as human beings and chimpanzees, and points to a common ancestor rather than one springing from the other.

Kaposi's Sarcoma-Associated Herpesvirus

•Caused by human herpesvirus-8 (HHV-8)
•Rare skin cancer
•Kaposi's sarcoma occurs most often in:
>>Elderly men of Mediterranean, Middle Eastern, or Eastern European decent
>>AIDS patients (50% risk in homosexual males with AIDS)
-No single viral oncogene identified,but GPCR and Bc1-2 are thought.

KSHV infection

- In a healthy host, KSHV infection of a KS progenitor is not oncogenic, since it leads to latent infection or to cytopathic lytic replication.
- Reactivation leading to oncogenic lytic gene expression is under immunological control.
- Scenario for AIDS-KS pathogenesis according to the Paracrine Oncogenesis and Abortive Lytic Hypotheses. In HIV/AIDS, decreased immunosurveilance, inflammatory cytokines and HIV Tat lead to KSHV reactivation and reinfection.
-This leads to increased, uncontrolled, early lytic oncogenic gene expression, with concomitant risk of cell transformation by somatic host cell oncogenic alterations.
-Upon transformation, cells shut down early lytic oncogenes. Latently infected transformed cells are stimulated in a paracrine manner by angiogenic and proliferative factors released from lytically infected or abortive lytic.
-In addition, lytically infected cells provide a constant source of virions for reinfection, while angiogenesis and inflammation recruit target KS progenitors. ART inhibits KSHV reactivation and lytic replication through immune reconstitution and decreased levels of HIV viral loads.

Life Cycle of coronavirus

Occurs in cytoplasm
1. Spike protein of SARS-CoV-2 binds to the receptor of the host cell, the virus enters the cell, and then the envelope is peeled off, which lets genomic RNA be present in the cytoplasm.
2. The ORF1a and ORF1b RNAs are made by genomic RNA (directly translated and make polyprotein), and then translated into pp1a and pp1ab proteins, respectively. Protein pp1a and ppa1b are cleaved by protease to make a total of 16 nonstructural proteins.
3. Some nonstructural proteins form a replication/transcription complex (RNA-dependent RNA polymerase, RdRp), which use the (+) strand genomic RNA as a template.
4. The (+) strand genomic RNA produced through the replication process becomes the genome of the new virus particle. Subgenomic RNAs ( from RdRp) produced through the transcription are translated into structural proteins (S: spike protein, E: envelope protein, M: membrane protein, and N: nucleocapsid protein), which form a viral particle. Spike, envelope, and membrane proteins enter the endoplasmic reticulum, and the nucleocapsid protein is combined with the (+) strand genomic RNA to become a nucleoprotein complex.
5. They merge into the complete virus particle in the endoplasmic reticulum-Golgi apparatus compartment, and are excreted to extracellular region through the Golgi apparatus and the vesicle.

LNP vaccines

- lipid nanoparticles are taken up by cells via endocytosis, and the ionizability of the lipids at low pH enables endosomal escape, which allows release of the cargo into the cytoplasm.
- In addition, lipid nanoparticles usually contain a helper lipid to promote cell binding, cholesterol to fill the gaps between the lipids, and a polyethylene glycol (PEG) to reduce opsonization by serum proteins and reticuloendothelial clearance.
>>Together encapsulate and protect the fragile mRNA core
- Pfizer-and Moderna SARS-CoV-2 mRNA vaccine (BNT162b2) encodes the full-length prefusion stabilized SARS-CoV-2 S protein with 2 proline substitutions

MMR vaccination

- MMR vaccination: All live attenuated vaccines to fight against the following:
-Measles: -ssRNA virus. "highly contagious", R nought 12-18
-Mumps: -ssRNA virus. "look like a chipmunk"
-Rubella: +ssRNA. "Rubella was eliminated from the United States in 2004"

mRNA vaccines process

(1)Injected mRNA vaccines are endocytosed by antigen-presenting cells.
(2) After escaping the endosome and entering the cytosol, mRNA is translated into protein by the ribosome. The translated antigenic protein can stimulate the immune system in several ways.
(3) Intracellular antigen is broken down into smaller fragments by the proteasome complex, and the fragments are displayed on the cell surface to cytotoxic T cells by major histocompatibility complex (MHC) class I proteins.
(4) Activated cytotoxic T cells kill infected cells by secreting cytolytic molecules, such as perforin and granzyme.
(5) Additionally, secreted antigens can be taken up by cells, degraded inside endosomes and presented on the cell surface to helper T cells by MHC class II proteins.
>>Helper T cells facilitate the clearance of circulating pathogens by stimulating B cells to produce neutralizing antibodies, and by activating phagocytes, such as macrophages, through inflammatory cytokines. BCR, B cell receptor; ER, endoplasmic reticulum; TCR, T cell receptor.

Neutralizing antibody approach

- Regen-Cov
- two monoclonal antibodies that have been developed
- Received inoculation of the spike protein
- Then screens for RBD specifics, or precise B cell that produces the antibodies and clone it
Steps:
1. IgG1 mAbs with unmodified Fc regions.
2. These two mAbs were chosen from a pool of more than 200 neutralizing mAbs present in the initial isolation of thousands of antibodies and were derived from parallel efforts using humanized mice and the sera of patients recovering from COVID-19.
3. Bind two distinct and non-overlapping sites on the RBD
4. The rationale for this antibody combination is that it is unlikely that a mutation in the S protein of SAR-CoV-2 will simultaneously render both antibodies ineffective.
5. In extensive in vitro testing, this combination retained its ability to neutralize all known S protein mutation

New viral outbreaks and how they occurred

- Usually due to untouched reservoir of virus that is capable of producing major genetic variants. This reservoir is then introduced into human population
- Examples of this include:
>Influenza
>Emergence of the H5N1 and H5N2 influenza A
>>Very fatal in domesticated birds. 53% fatal in human

Nipah virus

- Nipah virus: can go through malayan bat to pig and then to humans or DIRECTLY to humans
- Eating of fruit, pigs, oral fecal route
- High mortality rate

Norovirus

- Norovirus: Group IV Baltimore (+ssRNA, naked)
- The norovirus RNA-dependent RNA polymerase has a high error rate, with available evidence indicating that the RDRP of GII.4 human norovirus strains has a higher rate of mutation than other genotypes
- Increase in gastrointestinal illness caused by norovirus 10-fold between 2001 and 2004 on cruises (commonly called cruise virus)
-Noroviruses cannot be cultured in the lab
-No animal model for noroviruses
-Hard to determine the genetic changes in the virus that trigger more severe outbreaks
- acute and self-limiting
- Outbreaks after major disasters such as Hurricane Katrina
- common settings: healthcare facilities, restaurants and at catered events, schools and child care center, cruise ships

ORFs of coronavirus

- about 14 ORFs coding for 27 proteins
•Directly translated from +ssRNA from 5' cap
•Ribosomal Frameshift producing 2 polyproteins
•Generates polyprotein
•Within polyprotein are 2 proteases which will process
•Nsp 3/5- proteases

Origin of SARS-CoV-2 Horseshoe Bat

- Coronavirus ancestors seem to be bat and bird
- Leads to the basic evolvement of four subgenuses: alpha, beta, gamma, and delta
>>Gamma and delta is usually the bird as common ancestor
>>Alpha and beta is bats
- At present, there is seven known human corona viruses.
>>Four of them are the common cold. 10-30% of common cold is due to circulating corona viruses
>>Alpha can infect cats, dogs, pigs, bats
>>Two human coronavirus in this that are cause of common cold
>> Beta has four that infects humans. Two that cause benign infctions. Can infect bats, horse, dog, cats. MERS, COVID, are part of this

Pathogenesis of COVID-19

- The widespread presence of injury and the ability of the virus to infect endothelial cells in the microcirculation of many organs may account for the multi-organ damage observed in late stage COVID-19 patients.
- Stage 1: Asymptomatic state (intial 1-2 days after infection)
> Inhaled virus SARS-CoV-2 likely binds to epithelial cells in the nasal cavity and stats replicating. ACE2 is the main receptor for both CoV and CoV2
> single cell RNA indicates low level of ACE2 expression in conduction airway cells
- Stage 2: Upper airway and conducting airway response (next few days)
> The virus propagates and migrate down the respirtory tract along the conducting airways, and a more robust innate immune response is triggered
> for about 80%, the disease is mild and restricted to upper airways
- Stage 3 (also known as cytokine storm): Hypoxia, ground glass infiltrates, and progression to ARDS
>>Acute respiratory distress syndrome (ARDS) is a serious lung condition that causes low blood oxygen. People who develop ARDS are usually ill due to another disease or a major injury. In ARDS, fluid builds up inside the tiny air sacs of the lungs, and surfactant breaks down
> about 20% of individuals will progress to stage 3
> the virus now reaches the gas exchange units of the lung and infects alveolar type II cells. Both SARS and influenza preferentially infect type II cells compared to type I
> SARS-CoV propagates within type II cell, large number of viral particles are released and the cells undergo apoptosis and die

PP1A and PP1B

- They encode for these that contain enzymes and virulence factors. They are cleaved by viral proteases into bite-sized pieces
- enzymes: proteases such as nsp3 and nsp5 that are involved in cleaving on the polyprotein in critical parts
- pp1b is longer
- coding for about 15-16 non-structural proteins
> 15 compose the viral replocation complex as well as RNA processing and modifying enzymes needed for integrity
- NSP4 (double mebrane vesicle formation): important for replication trasncription complex also

Programmed ribosomal frameshifting

- generates 2 polyproteins encoding the replicase proteins
> slippery heptanucleotide that may cause read thru past Stop codon
> pseudoknot can cause ribosome to pause and frameshift

R naught

- Estimating the ability of a new pathogen to spread is a key measure in an emerging disease outbreak.
- A metric used to describe this spread is the basic reproductive rate (R0).
- R0 is defined as the average number of secondary transmissions from one infected person; when R0 is greater than 1, the epidemic is growing

Reemerging Virus

- A virus under control from a public health perspective but is making a comeback or reappearance, and increasing in incidence and geographical range of exposed human populations
- It can happen because of decrease in vaccination compliance and immigration of the unvaccinated
- Virus makes a comeback
- Examples: Measles, Mumps, Rubella, Polio

SARS-CoV-1

- First reported in 2003 as cause of disease
- Enveloped viruses, nonsegmented
- Baltimore Category IV (large RNA viruses, about 30kg)
- helical symmetry
- Spread of two previously unknown coronaviruses
- Causative agent for SARS and MERS
- SARS- Severe acute respiratory syndrome
- MERS- Middle eastern respiratory syndrome
>> Infection in your nose, sinuses, or upper throat
- Agricultural practices and deforestation can lead to exposure to bats and/or zoonotic diseases
- Corona virus in general is a RNA virus due to part of polymerase and part of the fact of recombination events that can occur

SARS-CoV-2 development

- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection starts with the infection of ciliated cells in the upper conducting airways, from where the virus can spread down the bronchiotracheal tree to the alveoli, likely as a result of poor or mistimed immune responses, in particular type I and type III interferon (IFN) responses.
- Alveolar damage may be a direct effect of the infection of alveolar type 2 (AT2) cells or an indirect effect caused by local inflammatory responses, which can result in endothelial activation. AT2 cells adopt a damage-associated transient progenitor (DATP) phenotype, an AT2 cell state associated with lung injury and that is characterized by failure to fully differentiate into AT2 cells.
- The disrupted epithelium and endothelium allow fluid to leak into the alveoli. The exposed subendothelial extracellular matrix attracts and activates platelets and initiates the coagulation cascade, leading to fibrin deposition. At the same time, immune cells, such as monocytes and neutrophils, are attracted, and these have dysfunctional phenotypes and can further promote inflammation and coagulation.
- Immature neutrophil populations are increased in severe COVID-19. Neutrophils, activated by platelets, release neutrophil extracellular traps (NETs) containing tissue factor, promoting the formation of microthrombi.
- The upregulation of plasminogen activator inhibitor 1 (PAI1) may further promote microthrombus formation by inhibiting fibrinolysis. Eventually, platelets may be used up, leading to thrombocytopenia.
- Macrophages in the alveoli may adopt a pro-inflammatory profibrotic phenotype and when infected may go into pyroptosis, while hyperinflammation may promote PANoptosis of T cells. CD16+ T cells are induced by complement activation and promote microvascular endothelial cell injury and the release of chemokines.
- 'Armed' natural killer cells expressing high levels of cytotoxic proteins are also associated with severe disease.
- The end result is a focal pattern of highly inflamed and flooded lung tissue, impairing oxygen exchange and leading to hypoxaemia. ARDS, acute respiratory distress syndrome.

SARS-CoV-2 Drug targets

- viral RdRP inhibitors
- Viral protease inhibitors
- Host protease inhibitors
- Attachment and virus neutralization
- Viral maturation inhibitors
- Only viral protease inhibitors and RdRP inhibitors have been successful so far in medications

SARS-CoV-2 genome

Genomic organization:
> 5' cap structure and 3' poly-A tail
> NO IRES because of the cap
- at 5' end there is two coding regions:
ORFIA and ORFIB (they produce PP1A and PP1B).
>>> this makes up 2/3 of the genome
>>> pp1a is cleaved into NSP1 to NSP11
>>>ORF1b requires a -1 programmed ribosomal frameshift to synthesize pp1b. pp1b is sliced into NSP1 to NSP10 and NSP12 to NSP16
- structural proteins such as S, E, M, and N. (encoded by 11 subgenomic RNAs) as well as accessory proteins
>>S is one of the most important ones (spike).
>>Make up just 1/3 of the genome (on 3' end)

SARS-CoV-2 structure

•Enveloped
•Spherical
•Very large +RNA genome (30 kb). Baltimore Category 4
•125 nm in diameter
•Large for an RNA virus with an Rdrp that has exonuclease activity
- considered to have maximum genomic capacity without becoming unstable

SARS-CoV-2 vaccine

- Pfizer: mRNA type
- Moderna: mRNA type
>> lipidnano particles (mRNA vaccine): cell-free manner, rapid, scalable and cost effective. s single mRNA vaccine can encode multiple antigens. Problems with mRNA delivery: very dense negative charge, so need a delivery vehicle to protect RNA and let it into cells.
- J&J: Viral Vector

SARS-CoV-2 viral entry

- S-protein binds to ACE2 intially through the S1 receptor binding domain
- S1 domain is then shed from the viral surface, allowing the S2 domain to fuse to the host cell membrane
- Transmembrane serine protease 2 (TMPRSS2) is a cell surface protein primarily expressed by endothelial cells across the respiratory and digestive tracts

Small percentage of cancers

- Pathogenic hallmark of viruses that induce cancer is that they carry a viral oncogene
- At least 6 viruses are thought to contribute to 20% of cancers:
>Human papillomavirus (HPV)- Major one
>Hepatitis B virus (HBV)
>Hepatitis C virus (HCV)
>Epstein-Barr virus (EBV)
>Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)
>HIV associated
>Human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2)

Spike protein of Covid

- Receptor binding protein
- has S1 subunit which facilitates ACE2 mediated virus attachment
- S2 subunit which promotes membrane fusion

Structural proteins (s, E, M, N)

- Nucleocapsid: protein coat that contains RNA genome and appears to have other function in immune inhibition
- Membrane: function to stabilize and maintain the viral envelope
- envelope (not standard): E is abundantly expressed inside the infected cell, but only a small portion is incorporated into the virion envelop. Role is viral assembly, release, and pathogenesis

Sub-genomic RNA transcription

- it is discontinuous and regulated by shared regulatory sequences (TRS)
- Template switching occurs as RdRp is transcribing the negative strand and encounters transcriptional regulatory sequences (TRS) preceding each gene called the body TRS (TRS-B)
- The TRS-B site has a 7-8-nt conserved core sequence (CS) which is thought to enhance the likelihood of RdRp template switching by hybridizing with leader TRS (TRS-L)
- The occurrence of this programmed template switching leads to the generation of sgmRNAs with identical 5′ and 3′ sequences
- Coronavirus transcription also involves template switching.
1. After a coronavirus infects a cell, it replicates its positive-strand RNA genome into a negative-strand genome with RdRp
2. The negative-strand genome subsequently serves as a template for the production of positive-strand genomes and subgenomic messenger RNAs (sgmRNAs), a set of 3′ coterminal RNAs encoding structural genes (Sawicki and Sawicki 1998).
3. The sgmRNAs share a common 5′ sequence, called a leader sequence, which is located at the beginning of the coronavirus genome (Zhang et al. 1994).
4. Template switching occurs as RdRp is transcribing the negative strand and encounters transcriptional regulatory sequences (TRS) preceding each gene called the body TRS (TRS-B) (Sola et al. 2015).
- The TRS-B site has a 7-8-nt conserved core sequence (CS) which is thought to enhance the likelihood of RdRp template switching by hybridizing with an identical or nearly identical CS in the leader TRS (TRS-L)
•The occurrence of this programmed template switching leads to the generation of sgmRNAs with identical 5′ and 3′ sequences,

The majority of emerging and reemerging viruses that are problematic contain

RNA genomes

Treatments (antiviral/immuno) for SARS-CoV-2

- three antivirals currently in use
>> Veklury remdesivir (injection): first and only anitviral treatment FDA approved. It inhibits the RNA depednant RNA polymerase. Phosphorylated by host and then by the virus. Known as a chain terminator
>> molnupiravir (oral antiviral): similar mechanism, but it gets incorporated into burgeoning RNA strands and induces errors.
>> Paxlovid (oral): protease inhibitor. Utilizes Nirmatrelvir which is a strong P450 inhibitor. Known as a booster agent. FDA EUA status

Unique characteristic about bats

- Evolution of tolerance to DNA damage and unique antiviral immune response in bats.
- Development of flight necessitated the evolution of bats with the ability to modulate the consequences of increased metabolic activity by suppressing inflammation.
> Inflammation was suppressed by dampening the activation of DNA sensors, such as STING, and reducing levels of inflammatory cytokines, such as TNFα (center). - These traits were positively selected but a reduced inflammatory response made it advantageous for virus replication
- Increased susceptibility of cells to virus replication was compensated by selection of more effective antiviral measures, such as higher constitutive expression of Interferons or unique ISG expressions
-Viruses persistently infect bats due to their reduced inflammation (reduced DNA sensor activation and decreased inflammatory cytokine levels) and their effective antiviral immune response (increased constitutive expression of interferons and unique ISG expressions).
- Stressful events alter the balance between host and virus and lead to an increase in virus replication, thereby leading to viral shedding.

Viral evolution and adaptation

- Mutation rate of RNA viruses: 1 mutation per every 104 to 105 nucleotides
- Mutation rate of DNA viruses: 1 mutation per every 108 to 1011 nucleotides
>> RNA viruses (HIV, Influenza) evolve approximately 1 million times faster than human DNA
- Influenza viruses: Antigenic drift/ Antigenic shift
- Reassortment- "Gene swapping", occurs with segmented viruses and coinfection of same cell- "antigenic shift"
- Recombination- can occur in segmented or nonsegmented genomes. The polymerase complex switches, midreplication from one RNA molecule to the next.
>>Coronavirus (SARS, MERS) does this frequently.

Viral transmission by mosquitoes

- The mosquito is a glorified needle.
- The needles, they actually amplify the virus after they feed and then they can transmit. The saliva actually blocks or inhibits the host response. They can block interference signaling of inflammatory response and they end up changing the immune microenvironment.
- What blood does to the mosquito response, this seems to block the toll-like pathways and gut immunity to propagate the virus within the mosquito itself.

Viral transmission Via mosquito

-Aedes and Culex spp., are spread throughout the world and are the dominant arthropod vectors for numerous arboviruses
- Mosquito incidentally feeds on a virus-infected host to acquire viruses circulating in the host blood
>>Viruses infect the epithelial cells in the mosquito gut and then spread into the mosquito
>> viruses subsequently disseminate into the salivary glands, thus enabling viral transmission by the infected mosquito to naive hosts through blood feeding
- 104 plaque-forming units (PFU) of viruses are required to achieve productive mosquito infection
- Tissues in the mosquito such as haemolymph, salivary glands, and fat body, are highly permissive to arboviral propagation

Viral Vector Vaccine

- uses human adenovirus
- naked
- DNA virus
- Spike protein cloned in Ad

Viruses that can cause cancer

- Pathogenic hallmark of viruses that induce cancer is that they carry a *viral oncogene* (and typically integrate in host genome)
- At least 6 viruses are thought to contribute to 20% of cancer:
1. Human papillomavirus (HPV)- Major one- DNA Virus
2. Hepatitis B virus (HBV)
3. Hepatitis C virus (HCV)
4. Epstein-Barr virus (EBV)
5. Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)
>>>HIV associated or immunosuppression
6. Human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2)
-80% of viral-associated cancers are cervical cancer (HPV)
- Liver cancer (HBV and HCV)- kills more than HIV

West Nile

- West Nile virus (WNV): enveloped +ssRNA virus, Group IV of baltimore
-Typically not in western hemisphere
- WNV was first isolated from blood of a woman in West Nile district, Uganda in 1937
- Leading theory for spread of WNV: Mosquitoes flew across the Atlantic Ocean from Israel on aircraft
>> Aircraft cabin spaces were not routinely sprayed with insecticides on intercontinental flights entering the U.S.
- Aircraft moves: Virus-infected animals, Arthropod vectors (e.g., mosquitoes)
-Main reservoir is birds
-Mosquitoes pick up from feeding on birds
-From Mosquitoes to humans and horses ("dead-end host")
-Dead end host: cannot transmit due to low viremia
> Horses do have a vaccine, we do not

Why cant we control SARS-CoV-2?

- The spillover event is unknown
- There is asymptomatic transmission and mild symptoms

Zika

- Zika is a +ssRNA, enveloped virus. Group IV Baltimore
> Spread mostly by day-biting Aedes spp. Mosquitoes
> Flavivirdae: +ssRNA. mosquito-born
-First human case was reported in Nigeria 1954
- First major outbreak of Zika virus infections occurred in Yap
- Spread rapidly
- Brazil to PR to Mexico to US
>Guillain-Barré syndrome
- Has similar presentation as other arbor viruses in humans
- Main symptom: highest risk was really in infants, microcephaly or abnormally small heads. This is because the virus would have an unusual presentation of neuro tropism and this would inhibit the brain and its development
- A mosquito would bite pregnant woman, transmit to the fetus through numerous ways (placental macrophages or it crosses through barrier), once in fetus, it uses a axel receptor that is expressed at high level, and can cause apoptosis of the neuronal cells and inhibit growth factors
- It has been mostly unseen recently because of herd immunity

What is a Virus?

Infectious, obligate intracellular parasite composed of genetic material surrounded by a protein coat and/or envelope derived from host cell

Characteristics of a virus?

Infect all living things
Smaller than bacteria
Has DNA or RNA
Can be split into 7 groups
"Freeloaders"

Who discovered viruses and why?

Ivanosky and Beijerinick found the idea of viruses through the discovery of Tobacco Mosiac Virus.
This was done by taking the sap from an infected plant and transferring it to another. Proved virsuses could pass through filter; resulted in Beijerinick calling it "contagious living fluid"

Hallmarks of virus discovery

-TMV - Pasteur filter
-bacteriophage found in human sewage, bacteriophage plaque assays used for replication
-Loeffler and Frosch: discovered foot and mouth disease, found it replicated in only the host (NOT in broth)
-TEM invented to see viruses
-PCR

Relative abundance of viruses

Viruses make up most of the biodiversity on Earth and are 94% abundant when compared to prokaryotes and protists.
-Humans Virome contains about 67.7% viruses
-Mostly non-pathogenic

Viruses are permanently within our DNA...why?

about 8.3% of out DNA is virus
-Not all viruses make you sick
-Relationship between viruses and humans is both pathogenic and symbiotic
-Large portion of our immune system is designed against viruses and majority do not/have little affect on well-being
- Why? - Most of the viral genes come from retroviruses that inserted copies of their DNA when infecting the cells.

How did Virology shape Immunology

Smallpox is the earliest recognized attempt to induce immunity, gave rise to the process of "variolation". This then resulted in Jenner inoculating a person with a cowpox lesion. This resulted in the cowpox creating immunity against the smallpox.
Virology gave people the idea to introduce the virus into other individuals who were healthy, to hopefully create immunity to said sickness in the future

The two phases of a virus

Dead and alive (virion vs infected cell)
"sleep, creep, then leap"

Beneficial viruses

latent herpes viruses (help the body to recognize cancer cells)
Adenoviruses

Why do we study viruses?

-Viruses outnumber cellular life by at least 10:1
-Viruses drive global cycles
_most cases they are beneficial to host
- Viruses have the greatest biodiversity on earth

Application of viruses in health problems?

Bacteriophage therapy
-Gene therapy: delivery of functional copies of dysfunctional genes via retroviruses and adenoviruses
... there has been a gene therapy approved for a type of blindness

2 fundamental basics of viruses?

- All viral genomes are obligate molecular parasites that can only function after they replicate in a cell
- All viruses must make mRNA that can be translated by host ribosomes: they are all parasites of the host protein synthesis machinery

Properties of Viruses and why are they formed?

Protection: without protection, the viral genome would degrade very quickly. Capsid serves this function
Recognition: Outer surface must recognize and interact with host cell during life cycle, survival depends on infecting same cell each time. Viral receptor regulates this
Self-assembly: simplest type of particle, can form spontaneously, little energy and highly stable
Fidelity: genes code for small proteins and reduce error. compromised of small proteins
Economy: Most volume for least surface area. Simplistic and repeated. Same protein subunit is essential for genome coding cpacity

Virus Shapes

3 types:
-Icosahedral - 20 identical triangular faces
-Helical - shaped like a rod or cylinder
-Complex - brick shaped

How viruses are classified

Viral capsid shape
Naked or enveloped
content of viral genome (DNA or RNA, double or single stranded)

naked vs enveloped virus

Naked: no lipid bilayer
Enveloped: lipid bilayer stole from host cell

Viruses that challenge the definition of virus?

Complex, poxviridae family

Viral taxonomy (early attempt)

Based on appearance, site of isolation, size, who discovered, and/or pathogenetic potential

ICTV

International Committee on Taxonomy of Viruses
-Charged with developing a universal taxonomic scheme to name and classify viruses
-Based on physical and chemical properties
-Includes the order, family, subfamily, genus, ect.

Baltimore Classification

-Based in type of nucleic acid, double stranded or single, polarity, and reverse trasnsciptase

Seven classes of Baltimore System and an example virus

ALL VIRUSES MUST GENERATE +ssmRNAs FROM THEIR GENOME
1. dsDNA (Herpes)
2. ssDNA (Parvovirus)
3. dsRNA (Rotavirus)
4. +ssRNA (Noro)
5. -ssRNA (Rabies)
6. RNA reverse transcribing (HIV)
7. DNA reverse transcribing (Hep. B)

Additional Hierarchical levels not determined by ICTV

Variant: difference in genome sequence
Strain: causes significantly different, observable, phenotype
Viral Subtype: based on receptor binding proteins on surface

Variants

All strains are variants but not all variants are strains
Viruses with mutations become variants

Viral Parts

-Lipid bilayer
-Matrix protein
-Genome
-Polymerase
-Receptor-binding protein

Enveloped virus

-Lipid bilayer wrapped
-obtained from host cell upon budding
-derived from PM or trans golgi network
-contain matrix
short time survival outside
-sesitive to desiccation, heat and detergents

Non-Enveloped virus (Naked)

-No lipid bilayer/envelope
-No matix
-Persist for weeks on surface
-Rhinovirus/poliovirus

Genes required to build viral particle (3)

3 categories of genes that code for:
Structural, Replication, and Binding
-Structural proteins that make up capsid
-Replication Enzymes (polymerase) needed in lifecycle
-Receptor binding proteins aka coat proteins