Virology Exam 4

Emerging Virus

A new recently identified virus to humans that has not been observed before. Usually zoonotic

Emerging Viruses (examples)

HIV, Zika, SARS, MERS-CoV, West Nile, SARS-CoV-2, Noroviruses

Reemerging Virus

A virus under control from a public health perspective but is making a comeback or reappearance, and is increasing in incidence and geographical range of exposed human populations.

Reemerging Viruses (ex)

Measles, mumps, Ebola, rubella, polio, enterovirus

Human factors that leave us susceptible to new viruses

Human demographics drive viral disease emergence more than virus evolution or ecological factors. Increasing human population, which leads to urbanization which leads to higher population densities which favors the spread of diseases. Travel

Mechanisms of exposure to emerging viruses?

crowding, sanitation, contamination of drinking water, healthcare facilities, air travel

Where has mortality shifted from and to in recent years

Mortality has shifted from infectious diseases to cardiovascular/cancer

Where and when was SARS first discovered

Hong Kong as of March 28, 2003. Patient zero of the SARS pandemic stayed at the Metropole Hotel.
Eventually spread to about 20 countries

How quickly did COVID travel the world?

Three months

What type of genomes are the majority of problematic emerging and remerging viruses?

RNA genomes

Reassortment

Gene swapping. Occurs with segmented viruses and coinfection of the same cell- antigenic shift.

Recombination

Can occur in segmented or non segmented genomes. The polymerase complex switches, mid replication from one RNA molecule to the next. Coronavirus (SARS and MERS) do this frequently.

Mutation rate of RNA viruses

Evolve approximately 1 million times faster than human DNA

What type of viral evolution happens with influenza?

Antigenic drift, antigenic shift, reassortment

Why can influenza be transmitted and mutate so rapidly?

Influenza has a reservoir that is capable of producing major genetic variants. The reservoir is then introduced into the human population (H5N1 and H5N2)

Reservoir is found wild waterfowl and shorebirds (as well as a range of domesticated animals who maintain their own influenza A viruses. Spillover events occur from wild birds (such as waterfowl). These birds have flyways and migration patterns over the world or regions of the US that can cause them to spread the disease to different areas. Genetic shift occurs when genes are passed within the same species while genetic shift happens when these birds pass these viruses to different species.

Norovirus' RNA-dependent RNA pol

High error rate and high rate of mutation

Why does norovirus have a high rate of transmission?

Highly contagious and spreads through contaminated food and water

healthcare facilities, restaurants and catering, schools and childcare centers, cruise ships

West Nile Transmission

Main reservoir is birds, mosquitos pick up WN from feeding birds and give it to humans and horses, who are dead-end hosts (cannot transmit due to low viremia)

How did the West Nile come to the US

Came to the US by mosquitoes flying across the Atlantic ocean on an aircraft

How did Zika spread to the US

From Brazil to Puerto Rico to Mexico to the US

How is Zika transmitted

Mosquito bites by Aedes spp. and vertical transmission

Why is Zika virus no longer relevant?

Immunity

Zoonosis

Infectious diseases transmissible from animals to humans or from humans to animals

What caused zoonosis in monkeypox

US exotic pet trading

What caused zoonosis in SARS

Open-Markets, horseshoe bat

Why coronavirus can be both transmitted and mutate so rapidly?

Highly transmissible because it is an aerosol. Mutates as it is zoonotic and infects many organisms quickly.

Nipah and Hendra Viruses Transmission

Nipah Virus Transmission

Bats can also contaminate fruit leading to human and pig infection

What environmental factors lead to outbreaks

Many outbreaks occur after environmental disruptions (hurricanes, floods, droughts)

Global climate changes are expected to increase
- Broadens the range of vector borne diseases
- Expands mosquito range

Arbovirus

Group of viruses that are transmitted by mosquitoes, ticks,
or other arthropods. They include encephalitis, dengue, and yellow fever. Heterogenous group of mainly RNA viruses

Aedes and Culex spp. are the dominant arthropod vectors for numerous arboviruses

togaviridae and flaviviradae

Togaviridae (alpha)

chikungunya, equine encephalitis, +ssRNA

Enveloped, non-segmented

Flaviviridae

Dengue, Zika, West Nile, Yellow Fever

Enveloped viruses containing a single-stranded positive-sense RNA gnome

Mosquitos that mainly spread disease

Viral transmission via mosquito

Mosquito incidentally feeds on a virus infected host to acquire viruses circulating in the host blood
Viruses infect epithelial cells in the mosquito gut and then spread into the mosquito
10^4 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
Viruses subsequently disseminate into salivary glands, thus enabling viral transmission by the infected mosquito to naïve hosts through blood feeding

Which vaccine preventable diseases are remerging?

Measles- -ssRNA virus- “highly contagious”, R nought 12-18 (per every infected person, how many people will it infect)
Mumps- -ssRNA virus- “look like a chipmunk” - lymphatic build up in parotid salivary glands
Rubella- +ssRNA- “Rubella was eliminated from the United States in 2004”

Amish community:
Poliomyelitis (+ssRNA)

Why do vaccine preventable diseases reemerge in general?

Decrease in vaccination compliance and immigration of unvaccinated

Telltale signs of measles

Facial rash and bad cough

Telltale sign of mumps

swelling of jaw

Telltale sign of Rubella

Rash that lasts 2-3 days

Structure of COVID-19

-Enveloped
-Helical
-Very large +RNA genome (cat IV)
- Nonseg
an Rdrp that has exonuclease activity (some proofreading ability)

COVID transmission

Origin of SARS-CoV-2

Horshoe bat

How many known coronaviruses

7
4 common circulating
3 emerging

Common ancestors of COVID are in which animals

bats and birds

Bats produce which coronaviruses

alphacoronavirus, groups a-d betacoronaviruses

can infect humans

Birds produce which coronaviruses

gammacoronaviruses and deltacoronaviruses

The seven types of human coronaviruses

Alpha:
- HCOV-NL63
- HCOV-229E (common cold cov)

Beta:
- HCOV-OC43 (common cold cov)
- HCOV-HKU1
- SARS
-MERS
- SARS-COV-2 (common cold cov)

What receptor do problematic covs (sars cov 1-2) tend to use

ACE2

Transmission route of coronaviruses

Why are bats good reservoirs for diseases

Bats are known to carry many different strain of viruses but do not get sick from them
-Live around 30 years
-Shed in saliva, feces and aerosol
-Immune system tolerates and sustains high concentration of viruses
-A lot of contact with humans
-Bats are the only flying mammal
-Typically have high levels of IFNs and ISGS
-They can control their damage by increased HSP’s (heat shock proteins)
-Enhanced autophagy
- Dampened intracellular viral response
-Unique balance between host defense and immune tolerance allows them to be well while transmitting the virus

Possible origins of COVID-19 into humans

Two possibilities of COVID origin

natural origin (more plausable) or lab leak (no evidence)

Where was the early epicenter of COVID

Huanan Seafood Market
- live sars-cov-2 susceptible animals being sold
SARS-CoV-2-positive environmental samples were spatially associated with vendors selling live mammals

Why is COVID-19 so hard to control? (as compared to sars cov)

More transmissible, less pathogenic

What is R naught

R0 is defined as the average number of secondary transmissions from one infected person; when R0 is greater than 1, the epidemic is growing

Viral Entry of SARS-CoV-2

ACE2

Angiotensin-converting enzyme 2
-Present in many cell types and tissues including the lungs, heart, blood vessels, kidneys, livers and gastrointestinal tract
-ACE2 is present in epithelium in the nose, mouth and lungs as well as many other tissues including brain tissue. In the lungs, ACE2 is highly abundant in type 2 pneumocytes
-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
-Also, Type 1 progenitors

ACE2 function

Sars binding to ACEII causes activation of angiotensin II (regulates bp, etc), when SARS inhibits this it leads to inflammation, lung injury and leaky vessels

Entrance in sarscov2 vs sarscov1

CoV-2’s RBD hides more frequently instead of remaining standing up most of the time as CoV’s does. This allows for less immunological evasion and CoV-2 has an enhanced entry mechanism as well as being pre-activated by furin given more enhanced entry.

Sars-CoV-2 binding process

Stages of COVID

Asymptomatic State
Upper airway and conducting airway response
Hypoxia, ground class infiltrates, and progression to ARDS

Stage 1: Asymptomatic state (initial 1-2 days) COVID

Inhaled virus SARS-CoV-2 likely binds to epithelial cells in the nasal cavity and starts replicating. ACE2 is the main receptor for both SARS-CoV2 and SARS-CoV.

Stage 2: Upper airway and conducting airway response (next few days)

The virus propagates and migrates down the respiratory tract along the conducting airways, and a more robust innate immune response is triggered.

Stage #3 Hypoxia, ground glass infiltrates, and progression to ARDS

ARDS

The virus now reaches the gas exchange units of the lung and infects alveolar type II cells. Both SARS-CoV and influenza preferentially infect type II cells compared to type I cells .

SARS-CoV propagates within type II cells, a large number of viral particles are released, and the cells undergo apoptosis and die.

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. You can get pulmonary edema

COVID Pathogenesis

Affect of COVID on alveoli

loss of vascular integrity (1), activation of the coagulation pathway (2) and inflammation (3)

COVID Pathogenesis compared to other diseases

covid infects lower lungs

What causes multi-organ damage observed in COVID-19 patients

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.

Five main genes of COVID

S - Spike (receptor binding protein for ACE2)
E - Envelope
N - Nucleocapsid
M - Membrane
ORF - Open Reading Frame (2/3 of the genome)

What do ORF1a and ORF1b produce?

Two polyproteins pp1a and pp1ab

What non-structural proteins are made from pp1a

Nsp1-11

What non-structural proteins are made from pp1ab

Nsp1-10 and Nsp12-16

What is included in the NSPS

proteases, pols and endonucleases

Nucleocapsid

protein coat that contains RNA genome and appears to have other functions in immune inhibition.

Membrane

function to stabilize and maintain the viral envelope

Envelope

Not the standard envelope

E is abundantly expressed inside the infected cell, but only a small portion is incorporated into the virion envelope. Role in viral assembly, release and pathogenesis.

Spike Protein Structure

Accessory proteins of COVID

3a, 3c, 6, 7a, 7b, 8 and 9b

Where does the SARS lifecycle occur?

In the cytoplasm

What happens at/to the 5' end of the genome?

This is where the ORFs are.
Programmed ribosomal frameshifting generates 2 polyproteins encoding the replicase proteins.
- Slippery heptanucleotide make ribosome read thru past stop codon
- Pseudoknot can cause ribosome to pause and frameshift (moves back the reading frame by one and then continues reading, bypasses one stop codon)

What happens to the 3' end of the genome

As rdrp is reading through the genome it comes across TRS-B (preceding each gene) and jumps to synthesize TRS-L sequence so that each genome has a 5’ cap and 3’ poly-A tail

Subgenomic RNA step by step

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,

RTCS

Replication and Transcription Complexes (RTC’s)
- Double membrane vesicles
- Delivered from ER
- Induced by Coronavirus nsp 3, 4, and 6
- Role could be hiding in genome and concentrating factors for replication and transcription
- Elaborate network with RER
- Transcription and replication of covid genome
- Not assembly of virion
- Contains many NSPs

COVID Replication Cycle

When the 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. The ORF1a and ORF1b RNAs are made by genomic RNA, 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. Some nonstructural proteins form a replication/transcription complex (RNA-dependent RNA polymerase, RdRp), which use the (+) strand genomic RNA as a template. The (+) strand genomic RNA produced through the replication process becomes the genome of the new virus particle. Subgenomic RNAs 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. 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.

Early v. Late v. No IFN-I signaling

How does SARS evade the immune system?

SARS-CoV2 is very efficient at blocking immune response. NSP13 blunts intracellular signaling such as rig1 or the nucleocapsid itself. Other proteins are involved in blocking various downstream signals in the immune system which inhibits the immune system from recognizing SARS-CoV-2.

What antivirals exist

Nucleoside analogs: remdesivir and molnupiravir
Protease inhibitor: nirmatrelvir/ritonavir

How does remdesivir work?

it is a nucleoside analog

Molnupiravir

gets incorporated into burgeoning RNA strands and induces errors. The compound can shift its configuration, sometimes mimicking the nucleoside cytidine and sometimes mimicking uridine

Nirmatrelvir/Paxlovid

Nirmatrelvir is an oral viral protease inhibitor that is active against MPRO, a viral protease that plays an essential role in viral replication by cleaving the 2 viral polyproteins. Nirmatrelvir is packaged with ritonavir (as Paxlovid), a strong cytochrome inhibitor and pharmacokinetic boosting agent that has been used to boost HIV protease inhibitors. Coadministration of ritonavir is required to increase nirmatrelvir concentrations to the target therapeutic range

Positives of mRNA vaccines

mRNA vaccines can be manufactured in a cell-free manner
Rapid, scalable and cost-effective production
a 5 liter bioreactor can produce almost a million mRNA vaccine doses in a single reaction
a single mRNA vaccine can encode multiple antigens
mRNA is three to four orders of magnitude larger than molecules that readily diffuse into cells;

Why do mRNA vaccines require lipid membranes?

the dense negative charge of mRNA electrostatically repulses the anionic cell membrane, preventing its uptake.
Therefore, mRNA vaccines require a delivery vehicle that not only protects the nucleic acid from degradation but allows the mRNA to get into cells.

Available vaccines and their type

mRNA vaccines consist of...

In vitro-transcribed (IVT) mRNA contains five structural elements: a 5′ cap containing 7-methylguanosine linked through a triphosphate bridge to a 2′-O-methylated nucleoside, flanking 5′ and 3′ untranslated regions (UTRs), an open reading frame (ORF) and a poly(A) tail.

How are mRNA vaccines put into lipids

(1) Once the genome of a pathogen has been sequenced, a sequence for the target antigen is designed and inserted into a plasmid DNA construct. (2) Plasmid DNA is transcribed into mRNA by bacteriophage polymerases in vitro and (3) mRNA transcripts are purified by high performance liquid chromatography (HPLC) to remove contaminants and reactants. (4) Purified mRNA is mixed with lipids in a microfluidic mixer to form lipid nanoparticles. Rapid mixing causes the lipids to encapsulate mRNA instantaneously and precipitate as self-assembled nanoparticles. (5) The nanoparticle solution is dialysed or filtered to remove non-aqueous solvents and any unencapsulated mRNA and (6) the filtered mRNA vaccine solution is stored in sterilized vials.

How do LNP Vaccines work

•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

how do mRNA vaccines work

(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. (6) 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.

How do viral vector vaccines work

Human adenovirus (harmless) has spike protein

Regen-Cov process of creation

•IgG1 mAbs with unmodified Fc regions.
•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.
•Bind two distinct and non-overlapping sites on the RBD
•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.