Virology Exam 2 Review

Ways to confirm viral infection (laboratory)

\-antigen/nucleic acid detection

\-serology

\-viral culture/cell culture

\-viral replication (cytopathic effects (CPE) or hemadsorption/hemagglutination

\-electron microscopy/light microscopy

Quality of Viral Diagnosis

depends on the specimen:

\-timing of specimen in relation to the patient’s illness

\-type of specimen (site of disease)

\-quality and amount of specimen

\-time and conditions of transport to the laboratory

Diagnosis by Viral Culture

\-traditionally relied on isolation of viral pathogens in cell cultures

\-slow process

\-was considered the “gold standard” for laboratory diagnosis of viral disease for decades

\-useful approach for testing when:

* viral titration is needed
* when viable isolate is needed
* if viable and nonviable virus must be differentiated
* when infection is not characteristic of any single virus (ex. when testing for only one virus is not sufficient)
* when available culture-based methods can provide a result in a more timely fashion than other molecular methods

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Systems for Viral Propagation

1. Animals
2. Embryonated eggs
3. Tissue culture (primary, secondary, and cell lines)

* cell culture = most common method

Steps in Viral Culture in Cells

1. Processing of specimen and inoculation in cell culture
2. Maintenance of the inoculated cell culture
3. Detection of viral growth

Detection of Viral Replication

\*how to detect the viral growth to determine if a virus is present and causing infection

\-detect cytopathic effects (CPE)- structural changes in a host cell, specifically in tissue culture resulting from viral infection

* generally, microscopically visible changes to the infected cell
* however some viruses grow to high titers and don’t produce visible CPE (ex. Rubella)

Ex. of Visible Changes

\-change in cell morphology

\-cell death (apoptosis, necrosis)

\-cell fusion (syncytia)

\-histologic changes (inclusion bodies)

\-cell surface changes (hemadsorption)

\-change in growth or lifespan

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\*Some CPEs are specific to a virus, which helps with identifying a specific virus

Viruses Causing Syncytium CPE

Paramyxoviruses

Herpes simplex virus (HSV)

Varicella-zoster

HIV-1

Virus Titration

\*quantification method of cytopathic effects in a local area of cells

\*cytopathic effects can be observed as a plaque or focus of infection

%%-Plaque assays- count infectious particles of cytolytic viruses (limited to a subset of animal viruses that can lead to cell lysis)%%

%%-Quantifies viruses that lyse or kill their host cells%%

Detection of Viruses by Electron Microscopy

\-not a standard clinical laboratory technique, used to detect certain viruses present in sufficient quantity

%%-useful when there is uncertainty regarding the identity of the virus%%

\-Advantages: speed, lack of requirement for viral viability, many different kinds of viral particles can be visualized

\-Disadvantages: cost and complexity of maintenance, need for a skilled operator, relative lack of sensitivity (need a high concentration of virus)

Detection of Viral Proteins and Nucleic Acid

\-standard of detection of viruses today

%%Proteins:%%

\-protein patterns (electrophoresis)

\-enzyme activities

\-hemagglutination and hemadsorption

\-antigen detection (ELISA, western blot, immunofluorescence, antigen rapid tests)

%%Nucleic Acids:%%

\-restriction endonucleases cleavage patterns

\-electrophoresis (segmented RNA viruses)

\-DNA genome hybridization (in situ hybridization)

\-Southern, Northern, and dot blots

\-Polymerase chain reaction (PCR, RT-PCR)

\*RT-PCR is either reverse transcriptase PCR or real time PCR, don’t confuse them

Antigen Detection of Proteins

\-detect the presence of a specific viral antigen which indicates current viral infection

used when viral antigen is expressed

\-an antibody is available

\-the antigen being detected is stable and is not degraded during transport and processing of the specimen



Ex. of a good viral antigen to detect = capsid antigen



\*Serological test = test of the serum in the blood; in the serum antibodies are present and the type that is present indicates what kind of infection a person may have

Hemadsorption/Hemagglutination

\-cells infected with influenza virus, parainfluenza virus, mumps virus, and togavirus express a viral glycoprotein (hemagglutinin, HA) on their cell surface that binds erythrocytes (hemadsorption)

\-in cell culture mediums, these viruses can cause agglutination of erythrocytes (hemagglutination)

\-the strain of virus can be identified by a specific antibody that blocks agglutination (hemagglutination inhibition)

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\*HA is the ligand protein for the receptor in a host cell

Immunofluorescence

\-labeling of antibodies or antigens with fluorescent dyes

\-substances labeled are studied with fluorescent microscope

\-fluorescein is a dye which emits greenish fluorescence under UV light

\-Two ways:

* direct immunofluorescence
* indirect immunofluorescence- uses a secondary fluorochrome antibody that recognizes the first unlabeled antibody that is specific for the viral antigen; better than direct immunofluorescence because it creates amplified results that are much easier to detect

Fluorescent Focus Assay (FFA)

\-indirect immunofluorescence assay; uses monoclonal antibodies (MAb)

\-employs __immunostaining__ techniques using __fluorescently labeled antibodies specific for a viral antigen__ to detect infected host cells and infectious virus particles

\-particularly useful for __quantifying viruses that do not lyse the cell membranes__, so detection isn’t just limited to lytic viruses like in the case with CPE

Western Blot

%%-laboratory method used to detect specific protein molecules from among a mixture of proteins%%

\-mixture can include all of the proteins associated with a particular tissue or cell type

\-Advantage = you can study size of viral proteins

\*proteins should run towards the positive pole in gel electrophoresis, not negative

Enzyme-Linked-Immunosorbent Assay (ELISA)

\-method of target antigen (or antibody) capture in samples using a specific antibody (or antigen)

\-method of target molecule detection/quantification using an enzyme reaction with its substrate

\*more sensitive than western blot, but western blot still gives you the size of the proteins (ELISA just detects presence of viral proteins)

Viral Serology

important component of diagnosis for certain viruses, particularly for viruses that cannot be readily cultured

\-used to identify the virus, evaluate the course of an infection, or determine the nature of the infection (primary or reinfection)

Ex. EBV, Rubella, HIV, HTLV, CMV, encephalitis viruses

\-chronic infections: the detection of antibodies of any isotype to these viruses indicates current infection__ and can identify the stage (early, late, etc.) of disease of the infection

Nucleic Acids: In situ Hybridization (ISH)

\-annealing of a labeled nucleic acid in fixed tissues, followed by visualization of the location of the probe

\-used to locate DNA sequences on chromosomes, or detect viral DNA or RNA

\-useful to detect latent viral infections

Southern Blot

detects viral %%DNA%%; studies %%structure%% of genes

Northern Blot

detects viral %%RNA%%; studies %%expression%% of genes

Blot Comparisons

Northern, Southern, and Western

Polymerase Chain Reaction (PCR)

%%-method to detect viral nucleic acid%%

\-amplified a piece of DNA very rapidly outside of a cell

\-three steps:

* DNA denaturation at 95 degrees C
* Primer annealing at 50-60 degrees C
* DNA polymerization by a thermostable DNA polymerase at 72 degrees C

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%%\*PCR only amplifies DNA so with RT-RNA viruses, the RNA is converted to cDNA which can then go through RT-PCR and study the genes in the new DNA strand%%

PCR Terminology

PCR = DNA



qPCR = quantification of DNA



RT-PCR = RNA to start and then conversion and analysis of cDNA



qRT-PCR = quantification of your RT-PCR

Discovery of Interferon

\-1957

\-Issacs and Lindenman were studying viral interference: mechanism whereby infection with virus A inhibits infection with virus B

\-Experiments revealed natural substance with antiviral activity (“interferon”)

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Isaacs and Lindeman’s Interferon Conclusion

\-a soluble factor, “interferon'“, was produced by cells as a result of virus infection

%%-interferon could prevent or interfere in the infection of other cells%%

%%-the inactivated virus triggered the infected cells to produce something that suppressed replication of the live virus%%

\-since the virus was not active, it was simply the detection of a viral presence that induced the production of interferon

\-interferon soluble factor was actually a %%protein%%

%%-first discovered cytokine%%

Cytokines

%%-broad category of peptides (small proteins) that allow cell-cell communication in immune responses%%

\-enter cell cytoplasm by specific membrane receptors

\-labeled as immunomodulating agents

%%-direct the development, maturation, localization, interactions, activation, and life span of immune cells%%

\*among them there are chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor

Types of Interferon

\*based on the type of receptor through which they signal

%%Type I:%% cytokines that %%play essential roles in inflammation, immunoregulation, tumor cell recognition and T cell responses;%% @@produced when the body recognizes that a virus has invaded it@@ (bind interferon-alpha and interferon-beta receptors); their release leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA

%%Type II: released by cytotoxic T cells and CD4 T helper cells%%, NK cells, macrophages, and dendritic cells (DC)(interferon-gamma)

Interferon Functions

\-signaling proteins released by host cells in response to the presence of microbes (ex. viruses and bacteria)

%%-alert nearby cells to increase their anti-viral defenses%%

%%-activate immune cells (ex. NK cells and macrophages); increase host defenses by up-regulating antigen presentation via increasing MHC antigen expression%%

* higher MHC 1 expression: increases presentation of viral and abnormal peptides from cancer cells to cytotoxic T cells (CD8 T cells) thereby increasing the recognition and killing of infected or malignant cells.
* higher MHC 2 expression: increases presentation of these peptides to helper T cells (CD4 T cells) that release cytokines (such as more IFNs and interleukins, among others) that signal and coordinate the activity of other immune cells.

\*some viruses have encoded elements that combat the IFN response and allow the virus to continue replicating

Biological Activities of Interferons

n/a

Interferon Signaling Steps

n/a

How Interferon Stops Viral Replication in Cells

n/a

How Viruses Resist Interferon

\*anti-IFN strategies include:

\-inhibiting IFN induction

\-blocking key mediators of the interferon response

Hepatitis

caused by HBV, HCV

Genital Warts

caused by HPV

Kaposi’s Sarcoma

caused by KSHV (HHV-8)

Characteristics of Antiviral Agents

Good:

\-metabolic stability

\-ready transport to infected target organs

\-chemical and thermal stability

\-good solubility at or close to physiologic pH

\-low cost of preparation

Bad:

\-acute toxicity

\-chronic pathogenesis effects (ex. teratogenesis, mutagenesis, carcinogens, etc.)

\-immunosuppression

\-incorporation into DNA of the noninfected cell

\-development of resistant mutant

\-activation of latent viruses

Antiviral Agents: Inhibitors of Attachment/Viral Fusion

\-neutralizing antibodies

\-HIV co-receptor binding inhibitor CCR5 inhibitor (Maraviroc): specifically blocks binding of HIV to cell receptor

\-Erfuvirtide (Fuzeon)- anti-HIV drug that inhibits the virus’ ability of fusing its envelope with the whole cell membrane, thus blocking its replication at the entry phase

Antiviral Agents: Block Penetration and Uncoating

For Influenza A:

@@-Amantadine and Rimantadine@@ buffer contents of endosomal vesicle, %%inhibit acid-mediated fusion of viral genome%%, block proton channels

@@-Xoflusa@@ inhibits cap-dependent endocnuclease activity of the viral polymerase, %%blocking the polymerase of the virus → thus blocking replication%%

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For Picornaviruses: Pleconaril, Arildone, Disoxaril, and other methylisoxazole compounds %%block uncoating by fitting into a cleft in the receptor-binding canyon of the picornavirus capsid%%

Antiviral Agents: Inhibiting Transcription

\-Interferon: inhibits transcription in Hep A/B/C and papillomaviruses

* degrades viral mRNA
* blocks mRNA binding to the ribosome
* triggers a cascade of biochemical events that blocks viral replication

\*interferon is species specific; antibodies are viral specific

\-Antisense Oligonucleotides: inhibit transcription in papillomaviruses and cytomegalovirus, which causes retinitis

Antiviral Agents: Nucleoside Analogues

%%-inhibit viral polymerases and thus replication processes%%

\-resistance to such drugs is usually caused by a mutation of the viral polymerase

Ex. Ribavirin = inhibitor of nucleoside biosynthesis; drug resembles guanosine monophosphate, and inhibits mRNA capping, and other processes important to viral replication

Antiviral Agents Against Hepatitis C

n/a

COVID Antivirals

Remdesivir: nucleotide analog;

\-Paxlovid: combination of two drugs

* nirmatrelvir- blocks protien in SARS-Cov-2 that makes the virus unable to reproduce itself
* ritonavir- stops the breakdown of nirmatrelvir so drug levels stay high

\-Molnupiravir: gets into the RNA of the SARS-Cov -2 and causes errors in the replication

Inhibitors of HIV Maturation

n/a

Inhibitors of Influenza Maturation

n/a

Types of Immunity

Humoral: most important for prevention of viral infections (antibodies)

Cellular: most important for eradication of an established viral infection and viral disease (T-cells)

Response to Viral Infection

%%-Interferon and IgA are the first responders%% to a beginning viral infection

\-As the virus travels throughout the body, antibodies are recruited to stop their spreading

\-When viruses reach their target organs, cells, complement, antibodies, and interferon are all present trying to stop the infection

Response to Viral Infection Over Time

\*NK cells typically kill tumor cells or virus-infected cells

Humoral Immunity

n/a

Cell-Mediated Immunity

Interactions Between Type 1 and Type 2 T Helper (CD4+) Cells

\-TH1 T cells release IL-2 to go activate and proliferate CD8 T cells that kill virus-infected cells

* IL-2 also activates more TH1 T cells to amplify the overall response; it also downregulates TH2 T cells

\-TH2 T cells releases IL-4 and IL-10 to trigger proliferation and maturation of antibody-producing B cells; antibodies neutralize viruses

* Il-4 and IL-10 downregulate TH1 activity

Viral Escape Mechanisms

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Mechanisms of Virus Induced Cell and Tissue Injury

\-direct injury

\-indirect injury

* immediate hypersensitivity
* antibody mediated cellular cytotoxicity
* immune complexes
* cell-mediated cytotoxicity

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Ex. lymphocytic choriomeningitis virus infection & glomerulonephritis

How Immune System Controls Viral Infections

%%-monoclonal antibodies:%% homogenous population of antibodies produced by a single clone of plasma B lymphocytes

* artificially made in laboratories against a specific antigen in order for them to bind to a particular antigenic molecule target

\*recall differences between monoclonal and polyclonal

%%-vaccines%%

Effective Vaccines

\-promote herd immunity

Inactivated Viral Vaccine

\-type of vaccine in which a virus is killed without its structure being destroyed

Live Attenuated Vaccines

\-pathogenic virus is isolated from a patient and grown in human cultured cells

\-cultured virus is used to infect monkey cells

\-virus acquires many mutations that allow it to grow well in monkey cells

\-virus no longer grows well in human cells (attenuated) and can be used as a vaccine

Other Types of Viral Vaccines

\-recombinant vaccines

\-%%subviral particle vaccines:%% instead of whole virus being given, only a portion of the virus is given (ex. only giving RNA or DNA of the virus)

\-%%anti-idiotypic vaccines:%% something binds to the something that would usually bind, to prevent it from doing so

\*idiotypic = binds to something

Problems with Vaccine Use

\-live vaccine can occasionally revert to virulent forms

\-interference by other other organisms’ biology may prevent live virus from producing “infection” to boost immunity

\-using live vaccine on immunocompromised person can be life threatening

\-side effects to vaccination can occur (ex. hypersensitivity, allergic reactions to antigen, etc.)

\-expensive development

\-viruses with many serotypes are difficult ot control w/ vaccination

First Vaccine

vaccinia for small pox

Modes of Delivery of Recombinant Subviral Vaccines

Picornaviruses (poliovirus)

\-naked, nonenveloped

\-single-stranded

\-perfectly icosahedral

\-single-stranded positive RNA genome (direct replication)

\-replications in cytoplasm of cells, which is common of RNA viruses)

\-mostly cytolytic (breaks host cell membrane to escape during proliferation)

\-include enterovirus (among which is poliovirus), rhinovirus, and heparnavirus (hep A virus)

Picornavirus Replication

\-the virus genomic RNA is like an mRNA but with no cap

\-virus shuts down host cellular protein synthesis by inactivating cap translation initiation factor

\-host translation system becomes cap independent and cell is unable to translate its own mRNA

\-viral genome contains internal ribosome entry site (IRES) that directs ribosomes to initiate translation that is independent of cap structure and allows viral mRNA to exclusively be translated

Poliomyelitis Pathogenesis

\-infection usually through ingestion, not as much through the nose

Prodrome

time before the major symptoms of a sickness occur (you’re starting to feel sick but haven’t developed full-blown symptoms)

Herpangina

\-type of clinical syndrome associated with Coxsackie a/b viruses (enteroviruses)

\-characterized by fever, sore, throat, pain on swallowing, anorexia, vomiting

\-vesicular lesions on mouth soft palate

Pleurodynia (Bornholm’s Disease)

\-type of clinical syndrome associated with Coxsackie A/B viruses

\-characterized by sudden onset of fever and unilateral low thoracic chest pain that may be excruciating

\-lasts approximately 4 days

\-”devil’s grip”

Hand-Foot-Mouth Disease

\-type of clinical syndrome associated with Coxsackie A/B viruses

\-vesicular lesions on the hands, feet, mouth, and tongue; subsides in a few days

Pathogenesis of Enterovirus Infection

\*review picture\*

\-transmission of enteroviruses is usually from human fecal matter to sewage, solid waste landfills, or to one’s hand and eventually into the person themselves

Diagnostic of Enterovirus Infections

\-isolation of virus in cell culture using appropriate specimens (ex. stool, throat swabs, cerebrospinal fluid)

\-serological testing (ELISA and neutralization assay)

\-molecular detection via PCR

Immune Responses to Enterovirus

Treatment, Prevention, and Control of Polio Infection

\*come back to review these bullet points

Rhinoviruses

\-interferon may limit the infection and contribute to the symptoms

\-immunity to rhinoviruses is transient and unlikely to prevent subsequent infection

* rhinoviruses are localized to the nose and throat which is why immunity is transient

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\*transient

Epidemiology of Rhinovirus Infections

n/a

Coronaviruses

\-enveloped

\-single-stranded,

\-non-segmented RNA viruses

\-Genomic RNA is capped (contrast with picorna)

\-genome acts as an mRNA and is infectious (positive-strand RNA)

\-spike protein = viral attachment protein

Detection of Coronaviruses

\-initially detected by EM

\-ELISA (detect antibodies)

\-nucleic acid hybridization

\-RT-PCR

\-plaque assay

\-immunofluorescence

Coronaviruses-Cell and Tissue Tropism

\*review this slide\*

Coronaviruses: Disease and Immunity in Humans

n/a

Severe Acute Respiratory Syndrome (SARS)

\-in the late winter and spring of 2003 a new illness (atypical pneumonia) emerged in China and Singapore known as severe acute respiratory syndrome (SARS)

\-SARS-CoV was identified as a novel coronavirus

SARS-Background

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Middle East Respiratory Syndrome (MERS)

\-type of coronavirus causing fever, cough, and shortness of breath

\-found in Middle East w/ first death recorded in 2012 in Saudi Arabia

\-camels suspected as primary source of infection for humans

\-at least 1026 recorded infections and 376 deaths since 2012

Coronavirus Pandemic (COVID-19)

\-Coronavirus appeared in Wuhan, China and spread around the world

\-105,394,979 cases about 1.15 million deaths

\-New strain is named SARS-CoV2

Signs of Disease w/ SARS-CoV2 strain

\-respiratory symptoms

\-fever

\-cough

\-shortness of breath

\-breathing difficulties

\-more severe cases: pneumonia, severe acute respiratory syndrome, kidney failure, and even death

Treatments Against COVID-19

\-monoclonal antibodies against the COVID-19 spike protein (mild to moderate cases)

* Bamlanivimab, Casirivimab and Imdevimab are monoclonal antibodies against different portions of the spike protein

\-antiviral agents

* remdesivir- nucleotide analog

More Drugs for COVID-19

n/a

Vaccines Against COVID-19 Approved in USA

n/a

What are the Arboviruses?

flaviviruses, togaviruses, bunyaviruses, and arenaviruses

Arboviruses

\-enveloped

\-single-stranded

\-segmented genome: bunyaviruses and arenaviruses

\-non-segmented genome: togaviruses, flaviviruses

Arboviruses: Flaviviruses and Togaviruses

\-positive-strand RNA viruses

\-enveloped

\-most enveloped +strand RNA viruses are transmitted by arthropods, thus the name __arbo__virus

\*__ar__thropod __bo__rn viruse = arbovirus

Togaviruses

\-enveloped viruses

\-icosahedral capsid

\-positive-sense single-stranded RNA genome

\-replication includes early and late (non-structural and structural) protein synthesis

\-replication occurs in the cytoplasm and the viruses bud at the plasma membrane

\-two subspecies: alphaviruses (causes disease in humans and animals) and rubivirus (rubella)

Arboviruses→Togaviruses→Alphaviruses

\-insect-borne

\-cause mostly mild and rare diseases in humans

\-sindbis virus is very well studied prototype

Arboviruses→Togaviruses→Rubivirus

\-rubella: one of the 5 childhood exanthems

\*exanthems = rash causing infections

Togavirus Replication

\-more complex than picornaviruses

\-cells need to maintain their structure to allow continuous budding of new viruses

\-less profound shutoff of host cell functions until a long time after infection

\-cells alter their surface (bud out or fold in different ways) to fuse with neighboring cells (syncytia)

* this allows virus to spread without lysing host cells

\*can be severe but have favorable prognosis

\*virus does not replicate in neurons but in cells of the brain’s protective surfaces; doesn’t kill neurons but can cause other effects

Many Alphaviruses are spread via which vector?

mosquitoes

Alphavirus: Chikungunya Virus

\-positive singe-stranded RNA genome

\-causes sudden onset fever (2-7), rash, and joint pains, lasting weeks or months and sometimes years

\-mortality rate is around 1 in 1000

\-transmitted by Aedes aegypti and Aedes albopictus mosquitoes

\-discovered to occur in Africa and Asia and spread since 2013 to America

Arboviruses: Flaviviruses

\-enveloped icosahedral viruses

\-linear positive-sense non-segmented single-stranded RNA genome

\-70 types, 13 can cause disease in humans (overall infect a broad range of species including vertebrates and invertebrates like mosquitoes and ticks)

\-disease ranges from febrile illnesses to life-threatening hemorrhagic fevers, encephalitis, and hepatitis

\-dengue, yellow fever, Japanese encephalitis, St. louis encephalitis = among the most important viral pathogens of the developing world

\-Dengue and yellow fever are the worst and most common types of flaviviruses; often more in monkeys

Flavivirus: West Nile Virus (WNV)

\-influenza-like illness endemic to Africa, the Middle East, and Western Asia

\-neurological illness more common in older patients (meningitis or encephalitis being the most frequent)

\-associated with polio-like illness with flaccid muscle weakness in severe cases

\-no available anti-viral treatment

Important Disease Associated with Flavivirus

\-encephalitis, fever, arthritis, and rash

\-yellow fever and dengue cause hemorrhagic fevers and potentially lethal syndromes

\*dengue hemorrhagic fever: bleeding from all body orifices

\*yellow fever: liver major target, massive necrosis of hepatocytes

\*flavi= yellow

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Flavivirus: Dengue Virus

hemorrhagic fever: generally observed during epidemics of dengue in a population with a previous history of infection with another dengue serotype

\-majority of cases: children who previously acquired antibodies against another type