FINALS 1_General Characteristics of Viruses, Viral Replication & Laboratory Diagnosis of Viral Infections

- most deadly viral pandemic in 1918

- also known as the Spanish flu

Influenza A virus (H1NI)

- causative agent of AIDS

- first identified in 1981

- continues to devastate entire continents

Human immunodeficiency virus (HIV)

➔ introduce into North America in 1999 ➔ resurgence in 2012

West Nile virus (WNV)

- causative agent of dengue fever

- more than 1 billion at risk of dengue infection worldwide

- one of the most concerning member of the family Flaviviridae

- mosquito bites - continuously spread dengue

Dengue virus

- began in late 2002 and spread rapidly worldwide in 2003

- high mortality rate

SARS-CoV-1-infections

- outbreak on the island of Yap (Micronesia) was reported in 2007

- In Brazil, the outbreak started in November 2015

- In 2016, subsequent spread to the Americas, there was a link to microcephaly in neonates

Zika virus

- first emerged in Mexico and rapidly spread to other countries

- Influenza variant, H3N2, affected humans and swine throughout the USA

- annual flu vaccine - successful prevention of flu pandemic outbreaks

Influenza A virus subtype H1N1 (2009)

- emerged in Saudi Arabia in 2012 and later spread to 27 countries - 2000 cases and 866 deaths

Middle East respiratory syndrome coronavirus (MERS-CoV)

- Caribbean countries and territories of the US in late 2013

- reported in Florida, Puerto Rico, and the US Virgin Islands

Chikungunya virus

➔ in West Africa started in 2014 to 2016 ➔ over 28,000 cases in 10 countries

Ebola outbreak

- China in late 2019

- cases of atypical pneumonia

- July 2022, WHO reported 544,324,069 deaths

SARS-CoV-2

- In 2022 caused outbreaks in several non-endemic countries including the USA

- Sporadic outbreaks reported in Africa since 2003

- closely related to smallpox virus, smallpox virus vaccine effective in preventing monkeypox infection

Monkeypox

- killed hundreds of children on the Asian continent

- Influenza virus and other viruses lead to hundreds of thousands of deaths globally

Enterovirus 71(EV71)

- In 2014, it caused severe illness among humans and horses in the southern Philippines

- Fatality rates among humans were high.

- Horse-to-human and human-to-human transmission occurred

- The most likely source of horse infection was fruit bats

Henipavirus infection

- DOH declared in August 2019

- over 350,000 cases of dengue and more than 1,300 related deaths have been reported

- It is the worst dengue epidemic the country has seen in a decade

Dengue Epidemic

more than 42,000 reported cases of and 560 deaths since the Department of Health (DOH) declared a epidemic outbreak in February 2019

measles

- March 2019, the DOH confirmed the re-emergence of 19 years after its eradication.

- A third case of __ was confirmed this week

polio

T/F:

VIRUS:

- Smallest known infective agents

- Simplest form of life

TRUE

VIRUS:

- possess cellular organization

- Fall strictly as unicellular microorganism

A. 1 only

B. 2 only

C. Both are correct

D. Neither is correct

B

VIRUS:

- Contain either RNA or DNA but NOT BOTH

- Obligate intracellular parasite

A. 1 only

B. 2 only

C. Both are correct

D. Neither is correct

C

VIRUS:

- Has enzyme necessary for protein and nucleic acid synthesis

- Multiply by a binary fission and NOT by complex process

A. 1 only

B. 2 only

C. Both are correct

D. Neither is correct

D

*lack enzyme

*complex process, binary fission

T/F

VIRUS: Unaffected by bacterial antibiotics

TRUE

Nuclear Material of virus

RNA or DNA, ss or ds

genome and its protein coat together

Nucleocapsid

entire virus particle

Virion

- sometimes surround the capsid

- originate from the host membrane, but proteins are virally encoded

- masks the shape of the virion

- phospholipid bilayer with adhesion molecules

Enveloped viruses

enveloped virus are typically susceptible to

- inactivation by high temperature,

- extreme pH,

- chemicals

morphology of virus

- Helical

- Icosahedral

- Complex

- Spherical

- Polyhedral

(morphology)

Tobacco mosaic virus

Helical

(morphology)

20 triangular sides

Icosahedral

(morphology)

bacteriophage

Complex

(morphology)

influenza

Spherical

(morphology)

adenovirus

Polyhedral

now, viruses are classified in:

- Orders

- Families

- Genera

- Genome type

- Number of strands

- Morphology

- Presence/absence of envelope

virales

a. orders

b. families

c. genera

A

virus

a. orders

b. families

c. genera

C

viridae

a. orders

b. families

c. genera

B

viral replication:

STEPS OF INFECTION

1. Absorption

2. Penetration

3. Uncoating

4. Eclipse/Replication

5. Assembly

6. Release

- Specific for cell receptors via viral adhesion molecules

- Most host cell receptors are glycoproteins

Absorption or attachment

determine the cell receptor of the poliovirus

immunoglobulin superfamily molecules

determine the cell receptor of the Rabies virus

acetylcholine

determine the cell receptor Influenza virus

sialic acid

determine the cell receptor HIV

CD4

determine the cell receptor Epstein Barr virus (EBV)

complement receptor c3d

determine the cell receptor SARS-CoV-2

angiotensin-converting enzyme 2

penetration of naked viruses

Direct penetration of the membrane

penetration of envelope virus

- Fusion with the plasma membrane

- Endocytosis

what steps/process of infection?

RNA viruses usually release the genome into the cytoplasm whereas most DNA viruses release their genome into the host nucleus.

uncoating

what steps/process of infection?

- Copies genome and produces protein - depending on the virus, metabolism of the host cell may be completely stopped (polioviruses) or may continue at reduced scale (influenza viruses)

Replication and translation

(assembly/maturation of the virus)

The capsid protein subunits aggregate to form ___

capsomers combine to form the ___

capsomers combine to form the ____

capsomers

capsid

nucleocapsid

(release or egress)

The new virions are then released by ___ if they are NAKED VIRUS or by ____ if they are ENVELOPED VIRUSES.

lysis, budding

Viral culture and identification using mammalian cell culture

Full service virology laboratories

- detects specific viruses

- These tests can involve the detection of viral antigens by using a number of methods such as:

1. immunofluorescence (IF)

2. enzyme immunoassay (EIA)

rapid tests

- Helps to identify variants of the same virus

- To identify the virulent and more dominant strains

Polymerase Chain Reaction (PCR)

- determining the patient's immune response to viruses— rather than detecting the viruses directly

- Very useful but the TAT is several weeks

Viral serology

viral serology usually takes ____ after infection before these antibodies are produced, which may mean that treatment would be too late or not needed

3 to 4 weeks

When are viruses most likely to be present?

Right before and at onset of symptoms

Sensitivity of viral culture can decrease rapidly_____ after the acute onset of symptoms

3 days

Swabs must be made of

Dacron or rayon

T/F:

NO calcium alginate swabs should be use bc it inhibits the replication of some viruses and can interfere with NAATs

TRUE

- Tissue samples should be kept moist and

- must be placed in transport media unless designed for viral preservation

A. 1 only

B. 2 only

C. Both are correct

D. Neither is correct

A

can be added to sterile containers to keep tissues from drying

Viral transport medium, saline, or trypticase soy broth

Most transport media consist of a buffered isotonic solution with a protein such as:

1. Albumin

2. Gelatin

3. Serum

Specimens are collected WITH Viral Transport Media are

Respiratory

Swab

Tissue Sample

Specimens are collected WITHOUT Viral Transport Media are

Blood

Bone Marrow

CSF

Amniotic Amniotic fluid Urine

Pericardial fluid

Pleural fluid

T/F:

Viral specimen for culture

Samples are best cultured immediately.

TRUE

(Viral specimen for culture)

if specimen not processed immediately, temp for storage

Refrigerate if necessary at 4C

(Viral specimen for culture)

if longer than 4 days

Freeze at -70°C

T/F

Viral specimen for culture should be stored at -20°C

FALSE

*never

(Viral specimen for culture)

Should never be stored at -20°C because it facilitates formation of ____

ice crystals

(Viral specimen for culture)

T/F:

Repeated freeze-thaw must be avoided it results in loss of viral variability

TRUE

Incorrect or poor specimen collection - ______ diagnostic result

a. false negative

b. false positive

A

identify the specimen for these site/infection:

1. Respiratory mucosa

2. Lesions

3. Intestinal mucosa

1. Respiratory secretions (NPS)

2. Aspirate or surface swabs

3. Stool

identify the specimen for these site/infection:

Systemic, congenital or generalized disease

Blood (buffy coat), CSF, Portals of entry (oral or respiratory tract) Exit (urine or stool)

(respiratory)

appropriate specimen for

Croup

Bronchiolitis

Pneumonia

Nasal aspirate

NP/Throat swab

BAL

(Cutaneous)

appropriate specimen for Gastroenteritis

Stool

Rectal swab

(Gastrointestinal)

appropriate specimen for

Lesion & exanthems (rashes)

Vesicle aspirate

Lesion swab

(Ocular)

appropriate specimen for

Conjunctivitis

Keratitis

Conjunctival scrapings

Corneal swabs

(CNS)

appropriate specimen for

Encephalitis

Aseptic meningitis

CNS

Blood

NP Swab Stool,

Urine

(Genital)

appropriate specimen for

Urethritis

Vesicle

aspirate

or swab

(Congenital)

appropriate specimen for Lesions Exanthems (rashes)

Throat

Urine

Serum

(Disseminated)

appropriate specimen for Multiple

Multiple sites

3 major methods in diagnostic in virology

Direct detection of virus in clinical specimen

Isolation of the virus in cell culture

Serologic assays to detect viral antibodies

Serologic assays to detect viral antibodies May take how long to make antibodies?

3-4 weeks

-are not as sensitive as culture methods but can offer quick results to allow rapid therapy

-many of these tests can be performed in a few minutes

Direct detection methods

- from cell scrapings (alteration, disruption, damage to cells)

- Can be detected using Bright-field microscopy

Cytopathic effect (CPE)

- most common method

- Can detect Cowdry type A bodies from HSV and VZV

- Scraping of an ulcer base

Tzanck smear

- can reveal Human papillomavirus (HPV)-associated koilocytes

Papanicolaou ( Pap) smears

sometimes diagnosed by detecting Negri bodies, which are eosinophilic cytoplasmic inclusions in neurons

Insensitive method for diagnosing rabies

Rabies

- Valuable tool to detect viral antigens

- Fluorophore-labeled antibodies against viral antigens

- Direct visualization of viruses and infected cells

- Some tests amplify signals to increase sensitivity

Antigen Detection

- Cells from the patient fixed to a microscope slide

- Fluorescence-labeled antibodies are added

- If the viral antigens are present in the sample, the labeled antibody will bind

- Fluorescence will be seen using Fluorescent microscopy

Direct Fluorescent Antibody (DFA) tests

direct visualization, amplify signals, enhance sensitivity

IF-labeled Abs

tests for viral detection are commercially available, with most using multiwell microtiter plate assays

Enzyme Immunoassays

(Enzyme Immunoassays)

what virus can you detect from respiratory specimen?

RSV and influenza A virus

(Enzyme Immunoassays)

what virus can you detect from serum/plasma specimen?

Hepatitis B virus (HBV) and HIV-1

(Enzyme Immunoassays)

what virus can you detect from stool specimen?

enteric adenoviruses

(Enzyme Immunoassays)

what virus can you detect from cutaneous lesions and conjunctival swabs specimen?

HSV

(Enzyme Immunoassays)

what virus can you detect from rectal swabs specimen?

Rotavirus, enteric adenovirus

advantages of nucleic acid-base detection

- Faster TAT

- Better sensitivity

- Quantitative

- For nonculturable (Norovirus and Hepatitis)

- Simultaneously (multiplex)

- Genotyping

disadvantages of nucleic acid-base detection

- Detect active and inactivated virus

- Higher cost

- Skill

- More complex

- Lack of approved assay