MICR5832 L21: Dengue and HIV 12/13/25

What is this?

- (+) ssRNA enveloped flavivirus

-Four serotypes that cause severe/fatal disease

-Acute viral infection resolves in 4-6 weeks

Dengue

What is this?

-Dengue

- (+) ssRNA enveloped flavivirus

-Four serotypes that cause severe/fatal disease

-Acute viral infection resolves in 4-6 weeks

Why does Dengue have such a high mutation rate?

-RNA virus has no proofreading mechanism

-Error prone, high mutation rate

-Exists as quasispecies, clades, genotypes

What are some symptoms of dengue?

-Flu, fever

-Hemorrhaging and vascular leakage

-Hypovolemic shock (severe dengue)

What causes the hypovolemic shock associated with severe dengue?

-Blood vessels made leaky by inflammatory response

-Net fluid movement into tissues

Who is at risk for getting severe dengue?

-Secondary infection with heterologous serotype

-High endemicity country

True or False: If you get infected with one dengue serotype, you will be protected against it lifelong but only transiently cross-protected from other serotypes

True

Why does the onset of severe illness coincide with the end of the viraemic period?

-Viraemia symptoms are from immune response

-Severe illness after immune response is finished

What happens during peak viraemia of dengue infection?

-High fever + high viraemia

-IgM and IgG production

True or False: The viraemia period will be longer during a secondary heterologous Dengue infection

False, it will be shorter

True or False: The viraemia period will be shorter during a secondary heterologous Dengue infection

True, due to crossreactive antibodies from the primary dengue infection

Why is the viraemia period shorter during a second heterologous dengue infection?

Preexisting antibodies from the previous dengue infection that crossreact with the secondary serotype

True or False: The T cell response is stronger and faster during a secondary heterologous Dengue infection

True

What makes the T cell response stronger and faster during a secondary heterologous Dengue infection?

-Cross-reactive memory T cells

-Increases risk of severe dengue

What is this dengue phase?

-1 to 7 days after symptom onset

-Confirmed via PCR, NS1 antigen, serology and tissue tests

Acute Phase

What would you use to confirm an acute phase dengue infection?

-PCR, NS1 antigen, serology and tissue tests

-RT-PCR

What is this dengue phase?

-7 or more days after symptom onset

-Confirmed via tissue and serology tests

Convalescent Phase

What would you use to confirm a convalescent phase dengue infection?

-Serology and tissue tests

-IgM ELISA

What specimens would you use for a dengue diagnosis?

-Serum

-Plasma

-Blood (whole)

-CSF

Why would you not test for IgG in a high endemicity country to confirm dengue infection?

Everyone has some IgG antibodies due to high endemicity

What would you look for during primary dengue infection, in addition to NS1 antigen?

Presence of IgM

True or False: In a secondary dengue infection, there may be some PCR RNA but no NS1 antigen or low IgM

True

True or False: If a patient from a low endemicity country has IgG, they most likely had a previous dengue infection

True

True or False: An acute dengue infection is confirmed if the patient any of the following (PCR, NS1, or IgM) even in the complete absence of IgG

True

True or False: A secondary acute dengue infection is confirmed if the patient any of the following (PCR, NS1, or IgM) even in the complete absence of IgG

False, secondary acute infection must have IgG

What is this?

-Titer

How much you can dilute a sample and still neutralize the virus using antibodies

What is this?

-Immune serum is titrated against serotypes

-Performed in Vero cell monolayers

Plaque reduction neutralization test (PRNT)

What are some signs of confirmed dengue infection?

-Virus isolation

-Genome detection

-NS1 Antigen detection via ELISA

-IgM or IgG seroconversion

What are some signs of probable dengue infection?

-IgM positive

-Elevated IgG titre (i> 1280) by hemagglutination inhibition test

What are some signs of previous dengue infection?

-IgG positive

-Neutralization assays

What is this?

-End stage disease caused by HIV

-Chronic, life-long infection

-Viral RNA remains detectable in blood in the absence of ART

AIDS

What is this?

-Enveloped, ss (+) RNA virus with a DNA intermediate

-Replicates using a reverse transcriptase

Human Immunodeficiency Virus (HIV)

What kind of polymerase is the HIV reverse transcriptase?

RNA-dependent DNApol

What is the first step of the HIV replication cycle?

-HIV attaches to CD4 receptor, CXCR4, CCR5 receptors

-Membranes fuse, virion enters cell

What receptors and co-receptors does HIV attach to during fusion/entry?

-CD4 receptor

-CCR4 and CCR5 receptor

What happens after this step of the HIV replication cycle?

1) HIV attaches to CD4 , CXCR4 or CCR5 receptors

-Membranes fuse, virion enters cell

Reverse transcription of vRNA by viral reverse transcriptase, to dsDNA

What happens after this step of the HIV replication cycle?

2) Reverse transcription from vRNA to dsDNA

Transport of vdsDNA into nucleus

What happens after this step of the HIV replication cycle?

3) Transport of vdsDNA into nucleus

-Integrase integrates DNA into chromosome by viral integrase, forms provirus

What happens after this step of the HIV replication cycle?

4) Integrase integrates DNA into chromosome by viral integrase, forms provirus

-Transcription of viral mRNA via RNA Pol II

-Export from nucleus

What happens to RNA after it gets spliced?

-Unspliced mRNA = Genomic RNA

-Spliced mRNA = Translation to viral proteins

What happens after this step of the HIV replication cycle?

5) RNA Pol II transcribes mRNA and exports it from nucleus to be spliced

Assembly and budding

What happens after this step of the HIV replication cycle?

6) Assembly and budding

-Viral protease cleaves structural polyprotein to mature Gag protein

-New infectious virions

List the steps of HIV replication

1) Fusion and entry

2) Reverse transcription RNA-> dsDNA

3) Transport dsDNA into nucleus

4) Integrase adds DNA into chromosome

5) Transcription via RNAPol II, export from nucleus

6) Assembly and budding

7) Viral protease cleaves polyprotein -> Gag protein -> virions

What contributes to HIV-1 diversity?

-High mutation rates

-Low fidelity of reverse transcriptase, APOBEC proteins

-Retroviral recombination, template switching

-High viral turnover, immune response selection

What Group is the dominant form of HIV-1 worldwide?

M Group

Which Clade is the dominant form of HIV?

Clade C

What are some examples of emerging variants of HIV-1 from recombined clades?

-Circulating recombinant forms (CRFs)

-Unique Recombinant Forms (URFs)

True or False: Higher set points for HIV infection will result in faster AIDS progression

True

What phase of HIV is this?

-Infection of first cells

-Systemic spread via lymph nodes

-Interferon response

-Viral reservoir established

-GALT destruction

Eclipse phase

What phase of HIV is this?

-First detection in blood

-Some flu-like symptoms

-CTL response

-Seroconversion (binding antibodies)

Acute phase

What phase of HIV is this?

-Viral setpoint established

-CD4+ T cell loss

-Chronic inflammation

-AIDS progression

Chronic phase

Is this set point high or low?

-More fit virus

-CTL escape

-Immune dysfunction

-High levels of inflammation

High

Is this set point high or low?

-Poorly fit virus

-Strong immune response

-CCR5 heterozygosity

-Low levels of inflammation

Low

How do you detect HIV antigen?

-Immunoassay (IA) to detect HIV p24 protein

-PCR test for viral RNA or proviral DNA

How do you detect HIV antibody?

-ELISA screen test for anti-HIV Ab

-Western Blot tests for HIV-specific proteins

List all possible tests for HIV

-IA for HIV p24 protein

-PCR for viral RNA/DNA

-ELISA for antibody

-Combination antigen-antibody simultaneous test

-Rapid tests for anti-HIV Ab and p24 Antigen

What is this?

-Fourth generation HIV tests

-Antigen/antibody combination test

-2-6 weeks after exposure becomes positive

What is required for a confirmed Western Blot diagnosis?

-2 bands of glycoprotein, core protein, enzye

-p24, gp41, or gp120/160

True or False: If you do not have HIV, there will be no p24 band

False, non-infected people may have strong p24 protein bands but no envelope glycoproteins

How does Western Blot work?

-Break virus up with detergents

-Current separates proteins by size

-Transfer onto solid matrix

-Large core glycoproteins at top

-Small proteins (p24, p18) at bottom

What are disadvantages of Western Blot?

-Mistake HIV-2 for HIV-1

-Takes longer to confirm

-Practical limitations

How often should you test HIV-exposed infants?

-14 to 21 days

-1 to 2 months

-4 to 6 months

Why should you PCR test for HIV DNA or RNA in infants <18 months, instead of looking at antibodies?

Persistent maternal antibodies can give false positive results

When should you apply an HIV antibody test to uninfected infants?

-12-18 months

-Document seroconversion

True or False: After 3-6 months, it is reasonable to exclude HIV infection from a non-breast fed infant if negative in 2 months or more

True

What are the defined markers for AIDS?

-HIV infection

-CD4+ T cell count of <200 cells per ul

-AIDS-defining complication

List some AIDS-defining conditions

Hints:

1) Found in bronchi, trachea, esophagus, and lungs

2) Retinitis-associated

3) Human Herpesvirus 8

4) Respiratory disease

5) Pneumonia from fungi

6) Cats

1) Candidiasis/thrush

2) Cytomegalovirus

3) Kaposi's Sarcoma

4) Tuberculosis

5) Pneumocystis jirovecii

6) Toxoplasmosis of brain

Which fungal infections are associated with AIDS?

-Candidiasis

-Coccidiomycosis, Cryptococcosis, Cryptosporidiosis

-Histoplasmosis

-Pneumocystis jirovecii

True or False: MAC Complex (Mycobacterium avium) is an AIDS-defining condition

True

What are some possible requirements for protection against HIV-1?

-CD4+ and CD8+ CTL activity

-Plasmacytoid DCs and innate immunity

-NK cells

-ADCC

-Neutralizing antibodies