ZIKV

Outline

• Introduction
• History & Epidemiology
• Dangers of ZIKV & Pregnancy
• Global Issue
• Current Diagnostics
• Current Sample Prep & Detection Methods
• Potential in ZIKV

Current Sample Prep. Techniques

One-Step RT-PCR

Saliva

Urine

• Method established in 2008, no applications thus far
• Purified RNA after extraction - RT-PCR - Gel electrophoresis
• Designed primers to target conserved regions across 37 strains, 92- 99% similarity to ZIKV parent strain (Uganda)

• Based on 182 patients exhibiting symptoms
• Serum + Saliva collected for comparison
• Viral RNA extraction + RT-PCR

• Based on 6 patient samples
• 200ul of serum and urine (comparison)
• Viral RNA extraction + RT-PCR
• Standard curve plot to estimate viral load
• DENV & CHIKV ruled out

• Higher viral load in urine
• Remains detectable for about 15 days upon onset of symptoms (>7days after it was no longer detectable in serum)
• Isolate and culture viruses from urine; failed

Introduction to ZIKV

• 19.2% positive in saliva but negative in serum
• 8.8% positive in serum but negative in saliva

• Immune system works to eliminate viruses through excretions (urine, feces etc)
• Live viruses present in urine??

• Detection of viral RNA for 1st week of onset
• No extension of detection window (unlike urine)
• Might be unreliable, can only serve as complement

• Mosquito-borne virus
• +ve single-stranded RNA genome
• Flaviviridae (same family as DENV)

• Detection limit similar to other flavivirus assays (337 pfu/ml)
• Low limit of detection from 10^3 to 10^6 pfu/ml

Gourinat, A.C. et al. (2015). Detection of Zika Virus in Urine. Emerging Infectious Diseases, 21(1), 84-86.

Musso, D. et al. (2015). Detection of Zika Virus in Saliva. Journal of Clinical Virology, 68, 53-55.

Faye, O. et al. (2008). One-Step RT-PCR for Detection of Zika Virus. Journal of Clinical Virology, 43, 96-101.

• Share the same vectors as DENV & CHIKV
• Transmission - primarily Aedes aegypti (others??)
• ~80% of ZIKV infected people are asymptomatic

Introduction to ZIKV

Symptoms of Infection

• Mild; self-limiting
• Acute onset of low-grade fever (<38.5)
• Maculopapular rash (face then body)
• Arthralgia (joint-swelling)
• Conjunctivitis
• Neurological complications - Guillain-Barré Syndrome

Very similar to dengue, misdiagnosis?

Current Methods of Diagnosis (CDC, WHO)

Current Methods of Diagnosis (CDC, WHO)

• Diagnosis for fetuses?
• Real-time RT-PCR of amniotic fluid possible (sensitivity & specificity unestablished)

• Amniocentesis associated with 0.1% pregnancy termination (<24 weeks)
• NOT recommended for <14 weeks
• Clinicians must weigh pros & cons
• Testing of asymptomatic women not adviced

Potential of ZIKV

• Absence of commercial kits (ZIKV-specific Ab)
• No gold-standard diagnostic tools except molecular diagnostics

• Brazil - public health emergency; diagnostic kits in development
• Lateral flow immunoassays are definitely possible although anti-ZIKV IgM are not commercially available (yet?)

Impending Challenges

• Rapid test kit
• Cross-reactivity of antibodies
• Extraction and purification of viral RNA outside a lab

The Danger of ZIKV & Pregnancy

• Infection can occur at ANY trimester
• Transplacental transmission or during delivery
• Spontaneous abortions possible
• Microcephaly (reduced brain size)

• Association of ZIKV with microcephaly and fetal deaths not proven (ongoing)

• Are pregnant women particularly susceptible to ZIKV?

ZIKV - A Global Issue

• No vaccines, anti-virals or prophylactics
• Non-fatal to mother; irreversible damage to baby
• High rates of undiagnosed or misdiagnosed cases
• Little is known about ZIKV (tip of the iceberg)

• Singapore - study found that local bred mosquitoes are just as susceptible (A. albopictus) - BAD NEWS
• Immunologically naive (no herd immunity)

History & Epidemiology

• Global - pandemic potential given forecasted distribution of mosquitoes (El Nino)
• Detrimental to economies

• First identified in Uganda, 1947
• 2007 epidemic - Yap Islands (Micronesia)
• 2013 & 2014 epidemic - Brazil & the Americas

• North-eastern Brazil; greatest hit due to high population
• 2015 Oct-Nov - Colombia, Guatemala, El Salvador and Mexico
• 2015 Dec - Puerto Rico reported first case

History & Epidemiology

• >4000 cases since July 2015
• Accelerating at 20x the normal rate
• Classic symptoms first, neurological complications later (viral mutations?)
• ZIKV - more neurotropic than DENV & CHIKV

• Wave of infections across the Americas
• S.E.A - sporadic cases in Indonesia, Cambodia & Thailand
• First imported ZIKV case - Thai man travelled to Taiwan (Jan 2016)

Zika Virus (ZIKV)