Molecular Detection and Characterization

Virus isolation and molecular characterization by real-time RT-PCR and RT-PCR and sequencing.

• Viral Isolate/Culture - = 0.5 mL of infected cells and media.
• CSF - Not recommended as polioviruses are rarely isolated by culture from CSF.
• Stool -  = 1.0 g (solid) or = 0.5 mL (liquid).  Preferred specimen type as the virus is present for longer than other specimen types.
• Rectal swab - Not recommended as the small quantity of faecal material obtained is inadequate for use.
• Serum/Plasma - Not recommended as this sample type is not likely to yield virus in culture.
• Throat Swab - Not recommended. Throat swabs are not as likely as stool to yield virus in culture.

• Viral Isolate/Culture - Collect in sterile leak proof container.
• Stool -Collect within 14 days of illness onset and place in sterile leak proof container, no special medium required. Isolation rates are
  increased by obtaining 2 samples collected 24 hrs apart.

Store samples frozen at =-20°C until shipped for testing. Ship frozen on dry ice. Package Polivirus samples in a separate shipping container from other samples being sent to the NML.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Cases of acute flaccid paralysis (AFP) in children less than 15 years of age and cases of suspected poliomyelitis in patients of any age.

Completed Enteroviruses and Enteric viruses requisition. If possible include relevant clinical findings, travel history, vaccination history, and any relevant lab results that have been completed at local or provincial laboratories.

N/A

Laboratory Surveillance for wild polioviruses and genetically divergent vaccine-derived polioviruses (VDPVs) involves several WHO accredited methods:

1. Virus Isolation in Cell Culture : Following the WHO isolation algorithm, two cell lines, L20B and RD, are inoculated simultaneously with processed stool samples from suspect polio or AFP cases. L20B positive isolates, which may be polio, are further typed by ITD and VDPV real time PCRs as described below.

2. Typing of poliovirus isolates, and intratypic differentiation to distinguish Sabin-like from wild polioviruses : Polio group, serotype, and vaccine strain-specific molecular reagents that target defining PV properties within the capsid region (VP1) are used in a series of real time RT-PCR assays to determine serotype and to distinguish Sabin-like polioviruses from wild polioviruses (intratypic differentiation). Sabin-like ITD result, the sample is tested by VDPV real time PCR; a non-Sabin-like ITD result, the sample is referred for VP1 RT-PCR and sequencing to confirm identity.

3. Screening Sabin-like polioviruses for potential vaccine derived polioviruses (VDPVs) : A real-time RT-PCR method targeting capsid region “hot spots” that typically revert in VDPVs is used to identify possible VDPVs once Sabin-like viruses have been identified using the intratypic differentiation methods described above.

   With a Sabin-like VDPV result, the sample result is reported accordingly; with a non-Sabin like VDPV result, the sample is referred for VP1 RT-PCR and sequencing to confirm identity.

4. Sequencing the VP1 region of non-Sabin-like and potential VDPV isolates : A series of degenerate primers are used for VP1 sequencing of non-Sabin like poliovirus isolates identified by either ITD or VDPV real time PCRs described above. Sequence data from the VP1 region is used to confirm the presence of a wild-type, a Sabin-like or a VDPV strains as follows:

4.1  Poliovirus are grouped into 3 categories of isolates based on the extent of divergence of the full VP1 region of the isolate from the corresponding Sabin reference strain (PV1 VP1 (AY184219); PV2 VP1 (AY184220); PV3 VP1 (AY184221):

4.1.1   Sabin-Like - < 1% divergent (PV types 1 and 3); < 0.6% divergent (PV type 2)

4.1.2   VDPV - = 1% divergent (PV types 1 and 3); = 0.6% divergent (PV type 2)

4.1.3   Wild Poliovirus (WPV) - = 15% divergent                  

5.  Further testing if required may be referred to the CDC in Atlanta, Georgia, which is the WHO GPLN Reference Laboratory.

The total TAT is 28 calendar days. Each component of the test algorithm has its own allotted TAT and it is broken down as follows:

• The TAT for PV Isolation is 14 calendar days from date the stool specimens are received into the lab.

• The TAT for PV ITD and PV VDPV real time RT-PCRs is 7 calendar days from the date the isolation is completed.

• The TAT for PV VP1 sequencing is 7 calendar days from the date that the real time RT-PCR results are completed.

1. World Health Organization. Polio Laboratory Manual. WHO/IVD/04.10,2004.
2. S1. Supplement to the WHO Polio Laboratory manual-an Alternative Test Algorithm for Poliovirus Isolation and Detection.
3. Kilpatick DR, Iber JC, Chen Q, Ching K, Yang S, De L, Mandelbaum MD, Emery B, Campagnoli R, Burns CC, Kew O. Poliovirus serotype-specific VP1 sequencing primers. J of Virol. Methods 174(2011) 128-130