Molecular Characterization and Phylogenetic Relationship of Wild Type 1 Poliovirus Strains Circulating across Pakistan and Afghanistan Bordering Areas during 2010–2012

<div><p>Pakistan and Afghanistan share a long uncontrolled border with extensive population movement on both sides. Wild poliovirus transmission has never been interrupted in this block due to war against terrorism, poor public health infrastructure, misconceptions about polio vaccines and inadequate immunization activities. All these issues complicate the eradication operations and reinforce the complexity of wiping out poliomyelitis from this region. This study illustrates the origins and routes of cross-border wild poliovirus type 1 (WPV1) transmission during 2010–2012 between Pakistan and Afghanistan. Sequence analyses were conducted based on complete VP1 capsid protein sequences for WPV1 study strains to determine the origin of poliovirus genetic lineages and their evolutionary relationships. Phylogenetic tree was constructed from VP1 gene sequences applying Maximum Likelihood method using Kimura 2- parameter model in MEGA program v 5.0. A total of 72 (14.3%) out of 502 wild-type 1 polioviruses were found circulating in border areas of both countries during 2010–2012. Molecular phylogenetic analysis classified these strains in to two sub-genotypes with four clusters and 18 lineages. Genetic data confirmed that the most of WPV1 lineages (12; 66.6%) were transmitted from Pakistan to Afghanistan. However, the genetic diversity was significantly reduced during 2012 as most of the lineages were completely eliminated. In conclusion, Pakistan-Afghanistan block has emerged as a single poliovirus reservoir sharing the multiple poliovirus lineages due to uncontrolled movement of people across the borders between two countries. If it is neglected, it can jeopardize the extensive global efforts done so-far to eradicate the poliovirus infection. Our data will be helpful to devise the preventive strategies for effective control of wild poliovirus transmission in this region.</p></div

Geographic distribution of patients with clinical cases of polio associated with isolates of wild type 1 poliovirus (indicated by colored arrows) from 2010–2012 in border areas of Pakistan and Afghanistan.

<p>A) Transmission Pattern in sub-genotype G1 illustrates five lineages (L1–L5); B) Transmission Pattern in sub-genotype G2 illustrates seven lineages (L6–12) and C) Transmission Pattern in sub-genotype G2 illustrates six lineages (L13–18). The arrows represent the lineages or transmission pathways between both countries. Transmission pathways of Wild poliovirus type 1 from Pakistan to Afghanistan are represented by brown arrows while yellow indicates the transmission from Afghanistan to Pakistan.</p

Wild Poliovirus type 1 (WPV1) distribution among Acute Flaccid Paralysis cases from Pakistan and Afghanistan during 2010–2012.

<p>In Pakistan, annual non-polio AFP rate (per 100,000 children aged <15 years) was 6.9, 7.2 and 6.3 in 2010, 2011 and 2012 respectively. In Afghanistan, this rate was 9.2, 10.5 and 9.5 in 2010, 2011 and 2012 respectively.</p><p>Wild Poliovirus type 1 (WPV1) distribution among Acute Flaccid Paralysis cases from Pakistan and Afghanistan during 2010–2012.</p

The role of supplementary environmental surveillance to complement acute flaccid paralysis surveillance for wild poliovirus in Pakistan – 2011–2013

<div><p>Background</p><p>More than 99% of poliovirus infections are non-paralytic and therefore, not detected by acute flaccid paralysis (AFP) surveillance. Environmental surveillance (ES) can detect circulating polioviruses from sewage without relying on clinical presentation. With extensive ES and continued circulation of polioviruses, Pakistan presents a unique opportunity to quantify the impact of ES as a supplement to AFP surveillance on overall completeness and timeliness of poliovirus detection.</p><p>Methods</p><p>Genetic, geographic and temporal data were obtained for all wild poliovirus (WPV) isolates detected in Pakistan from January 2011 through December 2013. We used viral genetics to assess gaps in AFP surveillance and ES as measured by detection of ‘orphan viruses’ (≥1.5% different in VP1 capsid nucleotide sequence). We compared preceding detection of closely related circulating isolates (≥99% identity) detected by AFP surveillance or ES to determine which surveillance system first detected circulation before the presentation of each polio case.</p><p>Findings</p><p>A total of 1,127 WPV isolates were detected by AFP surveillance and ES in Pakistan from 2011–2013. AFP surveillance and ES combined exhibited fewer gaps (i.e., % orphan viruses) in detection than AFP surveillance alone (3.3% vs. 7.7%, respectively). ES detected circulation before AFP surveillance in nearly 60% of polio cases (200 of 346). For polio cases reported from provinces conducting ES, ES detected circulation nearly four months sooner on average (117.6 days) than did AFP surveillance.</p><p>Interpretation</p><p>Our findings suggest ES in Pakistan is providing earlier, more sensitive detection of wild polioviruses than AFP surveillance alone. Overall, targeted ES through strategic selection of sites has important implications in the eradication endgame strategy.</p></div

Surveillance system circulation detection time of genetically-similar isolates* before polio case onset and difference in detection time between AFP and environmental surveillance overall, by year and by province environmental surveillance for Pakistan polio cases**– 2011–2013.

<p>*Genetically-similar isolates were defined as isolates with ≥99.0% identity in VP1 capsid nucleotide sequence to the polio case isolate. **28 of 361 total polio cases (11 pairs and 2 triplets) had 100% identity in VP1 capsid nucleotide sequence with one or two other isolates. These pairs and triplets were considered together, resulting in 346 genetically-unique polio case isolate sequences for analysis. <b>Abbreviations</b>: ES: Environmental Surveillance; AFP: Acute Flaccid Paralysis; AFP-S: Acute Flaccid Paralysis surveillance. ¹ Time (days) from first isolate within 99% of VP1 capsid detected by environmental surveillance to symptom onset date for polio case. ² Time (days) from first isolate within 99% of VP1 capsid detected by AFP surveillance to symptom onset date for polio case. ³ Number of days sooner that environmental surveillance detected circulation before AFP surveillance; Negative numbers indicate AFP detection preceding ES detection and positive numbers indicate ES detection preceding AFP detection. ⁴ Polio case was reported from a province conducing environmental surveillance (i.e. Balochistan, Sindh, Punjab, Khyber Pakhtunkhwa). ⁵ Polio case was reported from a province not conducting ES (i.e. Federally Administered Tribal Areas, Gilgit Baltistan).</p

Number of orphan isolates^* detected by acute flaccid paralysis surveillance (AFP) only, environmental surveillance (ES) only, and ES and AFP surveillance in conjunction–Jan 2011 –Dec 2013.

<p>Number of orphan isolates^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180608#t002fn001" target="_blank">*</a>} detected by acute flaccid paralysis surveillance (AFP) only, environmental surveillance (ES) only, and ES and AFP surveillance in conjunction–Jan 2011 –Dec 2013.</p

Type of surveillance system first detecting genetically-similar^* circulation for each Pakistan polio case^** by year, provincial environmental surveillance– 2011–2013.

<p>Type of surveillance system first detecting genetically-similar^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180608#t004fn001" target="_blank">*</a>} circulation for each Pakistan polio case^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180608#t004fn002" target="_blank">**</a>} by year, provincial environmental surveillance– 2011–2013.</p

Number of wild poliovirus (WPV) positive isolates by year, serotype, and surveillance system–Jan 2011 –Dec 2013.

<p>Number of wild poliovirus (WPV) positive isolates by year, serotype, and surveillance system–Jan 2011 –Dec 2013.</p

Percentage of Pakistan polio cases^* with genetically similar^** preceding circulation detected by surveillance system type and year–Jan 2011 –Dec 2013.

<p>Percentage of Pakistan polio cases^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180608#t003fn001" target="_blank">*</a>} with genetically similar^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180608#t003fn002" target="_blank">**</a>} preceding circulation detected by surveillance system type and year–Jan 2011 –Dec 2013.</p