Unrooted Phylogenetic tree of Human Enteroviruses was constructed by Neighbor-joining (NJ) method with Kimura 2-parameter (K2-P) model using MEGA version 5.0 software.

<p>This Phylogenetic tree based on A) VP1 and B) whole capsid (VP4-VP1) nucleotide sequences of PAK NIH isolates and other representative sequences of enterovirus serotypes retrieved from GenBank. AFP isolate is represented by ‘■’ taxon marker. The Prototype strain is labeled with taxon marker ‘▲’. Tree was evaluated with 1000 bootstrap pseudoreplicates. Bootstrap values greater than 50 are indicated at the respective nodes and the scale bar represents the evolutionary distance.</p

Map of the Federally Administered Tribal Areas (FATA) in north-west of Pakistan bordered by: Afghanistan to the north and west with the border marked by Khyber Pakhtunkhwa to the east and Balochistan to the south.

<p>The symbol ‘●’ shows the geographical positions of the acute flaccid paralysis isolates characterized in this study through molecular sequencing.</p

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

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

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

Nucleotide mutations and Amino acid substitutions identified in 5′UTR and P1 Coding region of Index case (1321) and cVDPVs.

<p>Nucleotide mutations and Amino acid substitutions identified in 5′UTR and P1 Coding region of Index case (1321) and cVDPVs.</p

Phylogenetic analysis of Human Astrovirus strains identified in this study based on the partial ORF2 region (348 bases) encoding outer capsid precursor protein.

<p>The reference strains and closest match isolates detected through BLAST are given for genetic comparison. The phylogenetic tree with 500 bootstrap replicates was reconstructed using neighbor joining method and the K-2P model through MEGA 4.0. Serotype-1 strains were compared to four prototype strains (USA-GenBank accession number L23513, isolated in 1994), United Kingdom (GenBank accession number Z25771, isolated in 1990), Japan (GenBank accession number AB009985, isolated in 1997) and Germany (GenBank accession number AY720892). Taxa with arrow head indicates prototype strains within each serotype.</p

Astrovirus detection rates in various age groups of children hospitalized during 2009–2010.

<p>The data labels indicate the percentage of positive samples among each age group (1a). Month wise distribution of astrovirus positive samples between January, 2009 to December, 2010 (1b).</p

Sequence and position of Primer pairs used for PCR and Sequencing.

<p>Sequence and position of Primer pairs used for PCR and Sequencing.</p

All cVDPVs type2 isolated from Nad Ali district of Helmand province are indicated with round circle filled in black color.

<p>Other globally reported cVDPVs type 2 has retrieved from GenBank to reconstruct phylogenetic tree from VP1 coding sequences using neighbor-joining algorithm with Kimura 2-parameter substitution model.</p