Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

Since 1988 the world has come very close to eradicate polio through global polio eradication initiative [1]. The objective of this initiative was to interrupt wild PVs as soon as possible to achieve certification of Global Polio Eradication and to strengthen the routine immunization and surveillance as well. Since its inception, this initiative has made remarkable headway worldwide and number of cases of poliomyelitis dropped from 35,000 in 1988 to 1650 in 2008 [2]. However, the Indigenous transmission of wild poliovirus (WPV) has never been interrupted in Afghanistan, Pakistan, India, and Nigeria [3‐5].

The Polio Eradication Initiative was launched in Pakistan in 1994 with remarkable success, the number of confirmed cases declined from 1015 in 1997 to 32 wild polio cases including 19 WPV1 and 13 WPV 3 in only 18 of 120 districts of Pakistan [6, 7]. In Afghanistan polio eradication activities started in 1997, [8] since then, significant progress have been made in both countries, wild type 2 poliovirus has been knocked out from Pakistan since 1997 and in Afghanistan 1999 [7, 9].

Despite intensified mass immunization activities, the two countries failed to interrupt poliovirus transmission. Persistent pockets of polio transmission along the border between Afghanistan and Pakistan are key epidemiological challenges due to insecurity and continued conflict [10, 11].

Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of family Picornaviridae [12]. It is composed of single-stranded positive-sense RNA genome that is about 7500 nucleotides long [13]. There are three serotypes of poliovirus, PV1, PV2, and PV3; each with a slightly different outer capsid protein which define cellular receptor specificity and virus antigenicity. Therefore infection with one serotype does not prevent infection with another serotype [14, 15]. PV1 is the most common type encountered in nature, however all three types are extremely infectious and can circulate independently [16, 17]. Long excretion periods and low population immunity also support its rapid evolution and spread in humans [18‐20]. The mutation rate in poliovirus is relatively high with a synonymous substitution rate of 1.0 × 10^-2 substitutions/site/year [21, 22] which creates a wide variety of mutants that are referred as different genetic strains or genotypes. Each serotype of wild poliovirus has many different genotypes that are distributed geographically and co-circulate worldwide [20, 23, 24]. Genetic diversity of wild polioviruses is determined by its genome sequencing that not only provide genetic relationship among wild Polioviruses as well as it helps to monitor the progress of Polio Eradication Program [25, 26].

In current study 111 wild type1 poliovirus isolates collected during 2005-2007 from Pakistan and Afghanistan were sequenced for complete VP1 gene to study their genetic diversity and to reveal the indigenous genotype in this region that would be helpful to track the transmission patterns in Pakistan and Afghanistan being a single epidemiological block.

The data presented here illustrates molecular characterization of wild type 1 polioviruses endemic in Pakistan and Afghanistan during last 3 years, 2005-2007. The sequence of VP1 gene was employed to determine the epidemiological links among the wild type 1 polioviruses circulating across the both countries. The VP1 gene comprised of 906 nucleotide bases with sufficient heterogeneous motifs that clearly discriminate between various genotypes. In 1993 Huovilianen et al., has been detected multiple genotypes of wild type 1 polioviruses in Karachi district of Sindh province in Pakistan [39]. However, in Pakistan and Afghanistan only one genotype (SOAS, South Asian) has been endemic for many previous years in contrast to African countries where endemicity of multiple genotypes have been reported [40, 41].

Despite this monophyletic character, distinctive reservoirs of wild type 1 polioviruses have been observed that confined to specific geographic regions in both countries. Based on sequence diversity of ≤ 5% among the VP1 nucleotide sequence, the wild type 1 polioviruses have been classified into certain groups called 'clusters'. A phylogenetic tree was reconstructed on the genetic relationship of VP1 gene sequence for the wild polioviruses detected during the 3 year period which clearly displayed the distribution of isolates among three clusters (A, B, C) (Figure 2). Within each clusters a definite pattern of virus distribution was found, representing a different lineage or transmission route in different geographic regions of Pakistan and Afghanistan that also highlights the epidemiological links during their evolution from 2005 to 007 time intervals.

The molecular data obtained from this study explicitly reveals the transmission pattern of wild type 1 polioviruses circulation in Pakistan and Afghanistan as a successful tool to monitor the AFP surveillance as well as to formulate the mop-up immunization activities established as a reference standard policy to interrupt the wild poliovirus multiplicity within population. The underlying factors posing a incessant threat to achieve the polio-free country status are the areas with poor security and hard to reach children communities leaving a number of children unimmunized despite a number of supplementary immunization campaigns in the country. Furthermore, parallel circulation of some lineages/multiple chains among both countries highlights the ongoing continuous presence of wild polioviruses.

The factors behind this worrisome situation are attributed to the security concerns and also to the unchecked cross-border movement as well. The similar concern has already been shown by Kew., et al. [26]. However, the synchronized immunization activities across the border will be helpful to stop the importation of wild polioviruses from each side of the border. Because immunization has been proved to be a successful approach for elimination of poliomyelitis by locating and maintaining a high level of immunity in children less than 5 years of age. Therefore extended use of type 1 monovalent oral poliovirus vaccine (mOPV1) and type 3 monovalent oral poliovirus vaccine (mOPV3) selected in areas of Pakistan and Afghanistan was adopt to knockdown the virus in 2005 and 2007 respectively [42‐44]. But transmission of virus could not be stopped in Khyber, South Waziristan, and the areas due to war and armed conflict which destroyed almost all public health infrastructures and health-care workers could not safely deliver vaccine and active surveillance was very difficult to carry out.

Current molecular data also explores the evolutionary pattern of wild type 1 poliovirus isolates in relation to their ancestral descent. Based on the 1% (in vivo) nucleotide evolutionary rate of VP1 gene, any wild poliovirus isolate indicating equal to or more than 2% nucleotide sequence divergence from its ancestor is recognised as an "orphan virus" [45]. Findings of this study also put emphasis on some silent multiple independent chains of transmission (orphan lineages) over a wide geographical areas involving cross-border population movement between Pakistan and Afghanistan. Regardless of the view that orphan viruses reflect some collapse of surveillance even with superfluous efforts reporting or long term circulation without causing overt cases of polio and gaps in immunization, the efforts of health care personnel working towards the Polio Eradication Initiative (PEI) under the prevalent difficult situation must be acknowledged.

Moreover, epidemiological records and results of this study clearly highlights that transmission of wild type 1 polioviruses is still uncontrolled. However, significant progress has been made to localize the wild poliovirus. The number of infected districts has been significantly decreased from 2005 to 2007 (Table 2) in both countries despite of war conflicts, poor access and a number of other reasons. In order to achieve polio free status, interruption of wild poliovirus transmission is the key target. Pakistan is under strict apprehension of policy implementation constrains required for high level population immunity. A concomitant locally appropriate communications and social mobilization strategy must be pursued to improve public awareness for effective oral polio vaccine coverage to susceptible population to ensure substantial progress.

In conclusion, this study provides requisite support to find out the locations, the extent of wild type1 poliovirus circulation in endemic areas, identify reservoir communities sustaining wild poliovirus endemicity, genetic relationships among these isolates and the source of imported wild poliovirus in Pakistan and Afghanistan. Moreover these analyses are valuable for monitoring the AFP surveillance and to target supplementary immunization activities with oral polio vaccine in order to interrupt chains of virus transmission.