Genetic Diversity of Non-Polio Enteroviruses Circulating in Pakistan

Abstract

Enteroviruses (EVs) are RNA viruses in the genus Enterovirus of family Picornaviridae having more than 100 distinct serotypes that were formerly sub-classified as polioviruses, group A coxsackieviruses, group B coxsackieviruses, echoviruses, and numberedenteroviruses. Later on, enterovirus taxonomy was up-dated by the International Committee on Taxonomy of Viruses and now the genus Enterovirus consists of 12 species: Enterovirus A (EV-A), EV-B, EV-C, EV-D, EV-E, EV-F, EV-G, EV-H, and EVJ; as well as Rhinovirus A (RV-A), RV-B, and RV-C. Enteroviruses are responsible for number of infections in humans and other mammals primarily caused via fecal-oral route. Majority of enterovirus infections in humans are asymptomatic with mild fever or skin rash but sometimes these infections may lead to a wide variety of acute and chronic diseases including acute flaccid paralysis (AFP). In Pakistan, polio is endemic and major focus has been on characterization of Polioviruses (PVs). Unfortunately, no attention was given to characterize the non-polio enteroviruses (NPEVs) isolated from AFP patients either by the polio eradication programme or other public health system. So, this study was proposed to explore the frequency, circulation patterns, and genetic diversity of non-polio enteroviruses isolated from AFP cases. To the best of our knowledge, this is the first study in Pakistan that gives a comprehensive overview on wide genetic diversity and diverse circulation patterns of non-polio enteroviruses. A total of 4437 stool samples were collected during the year 2013 (Jan-Dec) from AFP patients from different regions of Pakistan. The stool samples were processed and inoculated on RD and L20B cell lines and the cytopathic effect of NPEVs was observed under microscope. Only 17.7 % (n=786) samples were found positive for NPEVs and 6.7 % (n=298) for PVs while 75.6 % (n=3353) were negative. The highest proportion of NPEVs was detected in Punjab accounting for 44.0 % (n=350), followed by Khyber Pakhtunkhwa 22.8 % (n=179), Sindh 22.0 % (n=173), FATA 5.9 % (n=46) and Balochistan 4.8 % (n=38). Out of 786 NPEV cases, 491 (62.5 %) were male while 295 (37.5 %) were female with highest proportion (n=275; 35.0 %) of NPEV infections. Fever and progression of paralysis was found significant factor (p=<0.005) in all

XII

non-polio enterovirus infected patients. Microneutralization assay was performed on 786 NPEVs which successfully typed 43.9 % (n=345) samples into twenty enterovirus serotypes while 56.1 % (n=441) were untypeable. Furthermore, all 786 samples were screened for NPEVs by real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) and confirmed as NPEVs. These confirmed NPEV isolates were subjected to molecular characterization based on nucleotide sequencing of viral protein 1 (VP1) gene which successfully characterized all (n=786) isolates into 62 enterovirus serotypes that belonged to Enterovirus B and Enterovirus C species. Furthermore, these 62 enterovirus types were classified under coxsackievirus A (n=19; 2.4 %), coxsackievirus B (24; 3.0 %), numbered enteroviruses (n=162; 20.6 %) and echoviruses (n=581; 74 %). In this study, E-1, E-3, E-6, E-11, E-13, E-19, E-20, E-29 and EV-B75 were the most frequent serotypes and were isolated throughout the study period. It was also observed that E-19 (n=71; 9.0 %) and E-13 (n=51; 6.5 %) were marked with high isolation frequency which might suggest that there was a circulation of these two enteroviruses in the community due to a possible outbreak. On the other hand, CV-A1, CV-A11, CV-A13, CV-A19, CVA20, CV-B1, CV-B2, CV-B3, CV-B4, E-4, E-16, EV-B74, EV-B78, EV-B79, EV-B81, EV-B82, EV-B85, EV-B87, EV-B88, EV-B93, EV-B100, EV-B101, EV-B106, EV-B111 were the less frequent serotypes and were isolated sporadically during the study period. The study findings present an overview on the prevalence, molecular epidemiology and circulation patterns of NPEVs with high proportion of Enterovirus B species in Pakistani population. The one year molecular epidemiology data also confirms a significant genetic diversity in these viruses that allows emergence of new variants/genotypes that pose potential risks to the community. Moreover, the isolation of NPEVs from paralytic patients highlights the probable association of these viruses with paralysis but there is need for further studies to understand the role of these viruses in multiple enteroviral illnesses. Additionally, we assume that there may be increase in paralytic cases due to NPEVs after the post eradication of poliomyelitis. So, it is a need of time to monitor such viruses for early detection and better characterization which will help not only the pediatricians and neurologists for rapid diagnosis but will also support to implement a dynamic surveillance programme for non-polio enteroviruses in Pakistan