2. Molecular Epidemiology of Wild Poliovirus in India

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Sibyl Owen
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1 2. Molecular Epidemiology of Wild Poliovirus in India The Network of Polio Laboratories is three-tiered. The main responsibility of National Polio Laboratories (NPL) is isolation of polioviruses from stool samples of AFP cases reported in geographical area assigned to the particular laboratory. The NPL sends poliovirus isolates to a Regional Reference Laboratory (RRL) for differentiating the virus isolates into wild virus or Sabin vaccine-like. A RRL may receive poliovirus isolates from more than one NPL. In turn, wild viruses identified at the RRL are submitted a Global Specialized Polio Laboratory for genetic sequencing. Eight laboratories function as NPL in India. The Enterovirus Research Centre performs the functions of NPL, RRL as well as GSL Enzyme linked immunosorbent assay (ELISA) and RNA probe Hybridization method were used for differentiating poliovirus isolates in to wild or vaccine-like. RT- PCR was also used as the third method for some specimens. The ELISA looks for wild virus and vaccine virus specific antigenic sites whereas probe hybridization is targeted to specific nucleotide sequences. Intratypic differentiation of poliovirus isolates 2003 Timeliness of reporting No. Samples > % Cumm. Days Membrane showing results of nucleic acid probe hybridization test for ITD of poliovirus type 1 P1SL P2SL P3SL Sample 1 P1SL P2SL P3SL Sample 1 P1SL P2SL P3SL Sample 1 EV-group probe Vaccine virus probe Wild virus probe Enterovirus Research Centre, Mumbai 13 Annual Report

2 In 2003, a total of 1516 polioviruses isolated 776 cases were received from laboratories of national polio network for intratypic differentiation. Wild polioviruses were identified from a total of 225 cases i.e., 203 cases of poliovirus type1 and 22 cases of poliovirus type 3. State-wise distribution of poliomyelitis cases in India for the year 2003 is given in Table 3 and Figure 3. It can be seen that the wide spread transmission of wild poliovirus in 2002 was brought under control in most parts of the country. The polio cases in Gujarat, Rajasthan and West Bengal were reported in the first 3 months of However, there was a setback in the Karnataka (36 cases) and Andhra Pradesh (21 cases). Wild poliovirus type 3 was confined mainly to western Uttar Pradesh. Wild poliovirus type 2 has not been isolated since October Type 2 poliovirus has thus been eradicated. Table 3: Poliomyelitis cases in India Poliovirus Type 1 Poliovirus Type 3 Poliovirus Type 2 State Andhra Pradesh Assam Bihar Chandigarh Chhattisgarh Delhi Gujarat Harayana Jammu & Kashmir Jharkhand Karnataka Kerala Madhya Pradesh Maharashtra Orissa Punjab Rajasthan Tamil Nadu Uttaranchal Uttar Pradesh West Bengal Total In addition P1+P3 mix In 2001, wild poliovirus transmission was confined mainly to Uttar Pradesh and Bihar. In 2002, a total of 1600 poliomyelitis cases were reported in India. Poliomyelitis cases were reported in states that had achieved zero polio status in previous year(s). Orissa reported polio cases after 3 consecutive polio-free years. Gujarat, West Bengal, Rajasthan and Madhya Pradesh suffered from polio outbreaks in specific districts. Virus spread to adjacent districts causing additional cases was recorded in these States. Enterovirus Research Centre, Mumbai 14 Annual Report

3 Figure 3: Map Showing Geographical distribution of confirmed poliomyelitis cases in India in 2003 Molecular epidemiology of wild poliovirus is being studied since It was decided in 2001 that complete VP1 region sequences of all wild poliovirus isolates would be used for high resolution of virus transmission pathways. Table 4 shows the wild poliovirus sequences in the database. All wild viruses isolated in 2001 and more than 60% of isolates of 2002 were sequenced and genetic relationships were determined during The remaining isolates sequenced were studied in along with the new viruses isolated in Phylogenetic analysis was carried out using phylogeny programs in PHYLIP. DNA distances calculated for Kimura 2-parameter model were used to generate neighbor-joining trees. Virus isolates were grouped into genetic lineages (clusters). Table 4: Wild poliovirus complete VP1 sequence database. Year Poliovirus 1 Poliovirus /209 (99) 56 (100) /1484 (87) 110/113 (97) /203 (98) 20/22 (90) 2004 * 26/27 (96) 1 (100) Enterovirus Research Centre, Mumbai 15 Annual Report

Figure 4 shows the distribution of the different genetic clusters (color coded dots) of poliovirus type 1 in India in 2002 and 2003.

It was also found that in 2002 wild poliovirus spread from UP and Bihar to other poliofree States in the country.

4 Figure 4 shows the distribution of the different genetic clusters (color coded dots) of poliovirus type 1 in India in 2002 and A total of 14 genetic clusters of wild poliovirus type 1 and 4 of poliovirus type 3 were found to be circulating in India in It was also found that in 2002 wild poliovirus spread from UP and Bihar to other poliofree States in the country. West Bengal: In 2001, wild poliovirus seemed to be under control in West Bengal with only two confirmed poliomyelitis cases in the year. However, number of polio cases rose to outbreak proportions in Murshidabad district in Molecular studies identified that wild virus was introduced in Murshidabad from Bihar. Polio cases were also reported in Pakur district in the neighboring State of Jharkhand. The virus infections spread from Murshidabad to Pakur. Orissa was free from polio since In 2002 wild poliovirus type 1 of lineage from Murshidabad/ Pakur area was isolated from 3 cases in Orissa. The virus transmission continued until early Figure 4 :Distribution of genetic clusters of wild poliovirus type 1 in India, Enterovirus Research Centre, Mumbai 16 Annual Report

5 Gujarat: Only one case of poliomyelitis was recorded in Gujarat in We had confirmed that wild poliovirus responsible for that case was introduced from Sultanpur, Uttar Pradesh to Bharuch district in Gujarat. The wild virus did not establish transmission and no secondary cases were reported in Wild polioviruses were isolated from AFP cases in Bhavnagar district from June 2002 onwards and later on in several other adjoining districts. The 2002 isolates were genetically different from that isolated in Bharuch district in VP1 sequence analysis proved re-introduction of wild poliovirus 1 of two genetic lineages from western Uttar Pradesh to Gujarat in 2002 (Figure 5). The introduced viruses caused 24 cases in 2002 and 3 cases in Figure 5: Wild Poliovirus 1 Transmission in Gujarat. Example of importation and establishment of transmission in GUAML GURJC GUJUN GURJC GUBRH GUPAT GUBAN GUKTC GUKTC MHTHN RJJDP GUBVN GUBVC GUBVC GUBVC GUBVC GUBVN GUBVC GUBVC GUJUN GUBVN GUBVC GUBVN GUBVN GUBVN UPRMP UPMRD UPJPN UPJPN UPSUL GUBRH UPSUL UPSUL UPFER UPFER UPETA UPETA GUBVN RJUDP GUBVN GUBVN GUAMC00003 SAB1VP Madhya Pradesh and Rajasthan was polio free during Wild poliovirus was introduced from Uttar Pradesh in to these states causing 21 paralytic cases in MP and 40 in Rajasthan in 2002 and 11 and 4 cases in 2003 the two states, respectively. Intensive vaccination campaigns for six months to one year were required in these states to stop transmission of the reintroduced wild poliovirus. Enterovirus Research Centre, Mumbai 17 Annual Report

6 Maharashtra: The above examples represent those areas in which wild poliovirus could spread widely causing secondary cases. In contrast, wild polioviruses of 4 different genetic clusters were isolated in Maharashtra indicating multiple reintroductions of wild virus from Uttar Pradesh. However, no secondary cases occurred because of high vaccination coverage and the virus met with a dead end (Figure 6). Three poliomyelitis cases were reported in Maharashtra in Figure 6: Wild poliovirus 1 of different genetic clusters detected in Maharashtra C A UPAHB02061 UPBAL02007 UPAHB02048 UPAHB02029 UPBAL02008 UPPTG02020 UPAZG02079 UPAHB02036 UPAZG02072 MHBMC02107 UPAHB02038 CGBILC2015 UPAHB02044 UPFAI02017 UPFAI02018 MHNSK02027 UPFAI02023 UPFAI02033 UPABN02034 UPGND02041 UPAZG02106 UPGND02027 UPFAI02012 UPFAI KAGBG03008 UPABN02019 UPFAI02014 B UPABN02018 UPVRN02017 UPGND02167 UPSDN02019 BISAR02012 MPCTP02012 MHBMC02077 DLDLH02204 UPGBN02016 MPCTP02014 MPCTP02016 UPJNS UPHDO02008 D MHBMC02051 MHBMC Madhya Pradesh and Rajasthan was polio free during Wild poliovirus was introduced from Uttar Pradesh in to these states causing 21 paralytic cases in MP and 40 in Rajasthan in 2002 and 11 and 4 cases in 2003 the two states, respectively. Intensive vaccination campaigns for six months to one year were required in these states to stop transmission of the reintroduced wild poliovirus. A poliovirus genotype is defined as a group of isolates showing more than 85% sequence similarity within VP1. A cluster/ lineage is defined as a group of isolates showing more than 95% sequence similarity. The rate of nucleotide sequence divergence (Molecular clock) for poliovirus is about 1% substitutions per year. From the genetic distances between recent isolates it is possible to determine temporal relationship between poliomyelitis cases and virus transmission routes. Enterovirus Research Centre, Mumbai 18 Annual Report

7 Karnataka and Andhra Pradesh: In 2003, a new focus of intense transmission of wild poliovirus developed in Karanataka. The first case was recorded in May 2003, followed by an epidemic-like situation with a total of 36 cases in Karnataka and 21 cases in Andhra Pradesh. Genetic sequencing identified the origin of the virus in Western Uttar Pradesh, most likely Meerut district (Figure 7). Wild virus from Karnataka spread to Andhra Pradesh, Tamil Nadu and Maharashtra. Two poliomyelitis cases were reported from the States in south till the end of March 2004 one each from Karnataka and Tamil Nadu. Figure 7: Introduction of wild poliovirus type 1 from Uttar Pradesh to Karnataka, 2003 KEKZK00018 KAGBG00024 KABLK00002 KA GB G00022 KAKPP00004 UPM RT01029 UPM RT02021 UPM RT01041 UPM RT01038 UPMTR02012 UPM RT02017 UPM RT02056 UPPIL02044 UPMZ N02103 UPMZN02076 UPMZN02055 UPMZN02044 UPMZN02084 UPM RT02016 UPM RT02002 UPMZN02035 UPM RT01037 UPM RT01108 KABLY03006 UPM RT Indigenous genetic lineages were eliminated in 2000 SAB1VP1 First case in Karnataka. Wild virus was reintroduced from Meerut district of UP A total of 14 different genetic clusters of wild poliovirus type 1 were identified in Viruses of four clusters were not isolated in Additional three clusters stop transmission from February Three more clusters were eliminated by August Thus wild viruses belonging to only four genetic clusters continue transmission beyond Enterovirus Research Centre, Mumbai 19 Annual Report

Wild poliovirus type 3 isolated since 2001

Uttar Pradesh in 2003 wild viruses of the five

predominant and spread from Uttar Pradesh to

Viruses of cluster A were detected again in

Geographic distribution of wild poliovirus

8 Wild poliovirus type 3 isolated since 2001 have been classified into five genetic clusters. Transmission of wild type 3 was mainly in Uttar Pradesh in 2003 wild viruses of the five clusters were detected yet cluster D was predominant and spread from Uttar Pradesh to Bihar and West Bangal. Viruses of cluster A were detected again in 2003 after a gap of more than one year. Geographic distribution of wild poliovirus type 3 isolated since year 2000 is shown below PV3 Clusters Enterovirus Research Centre, Mumbai 20 Annual Report

ENTEROVIRUS RESEARCH CENTRE

ENTEROVIRUS RESEARCH CENTRE ANNUAL REPORT 2003-04 Enterovirus Research Centre ICMR Haffkine Institute Campus, Parel Mumbai 400 012 WHO GLOBAL SPECIALIZED LABORATORY FOR POLIO Enterovirus Research Centre,