Enterovirus Research Centre Mumbai. Highlights of work done

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1 Enterovirus Research Centre Mumbai Highlights of work done -07 was the most difficult year experienced by the Enterovirus Research Centre. In the early morning hours on 21 st April, fire broke out in the central equipment room. All the major equipments including genetic sequencers were completely destroyed. The fire also caused serious damage to the main electric supply cables. Hot smoke and soot spread in the laboratory giving ghastly looks. There was no structural damage to the building. Fortunately, none of the freezers storing virus isolates and clinical samples were affected in the fire. There was no loss of data. However, work was completely stopped and immediate repairs and renovation of the entire premises became the highest priority. ERC is housed in one of the buildings of the Haffkine Bio-pharmaceutical Corporation Ltd (HBPCL) occupying approximately 6000 sq ft area. Mrs. Gadgil, Managing Director and Mr. PS Chavan, General Manager HBPCL provided the much needed helping hand, providing space for locating our deep freezers (containing viruses and clinical samples) so that the virus isolates and samples are not affected. These freezers were started within 24 hours using electric supplies of HBPCL. Prof. NK Ganguly, Director General provided the most essential moral support preventing the total collapse of the Centre and immediately sanctioned funds required for renovation and restoration of the laboratory equipment. He turned the disaster into an opportunity. He also ordered that the scientific research support provided by the Centre to the Polio Eradication Programme must not be halted even for a single day.

2 Accordingly, genetic sequencing of poliovirus isolates and intratypic differentiation was restored immediately. Dr. K Ghosh, IIH Mumbai provided laboratory space some experimental work could be done. Dr. Mishra and Dr. Arankalle offered unlimited time (on priority) on DNA sequencer at NIV Pune. The DNA sequencers at NIRRH, IIH within ICMR and at ACTRECT, Navi Mumbai and NCCS, Pune were also availed to generate molecular epidemiologic information on wild poliovirus transmission in the country. We worked at 3 different locations to complete single experiments. Unfortunately, year turned out to be a bad year for the polio eradication programme as wild poliovirus cases started to increase from June onwards. This increased pressure on us for diagnosis and genetic sequence data. In 2005, more than samples collected from AFP cases were tested at ERC for wild poliovirus isolation. Virus culture required large laboratory with bio-safety level 2 and cell culture supply hence this aspect of laboratory work could not be started in a sister ICMR institute in Mumbai. It was therefore decided to transfer these samples to other polio network laboratories. Thrice a week, a staff of ERC took the stool samples of AFP cases from Maharashtra, Madhya Pradesh and Chhattisgarh to Polio Laboratory at BJ Medical College, Ahmedabad by road to make sure that samples are reached safely, in time and under cold chain. Every member of the staff of ERC showed enormous strength to sustain through the most difficult period. Most of the project staff had to be retrenched so we lost trained manpower. There was no space even to properly sit down and work. We were scattered. Many of us sat in groups, literally on the floor, to enter data in logbooks. Some of used spaces under the staircase or in passageway to the terrace to set up work tables. Some of

3 us did not have space at all so we sat in the shadows of the trees or what ever. Nobody complained and breached the discipline. Today we have a decent set up and we are happy again. Renovation funds were sanctioned and disbursed quickly. M/s HSCL was appointed as the agency. Funds for purchase of equipment were sanctioned and the equipment ordered. The WHO also ordered requisitioned equipment at the earliest. Building committee was established under the chairmanship of Dr. AN Bhisey, ex- Director ACTRECT. Mr. JS Arora, ex-director Buildings & Construction DAE provided expert guidance for making the renovation plans, identifying requisite materials. Noticing that we lack expertise in reconstructing the laboratory, Mr. Arora offered to guide us through. To put on the record, that we never imagined that so much of time and effort (apart for the expert technical guidance) would be required almost on a day-to-day basis. Therefore, we had not appointed him as a consultant. We shall be ever grateful to Mr. Arora for taking personal interest in every detail of the renovation and overseeing that high quality work in accomplished. Though some of the repairs works is still ongoing, we have now entered our new laboratories and are initiating work. We should be fully operational by the end of May The WHO also provided support for re-purchase of the equipment lost in the fire and restore the laboratory set-up. We have received and installed all the ordered new equipment from the ICMR as well as the WHO funds.

4 Research work New Algorithm for reducing time required for reporting virological results of AFP samples A total of 66 virologically confirmed poliomyelitis cases were reported in India in Again most of these were reported from either Uttar Pradesh or Bihar. Polio Eradication Programme therefore expected to get virus culture faster than usual so that field action could be undertaken quickly to stop spread of wild poliovirus. Combinations of the different methods available were discussed and some of these were chosen for testing. A new algorithm thus emerged bringing PCR upfront for simultaneously detecting and differentiating poliovirus isolates. This algorithm was tested along with the standard procedure on approximately 4000 stool samples received prospectively from December 2005 to March. Results of the study were presented at the Polio Virologists meeting at WHO Geneva in March. The new testing algorithm continues to use two cell lines for virus isolation, a passage of the isolate in L20B cells (genetically engineered murine cells expressing human poliovirus receptor) and PCR for virus typing and differentiation. Serological typing of poliovirus is completely omitted. The new algorithm would report poliovirus isolation, typing and differentiation within 14 days as against the 20 to 28 days required in the standard method. It was recommended that ERC would start reporting virus culture results using the new algorithm with immediate effect. Polio Laboratories in the WHO Network would be trained and by the end of or early 2007, the new method will become the standard

5 procedure for poliovirus isolation in the network. Unfortunately, due to the fire at ERC, virus isolation work had to be stopped since 21 st April. It was decided to train laboratory personnel from selected SEAR Polio Lab Network as quickly as possible at ERC. Accordingly, a WHO funded workshop was conducted at ERC during March 2007 to train personnel from 5 Indian Laboratories. International participants were from Sri Lanka, Indonesia, Thailand and DPR Korea. The new method is scheduled to become fully operational from 1 st June Laboratories not yet trained to use the new method would send untyped isolates to ERC for typing and differentiation. It is expected that the workload at ERC will again shoot up from June Workshop Photos shows the renovated laboratory facility

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7 Poliomyelitis in India Table 1 shows the wild poliovirus cases in reported in different states in India during 2004 to. Lowest numbers of cases were recorded in Poliovirus 1 as well as poliovirus 3 cases decreased significantly. Wild poliovirus 1 and 3 were detected in 8 and 1 States, respectively. Wild poliovirus 1 transmission was endemic in both Uttar Pradesh and Bihar. Wild type 3 poliovirus transmission was found in western Uttar Pradesh only. There was an increase in the number of poliomyelitis cases from 66 in 2005 to 674 in. Though Uttar Pradesh and Bihar reported most poliovirus 1 cases and type 3 poliovirus transmission was found in western Uttar Pradesh only (for third consecutive year), wild poliovirus 1 caused polio cases in as many as 16 districts. There was sevenfold increase in poliovirus type 3 cases in. Table 1. Poliomyelitis cases in India, STATE PV1 PV3 PV1 PV3 PV1 PV3 ANDHRA PRADESH ASSAM BIHAR CHANDIGARH DELHI GUJARAT HARYANA HIMACHAL PRADESH JAMMU & KASHMIR JHARKHAND KARNATAKA MADHYA PRADESH MAHARASHTRA PUNJAB RAJASTHAN TAMIL NADU UTTAR PRADESH UTTARANCHAL WEST BENGAL Total

8 Month-wise distribution of polio cases is shown in the graph in Figure 1. As can be seen, number of polio cases started increasing from May, reaching maximum during July to October and declined to levels by the end of the year. The distribution of polio cases showed the classical summer/ monsoon high peak Figure 1. Monthwise distribution of Poliovirus type 1 cases in India, Nos. of Polio Cases Jan-04 Feb-04 Mar-04 Apr-04 May-04 Jun-04 Jul-04 Aug-04 Sep-04 Oct-04 Nov-04 Dec-04 Jan-05 Feb-05 Mar-05 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 Jul-06 Aug-06 Sep-06 Oct-06 Nov-06 Dec-06 Jan-07 Feb-07 Mar-07 Molecular Epidemiology of wild Poliovirus in India Sequencing facility at ERC was lost in the fire incident. However, molecular characterization of wild poliovirus isolates was continued using sequencers at sister institutes in Mumbai and Pune. Two 16capillary Genetic Analyzers (one each from ICMR and WHO funds) were installed at IIH Mumbai in the month of April The two sequencers are now shifted and installed at ERC. Complete VP1 nucleotide sequence of wild poliovirus isolates is used for phylogenetic analysis of wild poliovirus isolates. Figure 2 shows the genetic relationship of wild poliovirus type 1 isolated in the year The poliovirus type 1 strains within the

9 transmission chain that has not been isolated in is marked the figure. This lineage seems to have been eliminated. Figure 2. Two genetic clusters of wild Poliovirus 1 found in The genetic lineage that caused large number of cases in in western UP is highlighted. Wild poliovirus infections spread from Moradabad and Meerut districts in Uttar Pradesh to increasingly larger geographical areas. These year-2005 Poliovirus 1 isolates from Moradabad and Meerut showed very high sequence similarity therefore the outbreak-like situation can be attributed to viruses in this transmission chain of 2005 (Figure 3).

10 Figure 3. Genetic Trees showing relationship of 2005 and wild poliovirus 1 isolates. Figure 4. Poliovirus 1 re-introduction from UP and Bihar to recently polio-free States

11 A total of 61 polio cases (all poliovirus type 1) were reported in Bihar in. Genetic analysis shows that the isolates are more closely related to the isolate from Munger, Bihar in 2004 than other isolates in Viruses of this transmission chain were isolated in eastern Uttar Pradesh. Notably, in 2005, 30 cases were reported in Bihar and 25 in Uttar Pradesh. Yet, surveillance failed to detect intermediate strains (cases) in Bihar as well as in Uttar Pradesh, in Figure 5. Sequence analysis highlighting genetic relationship of 2004 and wild poliovirus 1 isolates in Bihar.

12 As in previous years sequence analysis revealed virus importation in recently wild virus free geographical areas in. Relevant segments of a large neighbor joining tree showing genetic relationship of poliovirus isolates in some States other than UP and Bihar are shown in Figure 5. A total of 29 polio cases due to wild poliovirus 1 were reported up to March Figure 6 shows the origin of the virus that caused two cases in Andhra Pradesh (zero cases in 2004 and 2005) and one in Maharashtra. Figure 6. Wild Poliovirus 1 re-introductions in Andhra Pradesh and Maharashtra in early 2007

13 Wild Poliovirus type 1 importation in Bangladesh ERC is the sequencing laboratory for viruses isolated in Bangladesh. The first case of wild poliovirus type 1 (Bangladesh achieved zero polio status in 1999) was detected in Joypurhat district in January. Sequence analysis showed that the isolate was closely related to the Indian genotype and a source in western UP. A total of 18 cases were reported in, all belonging to a single importation. However, sequence diversity within Bangladeshi isolates indicated wide spread circulation of the virus before the cases were reported (Figure below).

14 Sequence analysis of wild poliovirus type 3 Wild poliovirus cases have remained confined to western Uttar Pradesh, predominantly in Moradabad. Sequence analysis shows presence of a single lineage since Environmental surveillance for wild poliovirus in Mumbai Screening of sewage samples collected weekly from three high-risk slum localities was started in We have generated wild transmission and repeated re-introduction of wild poliovirus in Mumbai environment. The work was halted from 21 st April. However, sewage samples were collected throughout this period for retrospective analysis. This activity will be restarted as soon as the laboratory gets ready. Hospital based surveillance for rotavirus disease and strains ICMR/ CDC network of laboratories was established in July 2005 with the aim to study rotavirus infections in hospitalized children suffering from acute gastroenteritis. NIE Chennai is coordinating the network with NICED, Kolkata, NIV Pune, CMC Vellore and ERC Mumbai as the collaborating laboratories. Pediatrics Department, Lokmanya Tilak Medical College and Hospital and Enterovirus Research Centre have collaborated for collection of samples and genotyping. The LTMC collects patient data and samples for testing. ELISA is used to detect rotavirus in fecal samples of the patients. Rotavirus positive stool samples are then provided to ERC for molecular typing (G and P typing) using PCR. During the period December 2005 to February 2007 a total of150 rotavirus positive samples were studied for G and P typing. Results are presented in the Table. During the

15 period under study, G2P4 (56/150) combination was found to be most predominant followed by G1P8 (26/150). Mixed genotype was detected in 12/ 150 samples. Table 2. Genotypes of rotaviruses detected in acute gastroenteritis patients in Mumbai G genotypes P genotypes P[4] P[8] P[4]+ P[8] P[6] P[6]+ P[8] Non- Typeable Total G G G G G1+G G9+G G1+G Non_Typeable Total Due to the fire incidence no testing was possible at ERC after 21 st April, rotavirus genotyping work was carried out in Rotavirus Laboratory at NIV Pune. Dr. Shobha Chitambar extended all the facilities as well as guidance to the SRF of ERC.

16 Papers Published Grassly NC, Fraser C, Wenger J, Deshpande JM, Sutter RW, Heymann DL, Aylward RB New Strategies for the elimination of polio from India Science ; 314: Dowdle W, van der Avoort H, de Gourville E, Delpeyroux F, Deshpande J, Hovi T, Martin J, Pallansch M, Kew O, Wolff C. Containment of polioviruses after eradication and OPV cessation: characterizing risks to improve management. Risk Anal. ; 26: Grassly NC, Wenger J, Durrani S, Bahl S, Deshpande JM, Sutter R, Heyman D, Aylward RB Protective efficacy of a monovalent oral type 1 poliovirus vaccine: a case control study Lancet 2007; 369: Workshop held at ERC WHO International workshop Hands on training on implementing alternate algorithm of poliovirus identification. Virologists and technical staff of WHO polio network laboratories from India, Sri Lanka, Indonesia, Thailand and DPR Korea attended the workshop March 2007

17 Details of Meetings/Visits/Training/Workshop attended During April to March 2007 Sr. No. Particulars Place Date Meetings attended by Dr. J.M. Deshpande 1 Meeting of AFP Expert Group for NPSP 10 April Case Classification New Delhi 2 Meeting of India Expert Advisory Group WHO, New Delhi May 3 Meeting of polio virologists for developing rapid diagnosis methods WHO, New Delhi May 4 WHO - 11 th Informal Consultation on the Global Polio Laboratory Network WHO HQ, Geneva June 5 Selection Committee Meeting for the ICMR HQ, 30 August post of Senior Research Officer New Delhi 6 Meeting on Indo-US Cooperation on emerging and re-emerging infections and disease surveillance ICMR HQ, New Delhi September 7 Meeting of investigators Hospital based surveillance of rotavirus disease and strains. NIE, Chennai September 8 Meeting of Expert Review Committee for AFP Case Classification NPSP New Delhi 13 October 9 WHO/ SEARO Meeting for identifying research areas to address polio WHO, New Delhi 19 October problem in UP 10 Meeting of Expert Review Committee for AFP Case Classification NPSP New Delhi 01 November 11 Negotiation committee meeting for equipment purchase ICMR, HQ, New Delhi 03 November 12 Indo US Symposium on viral diagnostics, epidemiology and pathogenesis New Delhi November 13 District level meeting of Rotary International: Invited speaker on polio Pune 26 November eradication strategies th Meeting of Virologists from SEAR Polio Laboratory Network Manesar, Haryana 4-5 December 15 Meeting of India Expert Advisory Group for polio eradication New Delhi December 16 Meeting of Directors/ OIC of ICMR Jodhpur 23-24

18 Institutes/Centres December Meeting of Expert Review Committee for AFP Case Classification NPSP New Delhi 22 January Meeting of WHO Polio Laboratory Network Coordinators Trivandrum February 200 Symposium/Conference/Workshop 19 Molecular Epidemiology of Poliovirus Invited Lecture IISc., Bangalore 20 Taxonomy of viruses- Invited lecture Agharkar Institute, Pune 21 Indo US Symposium on viral diagnostics, epidemiology and pathogenesis 22 Facilitator Hands on training on implementing alternate algorithm of poliovirus identification 6 July 22 December New Delhi November ERC, Mumbai March 2007 Visits 23 Assessing laboratory facility for taking up New Algorithm testing for Poliovirus: WHO/ SEARO 24 Assessing laboratory facility for taking up New Algorithm testing for Poliovirus: WHO/ SEARO SGPGI Lucknow KIPM Chennai December S.No. Particulars Venue Date Meetings attended by Dr. V.K. Saxena, SRO 1 Equipment Condemnation Committee Meeting Workshop attended by Dr. V.K. Saxena ICMR HQ, New Delhi 21 August 2 Workshop on Microarray Analysis Chennai February 2007

19 S.No. Particulars Venue Date Meetings attended by Mrs. SS Nadkarni, RA 1. Alternate Algorithm Methods for ITD of Polioviruses th Meeting of Virologists from SEAR Polio Laboratory Network 3. WHO Polio Laboratory Network Coordinators Meeting Workshop attended by Mrs. SS Nadkarni WHO, New May Delhi Manesar, 4-5 December Haryana Trivandrum February Facilitator -Hands on training on implementing alternate algorithm of poliovirus identification ERC, Mumbai March 2007

20 Acknowledgements The Director and staff of the Enterovirus Research Centre wish to express their deep sense of gratitude to Prof. NK Ganguly, Director- General ICMR for the enormous moral, scientific and financial support to the Centre. No words are enough to express our thanks to Shri. JS Arora for engineering and technical advice extended right from conceptualization of the renovation to its completion and Dr. David Heymann, WHO Geneva for recommending financial support for equipment purchase and to Mr. PS Chavan, HBPCL for providing shelter for equipments, materials and to some of the staff. We wish to thank Dr. AN Bhisey, Dr. Dipali Mukherji, Dr. Lalit Kant, Mr. AK Srivastava and other from ICMR for continued support. Special thanks are due to Dr. K Ghosh, Dr. AC Mishra, Dr. Vidya Arankalle & her staff for providing support so that laboratory work could continue. Each and every member of the Staff of the Centre deserves a big personal THANK YOU from me (JM Deshpande, Director ERC) for the untiring support given to me during these most difficult times. I cannot resist myself to mention the names of Mrs. Nadkarni, Mrs. Shaikh, Dr. Saxena and my wife Anuja whose undeterred support kept me going on.