|Year : 2015 | Volume
| Issue : 1 | Page : 143-146
Emergence of influenza A (H1N1) PDM09 in the remote Islands of India - A molecular approach
N Muruganandam1, D Bhattacharya2, IK Chaaithanya1, H Bhattacharya3, R Reesu1, A Maile1, GSJ Bharathi1, AP Sugunan1, P Vijayachari1
1 Regional Medical Research Centre (ICMR), Port Blair, Andaman & Nicobar Islands-744101, India
2 Regional Medical Research Centre (ICMR), Port Blair, Andaman & Nicobar Islands-744101; Regional Medical Research Centre (ICMR), Nehru Nagar, Belgaum-590010, India
3 Regional Medical Research Centre (ICMR), Port Blair, Andaman & Nicobar Islands-744101; KLE Dr. Prabhakar Kore Basic Science Research Centre, KLE University, Belgaum-590010, India
|Date of Submission||26-Jul-2013|
|Date of Acceptance||21-Mar-2014|
|Date of Web Publication||5-Jan-2015|
Regional Medical Research Centre (ICMR), Port Blair, Andaman & Nicobar Islands-744101
Source of Support: The study was supported by the intramural funds
from the Indian Council of Medical Research (Department of Health
Research, Ministry of Health and Family Welfare, Government of India), Conflict of Interest: None
Background: A disease outbreak of A (H1N1) PDM09 was reported in Andaman and Nicobar islands in 2009 with an attack rate of 33.5% among settler population and 26.3% among the aboriginal Nicobarese tribe. During the ongoing outbreak of A (H1N1) PDM09 disease in different parts of the world, a subject working in Dubai city of Saudi Arabia, came to Port Blair, following which the pandemic triggered for the first time in these Islands. Materials and Methods: During the period August 2009 to January 2011, 30 confirmed cases of Influenza A (H1N1) PDM09 virus infection was detected. To understand the genetic relationship, the NA gene sequences of the viruses were phylogenetically analysed together along with the virus sequence isolated from other parts of the world. Result: Formation of multiple clusters were observed, with the sequences of Andaman Islands, mainland India, Mexico, Saudi Arabia and few other counties clustering together. The sequence analysis data revealed that there was no specific mutation conferring resistance to oseltamivir among the Andaman A (H1N1) PDM09 virus isolates. The result of phylogenetic analysis have also revealed that the A (H1N1) PDM09 virus might have spread in these remote Islands of India via the subject from Saudi Arabia/Dubai. Conclusion: A (H1N1) PDM09 Influenza outbreak have highlighted the need to strengthen the region-specific pandemic preparedness plans and surveillance strategies.
Keywords: Genetic distance, H1N1, mutation, NA gene, pandemic, phylogenetic analysis
|How to cite this article:|
Muruganandam N, Bhattacharya D, Chaaithanya I K, Bhattacharya H, Reesu R, Maile A, Bharathi G, Sugunan A P, Vijayachari P. Emergence of influenza A (H1N1) PDM09 in the remote Islands of India - A molecular approach. Indian J Med Microbiol 2015;33:143-6
|How to cite this URL:|
Muruganandam N, Bhattacharya D, Chaaithanya I K, Bhattacharya H, Reesu R, Maile A, Bharathi G, Sugunan A P, Vijayachari P. Emergence of influenza A (H1N1) PDM09 in the remote Islands of India - A molecular approach. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 13];33:143-6. Available from: http://www.ijmm.org/text.asp?2015/33/1/143/148417
| ~ Introduction|| |
Influenza pandemics are typically characterised by higher, but widely varying, number of deaths than are seasonal epidemics.  In June 2009, WHO declared a global influenza pandemic,  the first for over four decades. Influenza A is unique among the major pandemic threats in that it could potentially infect 30% of the world's population within a matter of months. Respiratory and cardiovascular deaths associated with 2009 pandemic influenza A H1N1 resulted in 151700-575400 deaths during the first year of virus circulation in every country in the world.  The deaths caused by H1N1 pandemic flu in its first year (2009-10) could be 15 times higher than the number of laboratory-confirmed deaths previously reported to the World Health Organization.  Report of the first confirmed case of influenza A (H1N1) PDM09 was from Mexico on April 23 2009,  whereas in India first confirmed case was reported on 16 May 2009.  The Andaman and Nicobar Islands, a Union Territory of India, is an archipelago of 555 islands/islets stretching over 700 km from north to south in the Bay of Bengal. The territory is situated about 150 km (93 mi.) north of Aceh in Indonesia and is separated from Thailand and Burma by the Andaman Sea. Thirty-eight islands are inhabited, with a population of approximately 3.8 lakhs. Andaman and Nicobar Islands are divided into 3 districts namely North and Middle Andaman district (Headquarters: Mayabunder), South Andaman district (Headquarters: Port Blair) and Nicobar District (Headquarters: Car Nicobar). Health care in the islands is almost entirely under the government sector (provided by three district hospitals in the district and by Community Health Centres and Primary Health Centres in the more peripheral regions) and is provided by the Directorate of Health Services. 
The first outbreak of Influenza A (H1N1) PDM09 was reported in Andaman and Nicobar islands in 2009 with an attack rate of 33.5% among settler population and 26.3% among the aboriginal Nicobarese tribe.  However, no death occurred during the period and all the patients recovered without any complications.
During the ongoing outbreak of influenza A (H1N1) PDM09 in mainland India, influenza-like illness (ILI) was observed for the first time in a 14-year-old girl, residing in Mayabunder (North Andaman), who was visited by a relative from Dubai prior to the development of the illness. She was immediately quarantined and put on treatment with Tamiflu. Her sample collected and tested post treatment and was found negative by real time-polymerase chain reaction (RT-PCR) for Influenza A (H1N1) PDM09.
After the outbreak, several sporadic cases of ILI were observed among the inhabitants of Andaman and Nicobar islands, but all the samples were tested negative. Unavailability of proper testing facility, improper storage condition during the transport, and delay in transportation to the laboratory in mainland India might have played a role in negative diagnosis of the sample. Thereafter, an outbreak of ILI was reported among the inmates of a training camp of the Annual Naval National Cadet Corps (NCC) during the last week of September. Of the 319 camp participants, 107 (33.5%) suffered with ILI. Eleven samples were collected and tested, of which 7 (63.6%) samples were tested positive for influenza A (H1N1) PDM09.  Among the confirmed cases, few subjects belong to the locality, where the first case of ILI was reported. In the present study, an attempt was made to understand the emergence of this virus in these remote Islands of India based on molecular tools.
During the period between August 2009 to January 2011, Influenza A (H1N1) PDM09 virus infection was confirmed in 30 cases from various parts of Andaman and Nicobar islands with no history of visit to mainland India or other parts of the world prior to the infection. Throat swabs were collected from all these 30 patients [Table 1] and transported to Regional Medical Research Centre (RMRC) laboratory in viral transport media maintaining cold condition. Viral RNA was extracted from these 30 throat swabs and subjected to RT-PCR, using QIAamp viral RNA minikit (Qiagen, Hilden, Germany). RT-PCR was performed to amplify 298 bp of the neuraminidase (NA) gene using specific primers (forward: 5′-ACACAAGAGTCTGAATGTGCATGT-3′; reverse: 5′-GTCTCCGAAAATCCCACTGCATAT-3′).  Direct sequencing of PCR products was performed by using a Big-Dye Terminator v3.1 cycle sequencing reaction kit on an ABI PRISM 3130 DNA analyser (Applied Biosystems, Foster City, CA, USA). The sequence data was analysed using the SeqScape software (Applied Biosystems, Foster City, CA, USA). To understand the genetic relationship of these 30 viruses that were prevalent in mainland India and other parts of the world, the NA gene sequences of the viruses were phylogenetically analysed together employing MEGA 4.0.  Only the sequences of the strains isolated from Homosapiens were taken into consideration.
|Table 1: Demographic details of the patients from whom throat samples were collected and tested positive |
Click here to view
The pair-wise genetic distance (K2P) among isolates of Andaman was 0.00 percentage. No genetic distances (K2P = 0.00) were found between Andaman isolates and the sequence of the virus isolated from some part of the mainland India including Delhi, Mumbai and Nagpur. However, genetic distance was 0.007 for Bangaluru, 0.003 for Pune and Hyderabad.
Further analysis was done with sequences of the A (H1N1) PDM09 viruses isolated worldwide during two different years of 2009 and 2010 (retrieved from NCBI nucleotide database). Formation of multiple clusters were observed, with the sequences of Andaman Islands, mainland India, Mexico, Saudi Arabia and few other countries clustering together [Figure 1]. The pair- wise genetic distance between isolates of Andaman and Saudi Arabia (HQ698879, HQ698879) was 0.00 per cent. The result of phylogenetic analysis revealed that the influenza A (H1N1) PDM09 virus might have spread to these remote Islands of India through the infected person from Saudi Arabia/Dubai, who on his way home to Kerala visited the girl in Mayabunder. The infected person was working in Dubai city of Saudi Arabia, and visited Port Blair (Capital city of Andaman and Nicobar islands) during mid-August to meet his relatives residing in Mayabunder and travelled to his native, Kerala, after a stay of 2-3 days in this Island. Immediately after reaching Kerala, he had ILI and was diagnosed with A (H1N1) PDM09 and subsequently died. He might have got infected by the virus in Dubai and developed symptoms once he reached Kerala. Since he did not develop any symptoms immediately, he was not detected at the entry point in Chennai.
|Figure 1: Phylogenetic tree of Influenza A(H1N1)PDM09 virus isolated from Andaman and Nicobar Islands analysed with the sequences from worldwide|
Click here to view
Although the pair-wise genetic distance of virus sequences from several other countries was 0.00 per cent, there was no history of any travel to these islands made from these countries as the foreign nationals were thoroughly screened at the entry point in Chennai and Kolkata. In the present study, the NS gene sequence effectively helped in understanding the emergence of the virus in these remote islands of India.
The sequence analysis of NA region revealed that there was no specific mutation conferring resistance to oseltamivir among the Andaman Influenza A (H1N1) PDM09 virus from both tribal and non-tribal population. Majority of the drug resistant cases were reported where oseltamivir was given as preventive medication to people exposed to the flu but not tested positive themselves. Before the 2007/08 season, resistance of influenza viruses to the NA inhibitors (NAIs) oseltamivir and zanamivir was only detected rarely, as the NA amino acid substitutions that conferred reduced susceptibility or resistance to these drugs had deleterious effects on the function of the NA and hence on the viruses ability to replicate and transmit. ,, However, increased use of antiviral drugs in certain regions of the world and stockpiling of antiviral drugs for use during a pandemic, increased the need for systematic surveillance of anti-viral resistance. 
In the backdrop of re-emergence of the virus in mainland India, there is a possibility of the virus re-invading these remote Islands. Since the resistance towards oseltamivir has already been reported from several parts of the world like USA, Australia, and South east Asia ,, there exist an impending danger of circulation of these mutated virus strains in these remote islands. As use of anti-viral drugs continues to grow, further reports of drug-resistance viruses are certain to occur. All influenza strains, but especially A (H1N1) virus, need to be monitored for resistance to NAIs to evaluate the effectiveness of the treatment for both seasonal and potential pandemic influenza viruses.
The study also highlights the importance of the nonstructural (NS) gene sequence, in tracking the virus and understanding the evolution overtime. The Influenza A (H1N1) virus outbreak has highlighted the need to strengthen the region-specific pandemic preparedness plans and surveillance strategies. To delay or prevent influenza entry into these remote islands, influenza screening needs to be considerably more effective. During the inter-epidemic period, major international intervention is required for international air travel in the dissemination of emerging and re-emerging infectious diseases. It should consider system-wise approaches, including exit screening, standardised health declarations, active screening of individual passengers (using rapid laboratory tests and thermal scanning), and passengers tracking and circumstances, where airline should be suspended entirely.
| ~ Acknowledgements|| |
The study was supported through the extramural funds of Indian Council of Medical Research. The authors are thankful to Directorate to Health Service, A and N Administration. The authors are also thankful to Lady Tata Memorial Trust for providing financial support to NM and HB.
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