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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 1  |  Page : 42-49
 

Genetic characterisation of influenza A(H1N1)pdm09 viruses circulating in Assam, Northeast India during 2009–2015


Division of Virology, ICMR-Regional Medical Research Centre, N.E. Region, Dibrugarh, Assam, India

Date of Web Publication16-Aug-2019

Correspondence Address:
Dr. Dipankar Biswas
ICMR-Regional Medical Research Centre, N.E. Region, Post Box - 105, Dibrugarh - 786 001, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_416

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 ~ Abstract 


Introduction: Influenza A(H1N1)pdm09 virus, since its identification in April 2009, has continued to cause significant outbreaks of respiratory tract infections including pandemics in humans. In the course of its evolution, the virus has acquired many mutations with an ability to cause increased disease severity. A regular molecular surveillance of the virus is essential to mark the evolutionary changes that may cause a shift to the viral behavior. Materials and Methods: Samples of Throat/Nasal swabs were collected from a total of 3715 influenza-like illness cases and screened by Real-time Reverse Transcription-Polymerase Chain Reaction for influenza viruses. Nucleotide sequence analysis was done to identify changes in antigenicity of the virus strains. Results: The present study describes the molecular characteristics of influenza A(H1N1)pdm09 viruses detected in Assam of Northeast India during 2009–2015. Influenza A viruses were detected in 11.4% (425/3715), of which influenza A(H1N1)pdm09 viruses were detected in 41.4% (176/425). The nucleotide sequencing of influenza A(H1N1)pdm09 viruses revealed a total of 17 and 22 amino acid substitutions in haemagglutinin (HA) and neuraminidase (NA) genes of the virus, respectively, compared to contemporary vaccine strain A/California/07/2009. The important mutations detected in HA genes of A/Assam(H1N1)pdm09 strains included E391K, K180Q and S202T. Mutation 'N248D' which has an ability to develop oseltamivir resistance was also detected in NA gene of A/Assam(H1N1)pdm09 strains. Conclusions: Regular molecular surveillance of influenza A(H1N1)pdm09 is important to monitor the viral behavior in terms of increase virulence, drug resistance pattern and emergence of novel strains.


Keywords: Haemagglutinin, influenza A(H1N1)pdm09, neuraminidase, Northeast India, pandemic


How to cite this article:
Biswas D, Dutta M, Sarmah K, Yadav K, Buragohain M, Sarma K, Borkakoty B. Genetic characterisation of influenza A(H1N1)pdm09 viruses circulating in Assam, Northeast India during 2009–2015. Indian J Med Microbiol 2019;37:42-9

How to cite this URL:
Biswas D, Dutta M, Sarmah K, Yadav K, Buragohain M, Sarma K, Borkakoty B. Genetic characterisation of influenza A(H1N1)pdm09 viruses circulating in Assam, Northeast India during 2009–2015. Indian J Med Microbiol [serial online] 2019 [cited 2019 Oct 14];37:42-9. Available from: http://www.ijmm.org/text.asp?2019/37/1/42/264483





 ~ Introduction Top


Influenza virus, a single-stranded negative-sense RNA virus of the family Orthomyxoviridae, is a major cause of acute respiratory illness. Influenza viruses infect 10%–20% of world population and cause 250,000–500,000 deaths annually.[1] Among the three types of influenza viruses (A, B and C), influenza B and C are restricted to human host only while influenza A virus infects a variety of avian and mammalian hosts and carries zoonotic significance. Aquatic avian species such as geese, shorebirds and wild ducks act as the natural reservoirs of influenza A viruses of all subtypes. On the other hand, pigs are susceptible to both human and avian influenza viruses and act as an intermediate host playing vital role in epidemiology and its evolution.[2] Influenza A virus possesses eight segmented genomes encoding 11 proteins. Based on variation of surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA), influenza A viruses are categorised into 18 HA (H1–H18) and 11 NA (N1–N11) subtypes.[3] Influenza viruses have their ability to undergo rapid and consistent genetic and antigenic evolution due to point mutations in genome, especially HA and NA genes and reassortment of gene segments from intra- and inter-species influenza viruses.[4] Consequently, there is emergence of novel variants with increased virulence and pathogenicity that escape the immune system of their hosts resulting in annual outbreaks, epidemics and occasional pandemics. The novel swine origin influenza virus A (H1N1) was responsible for the recent outbreaks and epidemics in various parts of the world which emerged as reassortment between swine influenza viruses of two distinct lineages and Eurasian avian-like swine (triple reassortment).[5],[6] Influenza A(H1N1)pdm09 virus was first reported from Mexico in April 2009, and later, the infection led to spread of disease across 207 countries around the globe, affecting more than 318,925 cases and 3917 deaths.[7] During influenza pandemic wave, the first case of influenza A(H1N1)pdm09 in India was reported from Hyderabad on 16 May 2009 and continued till 2010 with 20,604 cases and 1763 deaths by the end of the year.[8] After the pandemic, the virus continued to reappear in several northern and western states of the country during 2012–2013 with 5044 cases including 405 deaths in 2012[2] followed by 5250 cases and 692 deaths in 2013.[9] The latest resurgence of the influenza A(H1N1)pdm09 in December 2014–March 2015 caused outbreaks in several Indian states accounting for 30,000 cases and 2000 deaths.[8] The susceptibility of human population is said to be directly proportional to the degree of changes in HA and NA genes of H1N1 that are under constant selective pressure to evade the host defense system.[10],[11] Thus, continuous monitoring of influenza A(H1N1)pdm09 virus is essential due to its rapid transmissibility and pathogenicity in humans.[12] In India, the available influenza A(H1N1)pdm09 sequence information shows continuous evolution of the virus.[13] The genetic diversity of influenza A(H1N1)pdm09 viruses remains to be a public health concern and needs year-round monitoring as studied earlier.[14] North Eastern region of India has a different topographical and climatic condition in relation to other parts of India. The North Eastern region of India is also important as it shares 5 international borders, and the transmission of viruses across the border cannot be ruled out. Although the region has experienced every bit of the waves of influenza A(H1N1)pdm09 infection the country underwent, there is lack of information on influenza A(H1N1)pdm09 strains circulating in Northeast India since 2009. Thus, the aim of the present study was to monitor the activity and molecular characterisation of influenza A(H1N1)pdm09 viruses from Assam, Northeast India during 2009–2015.


 ~ Materials and Methods Top


Study site

The present study was carried out at Regional Medical Research Centre, Dibrugarh, Assam, Northeast India during 2009–2015. Patients with symptoms of Influenza-like illness (ILI) attending outpatient department of primary health centres as well as hospitalised cases in Dibrugarh district of Assam, Northeast India were recruited.

Patients and samples

Patients of all age groups meeting the WHO case definition of ILI were enrolled in the study. A case of ILI was defined as a person with sudden onset of fever >38°C and cough or sore throat in the absence of other diagnosis. The onset of fever should be within 3 days of presentation and fever should be measured at the time of presentation.[15] Clinical specimens of nasopharyngeal and throat swabs (nasal swab/TS) were collected in viral transport medium from the enrolled patients and transported to the laboratory in cold condition (+4°C). Clinical history was recorded in a structured case investigation form, and a written informed consent was obtained from all the patients or guardians before collection of samples. The study was approved by the Institutional Human Ethics Committee of Regional Medical Research Centre, Dibrugarh, Assam.

Sample processing and RNA extraction

The collected clinical specimens of nasopharyngeal and throat swabs were vortex mixed followed by centrifugation at 1000 g for 10 min at +4°C. The supernatant of the specimens were aliquoted and frozen at −80°C. Viral RNA was extracted from 140 μl of the specimens using QIAamp Viral RNA mini kit (Qiagen, GmbH, Hilden, Germany) according to manufacturer's instruction with proper biosafety measures.

Molecular detection of Influenza viruses

The extracted RNA was subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) for detection of influenza virus (Type A and Type B). Influenza A-positive samples were further subjected to detection of influenza A(H1N1)pdm09 utilising Taqman chemistry with four sets of primers and probes (Inf-A, SW-A, SW-H1 and RNase P).[16] The reactions were performed using SuperScript III Platinum One step quantitative RT-PCR system with Rox (Invitrogen, USA) following manufacturer's instructions. The RT-PCR was carried out at 50°C for 15 min, 95°C for 2 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 30 s. Influenza A-positive samples were also processed for influenza A/H3N2 and seasonal influenza A/H1N1 by nested RT-PCR using Qiagen one step RT-PCR kit (QIAGEN One Step RT-PCR kit, QIAGEN, USA) and primers as recommended by WHO.[16]

Isolation of viruses

The specimens found positive for influenza A in real-time RT-PCR were attempted for isolation of virus using Madin-Darby canine kidney (MDCK) cell lines. MDCK cells were maintained in minimum essential medium (supplemented with 10% fetal bovine serum, Penicillin [100 U/ml], Streptomycin [100 μg/ml] and Amphotericin B [0.25 μg/ml]) in T-25 flasks and incubated at 37°C with 5% CO2. The specimens (1 ml) were inoculated into MDCK cells with viral growth medium (minimal essential medium with 2 μg/ml TPCK-treated trypsin) and incubated for 1 h followed by addition of viral growth medium. Infected cells were monitored for 7 days and harvested when cytopathic effect was observed. Supernatants from all the T-25 flasks were subjected to HA test using guinea pig red blood cell as per standard method. Subtypes of the HA-positive isolates were identified by HA inhibition test.[17],[18]

Nucleotide sequencing

About 10% of samples found positive for influenza A(H1N1)pdm09 by quantitative PCR were attempted for nucleotide sequencing targeting HA and NA genes of influenza A virus using primers and protocols as recommended by WHO.[19] One Step RT-PCR was carried out in a total volume of 50 μl of master mix, containing 5.0 μl RNA template, 10 μl of 5X RT-PCR buffer, 10 μl 5X Q buffer, 2.0 μl enzyme mix (QIAGEN One Step RT-PCR kit, QIAGEN, USA), 2.0 μl dNTP mix (Promega, Madison, USA), 15 μl nuclease-free water and 3.0 μl each of forward and reverse primers as depicted in [Supplementary Table 1]. The RT-PCR was carried out at 50°C for 30 min, 94°C for 10 min, followed by 40 cycles of amplification at 94°C for 50 s, 55°C for 50 s and 68°C for 2 min, with a final extension of 68°C for 10 min. The amplicons were then separated by agarose gel electrophoresis (2%) and purified using PCR product purification kit (Roche, USA). DNA sequencing was carried out using Big Dye terminator V 3.1 cycle sequencing ready reaction kit (Applied Biosystem, Foster City, CA, USA) with the above primers. The sequences were analysed in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Massachusetts, USA). Raw sequence data were edited, and consensus sequences were constructed using Bioedit version 7.0 (Informer Technologies, Inc).[20]



Phylogenetic and molecular analysis

HA and NA gene sequences of influenza A(H1N1)pdm09 isolates from each period were compared with contemporary sequences available in GenBank. Multiple alignment of nucleotide sequences was done using MEGA 6.0 software (The Pennsylvania State University, University Park, PA.)[21] and the nucleotide and deduced amino acid sequences were used to calculate the respective diversities. Phylogenetic tree of both HA and NA gene sequences were individually constructed based on Maximum Likelihood (ML) method in MEGA 6.0 software with 1000 bootstrap replications. Hasegawa, Kishino, Yano (HKY) + G + I and HKY + G models were used for constructing the HA and NA gene tree, respectively. The antigenic and glycosylation sites of HA and NA gene segments were identified following their comparison to the established antigenic and glycosylation sites. The number and locations of the glycosylation sites on HA and NA were numbered according to the full length HA and NA sequence of A/California/07/2009 (KU933485.1). For prediction of putative glycosylation sites on HA and NA protein, the consensus N-linked glycosylation motif asparagine-X-serine/threonine (N-X-S/T, X can be any amino acid except proline)[22] was scanned manually. To visualise and determine the positions of the glycosylation sites, the three-dimensional (3D) structure of representative HA and NA proteins with different patterns of potential N-glycosylation sites were generated using PyMOL version 1.8.4.0 software (The PyMOL Molecular Graphics System, Schrödinger, LLC, New York).


 ~ Results Top


A total of 3715 (male 1862 and female 1853) ILI cases were recruited during 2009–2015. Samples of all age group ranging from 4 months to 85 years were referred. The influenza A and B viruses were detected in 11.4% (425/3715) and 5.49% (204/3715), respectively. Further subtyping of influenza A led to detection of H3N2 in 58.11% (247/425), influenza A(H1N1)pdm09 in 41.4% (176/425) and seasonal H1N1 in 1.17% (05/425). During the study period, influenza A(H1N1)pdm09 viruses were detected in the year 2009, 2010, 2012 and 2015. The year-wise distribution of influenza A(H1N1)pdm09 cases are shown in [Figure 1]. The males (54.54%, 96/176) were more infected by influenza A(H1N1)pdm09 viruses as compared to females with 45.45% (80/176). The mean age for influenza A(H1N1)pdm09-positive female patients was 15.2 years (standard deviation [SD] ± 11.3) and 17.3 years (SD ± 13.2) in case of males. Age-wise distribution of influenza A(H1N1)pdm09-positive cases are shown in [Table 1], where maximum cases were detected in the adults. The most common symptoms among influenza A(H1N1)pdm09-positive cases were fever (100%), rhinorrhoea (92.61%), cough (78.98%), headache (67.61%) and body ache (52.8%). The detailed clinical symptoms of the patients recorded during the study are listed in [Table 2].
Figure 1: Temporal distribution of A(H1N1)pdm09-positive cases in Dibrugarh, Assam (2009–2015)

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Table 1: Age group-wise distribution of Influenza-positive cases in Dibrugarh, Assam (2009-2015)

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Table 2: Clinical symptoms of influenza A(H1N1)pdm09 virus-positive cases from Dibrugarh, Assam (n=176)

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The sequence analysis of HA gene of influenza A(H1N1)pdm09 virus could be done for 13 representative samples corresponding to the study period. Of the available 13 HA gene sequences, 4 were complete (GenBank accession number: KU310626, KU310627, KU310628 and KU310638) and 9 were partial gene sequences (KU310629, KU310630, KU310631, KU310632, KU310633, KU310634, KU310635, KU310636 and KU310637). Likewise, 12 NA gene sequencing of influenza A(H1N1)pdm09 could be done, of which 5 were complete sequences (KU310639, KU310640, KU310641, KU310642 and KU310643) and 7 were partial sequences (KU310644, KU310645, KU310646, KU310647, KU310648, KU310649 and KU310650). The influenza A(H1N1)pdm09 strains detected during the study period are hereafter designated as A/Assam(H1N1)pdm09.

Sequence analysis of HA gene of all the 13 A/Assam(H1N1)pdm09 strains exhibited a nucleotide distance of 0.6%–2.1% with vaccine component (A/California/07/2009). The A/Assam(H1N1)pdm09 strains from 2009, 2010 and 2012 belonged to a large clade including A/California/07/2009 and contemporary strains. However, 2015 A/Assam(H1N1)pdm09 strains formed a distinct cluster with statistical significance (bootstrap >75%) and belong to contemporary strains from different geographical origin. Phylogenetic tree of HA is shown in [Figure 2]. Deduced amino acid sequences of HA gene of A/Assam(H1N1)pdm09 strains showed 0.7%–1.7% diversity when compared with A/California/07/2009 strain. Further, comparison of deduced amino acid sequences with 1918 strain suggests that the distance between all the circulating influenza A/Assam(H1N1)pdm09 ranged between 9.3% and 10%. A total of 17 amino acid substitutions were observed in A/Assam(H1N1)pdm09 strains as compared to A/California/07/2009 strain. Three conserved amino acid substitutions at positions P100S, S220T and I338V of HA gene were observed in all the sequences of A/Assam(H1N1)pdm09 strains [Table 3]. Amino acid substitution at position E391K was observed in all the A/Assam(H1N1)pdm09 strains except in 2010, where 2 additional substitutions at position T11P and S200P were observed.
Figure 2: Molecular phylogenetic analysis of haemagglutinin gene of Influenza A(H1N1)pdm09 from Assam, Northeast India during 2009–2015. The haemagglutinin gene sequence were obtained from National Center for Biotechnology Information GenBank and aligned with A/Assam(H1N1)pdm09 sequence by ClustalW in MEGA software version 6.0. The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model in MEGA software. The robustness of the tree was inferred by bootstrapping 500 replicates. The contemporary vaccine strain is shown as solid triangle (A/California/07/2009) and A/Assam(H1N1)pdm09 strains are shown as solid spheres

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Table 3: Amino acid substitutions of haemagglutinin gene of A/Assam(H1N1)pdm09 strains compared with A/California/09/HINIpdm (2009-2015)

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The NA gene sequence analysis of 12 A/Assam(H1N1)pdm09 strains showed 0.1%–0.76% nucleotide diversity compared to A/California/07/2009 strain. The A/Assam(H1N1)pdm09 strain from 2009, 2010, 2012 and 2015 belong to the clade containing A/California/07/2009 and contemporary strains. Phylogenetic tree of NA gene is shown in [Figure 3]. Deduced amino acid sequences of NA gene of A/Assam(H1N1)pdm09 strain showed a total of 22 substitutions at 21 different sites compared with A/California/07/2009 strain where substitution N248D was found to be conserved across A/Assam(H1N1)pdm09 strains. The details of all the substitution in NA protein of A/Assam(H1N1)pdm09 strains are given in [Table 4]. The amino acid substitutions of HA gene segment at position K180Q, S202T and S220T of A/Assam(H1N1)pdm09 strains fall at the antigenic sites Sa, Sb and Ca1, respectively[23] [Supplementary Figure 1]. While substitutions at positions N200S (2012 and 2015) and K432E (2015) of NA gene segments fall at the antigenic site 2 and antigenic site 7, respectively [Supplementary Figure 2]. The deduced amino acid sequences of NA gene with contemporary vaccine component showed histidine residues at 275 position exhibiting all the A/Assam(H1N1)pdm09 strains to be sensitive for the drug oseltamivir. The N-glycosylation sites observed in HA protein of A/Assam(H1N1)pdm09 strains were consistent with those observed in A/California/07/2009 strain. The N-glycosylation sites present in NA protein of A/Assam(H1N1)pdm09 strains were also consistent with contemporary vaccine strain besides a loss of an N-glycosylation site at position N386S and N386K in strains belonging to 2010 and 2015, respectively.
Figure 3: Molecular phylogenetic analysis of neuraminidase gene of Influenza A(H1N1)pdm09 from Assam, Northeast India during 2009–2015. The neuraminidase gene sequence was obtained from National Center for Biotechnology Information GenBank and aligned with A/Assam(H1N1)pdm09 sequence by ClustalW in MEGA software version 6.0. The evolutionary history was inferred using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model in MEGA software. The robustness of the tree was inferred by bootstrapping 500 replicates. The contemporary vaccine strain is shown as solid triangle (A/California/07/2009), and A/Assam(H1N1)pdm09 strains are shown as solid spheres

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Table 4: Amino acid substitutions of neuraminidase gene of A/Assam(H1N1)pdm09 strains compared with A/California/09/HINIpdm (2009-2015)

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 ~ Discussion Top


The present study mainly focused on monitoring the activity of circulating A/Assam(H1N1)pdm09 virus in Assam, Northeast India to unravel the molecular changes compared to contemporary vaccine and other circulating strains. During the study period, influenza A(H1N1)pdm09 viruses were detected in the year 2009, 2010, 2012 and 2015. The positivity of influenza A(H1N1)pdm09 virus was found to be mostly in the adults, as observed in other similar studies.[24],[25] The common influenza symptoms such as fever, cough, sore throat and rhinorrhoea are strongly associated with positive cases. Among other clinical symptoms, breathlessness, headache and body ache were mostly common among the adult-positive cases.

The HA gene is the highly variable gene among the eight genes of influenza virus. Consequently, most of the study target the HA gene for molecular analysis of influenza virus.[26] There were altogether 17 amino acid substitutions detected in HA protein of A/Assam(H1N1)pdm09 strains deduced from HA gene. The substitution in HA gene at E391K was found in all the A/Assam(H1N1)pdm09 strains, except 2010 which has been found to be an important site for membrane fusion.[27] Influenza A(H1N1)pdm09 variant with HA mutation E391K along with changes in certain markers of NA gene has been found to be associated with some fatal cases and linked with vaccine breakthroughs.[28] It was noted that the substitution at T11P of 2010 and A152T of 2015 strains were unique in A/Assam(H1N1)pdm09 strains when compared with contemporary influenza A(H1N1)pdm09 strains around the globe.

The NA gene of influenza virus is known to be a drug target for the prevention of influenza virus infection.[29] Thus, mutation in the NA gene may reduce the drug susceptibility during infection. A total of 22 amino acid substitutions detected in NA protein of A/Assam(H1N1)pdm09 strains deduced from NA gene. On the other hand, NA gene sequences of A/Assam(H1N1)pdm09 with respect to that of A/California/07/2009 showed a single significant conserved substitution at position N248D. The N248D mutation has been found to alter an antibody recognition site and thus is important for vaccine development.[30] The H275Y mutation in NA gene has been reported for oseltamivir resistance.[31] However, in our study, deduced amino acid sequences of NA gene of A/Assam(H1N1)pdm09 strains with contemporary vaccine component did not show H275Y mutation unlike other circulating influenza A(H1N1)pdm09 strains of India, as previously studied.[32]

Among all the substitutions detected in the HA gene of A/Assam(H1N1)pdm09 strains, three substitutions – K180Q, S202T and S220T belong to the antigenic sites Sa, Sb and Ca1, respectively [Supplementary Figure 1], which may affect the interaction of HA with its receptor as has been reported previously.[33],[34]

The 3D structure of HA protein reveals that the receptor-binding sites comprise of three structural elements, namely an alpha helix and two loops (130 loop, HA1 134–138 and 220 loop, HA1 221–228).[35] Incidentally, mutation S220T of A/Assam(H1N1)pdm09 strains localised in the defined antigenic site falls at the receptor-binding pocket in 220 loop. Therefore, this amino acid substitution will influence both the specificity of receptor recognition as well as antibody binding.


 ~ Conclusions Top


The molecular characterisation of influenza A(H1N1)pdm09 viruses in the region over a period of 6 years (2009–2015) has revealed substitutions in HA and NA genes, some of which were conserved in A/Assam(H1N1)pdm09 strains. There is paucity of information regarding the molecular epidemiology of influenza A(H1N1)pdm09 strains circulating in Northeastern region of India. This is important as the region is porous being shared by international boundaries with China, Bangladesh, Bhutan, Nepal and Myanmar. This study along with others will help to understand the global epidemiology of influenza A(H1N1)pdm09 virus. The phylogenetic analysis in the present study might also serve as an important tool to understand the evolutionary dynamics of circulating influenza virus strains. However, further studies will be required to monitor the phenotypic changes associated with such strains.

Financial support and sponsorship

The study was supported by intramural fund of ICMR-Regional Medical Research Centre, Dibrugarh, Assam, India.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04