sábado, 30 de agosto de 2014

Ahead of Print -Detection of Measles Virus Genotype B3, India - Volume 20, Number 10—October 2014 - Emerging Infectious Disease journal - CDC

Ahead of Print -Detection of Measles Virus Genotype B3, India - Volume 20, Number 10—October 2014 - Emerging Infectious Disease journal - CDC


Volume 20, Number 10—October 2014


Detection of Measles Virus Genotype B3, India



Technical Appendicies

To the Editor: Molecular epidemiologic investigations and virologic surveillance contribute notably to the control and prevention of measles (1). Nearly half of measles-related deaths worldwide occur in India, yet virologic surveillance data are incomplete for many regions of the country (2,3). Previous studies have documented the presence of measles virus genotypes D4, D7, and D8 in India, and genotypes D5, D9, D11, H1, and G3 have been detected in neighboring countries (3,4).
Kerala, India’s southernmost state, has high measles vaccination coverage compared with many other states in the country; however, the disease is still endemic in the region. Two districts, Thiruvananthapuram and Malappuram, report the highest numbers of cases (5). Baseline data on circulating measles virus genotypes are needed for measles elimination, but such data are not available for Kerala. In this context we performed a pilot genetic analysis of the measles virus strains circulating in Thiruvananthapuram, the capital of Kerala. We used throat and nasopharyngeal swab and serum samples from children admitted to Sree Avittom Thirunal Hospital during measles outbreaks occurring March–August 2012 in this study.
We used the Vero/human-SLAM cell line (Ono et al. J. Virol. 2001;75:4399–4401). for isolation of measles virus from throat and nasopharyngeal swab samples. For serologic confirmation of cases, we used a commercial measles IgM ELISA kit (IBL International GmbH, Hamburg, Germany). Virus genotyping was based on the 450-nt coding sequence for the carboxyl terminus of nucleoprotein (N) of measles virus, as recommended by the World Health Organization (3,6). We extracted RNA from the samples using TRIzol reagent (GIBCO-BRL, Grand Island, NY, USA). We performed reverse transcription PCR using a SuperScript One-Step RT-PCR kit with a Platinum Taq system (Invitrogen, Carlsbad, CA, USA) and previously described primers (3,6). Amplicons were subjected to bidirectional sequencing using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). We edited and aligned nucleotide sequences using Bio Edit 7.1.11 software (7). Phylogenetic analysis was performed by using the maximum-likelihood method implemented in the MEGA5 program (8) to compare the determined N gene sequences with the World Health Organization reference sequences of the 24 known measles genotypes.
PCR products could be amplified from 16 of the 24 samples analyzed. Ten samples provided high quality sequence reads for the N gene coding region, which were used for further analysis. Clinical and demographic data for these 10 cases, virus isolation status, and GenBank accession numbers of the sequences are summarized in the Table.
Thumbnail of Phylogenetic analysis of sequences coding for the carboxyl terminal end (450 nt) of nucleoprotein (N) from 24 reference strains and 10 clinical strains of measles virus. Maximum-likelihood analysis was performed with 1,000 bootstrap replications by using Kimura2-parameter correction with gamma-distributed settings. Solid triangles indicate strains from the current study in Kerala, India, 2012. Scale bar represents nucleotide substitutions per site.
Figure. Phylogenetic analysis of sequences coding for the carboxyl terminal end (450 nt) of nucleoprotein (N) from 24 reference strains and 10 clinical strains of measles virus. Maximum-likelihood analysis was performed with...
Phylogenetic analysis revealed 1 of the 10 measles virus strains to be of genotype D8 (Figure), a genotype previously found to be circulating in Kerala and in other regions of India (3,6,9,10). The other 9 virus strains were closely related to B3 genotype reference strains, indicating circulation of the B3 genotype in Kerala (Figure). The nucleotide sequences of 7 of the 9 strains were identical, indicating a single chain of transmission. The remaining 2 samples showed sequence divergence, indicating independent sources of infection. In a phylogenetic analysis comparing the Kerala B3 genotypes and a dataset of global measles B3 genotypes selected from GenBank, the strains from Kerala formed a separate cluster (Technical Appendix[PDF - 621 KB - 1 page]Figure). This cluster also contains a strain from Germany (MVs/Regensburg.DEU/37.12/). The strains in this cluster show close identity to a measles strain identified in the state of New York, USA. A search of the MeaNS (Measles Nucleotide Surveillance) database revealed that the Kerala B3 sequences had the closest match to the strain isolated in Germany mentioned above and also to a strain from the Sultanate of Oman (MVs/Muscat.OMN/38.11/).
The B3 genotype identified in this study could be a previously undetected genotype endemic to India or a recent importation. B3 is endemic to many countries in Africa, and its importation into Europe and North America and elsewhere has been described (4). Further studies with samples from a wider geographic area of Kerala are required to determine the spread and genetic diversity of these strains and ascertain their relationship to the global B3 strains. It would also be of interest to determine whether B3 strains co-circulate with D8 strains, whether they will eventually replace D8 as the predominant genotype in Kerala, or if they will cease to exist as the outbreak diminishes. This report underscores the need for systematic nationwide measles virus surveillance in India to identify all endemic virus genotypes and to monitor importation of new virus strains from other countries.
This study was approved by the Institutional Human Ethics Committee of the Rajiv Gandhi Centre for Biotechnology. It was carried out as part of the National Virology Network Laboratory Program of the Indian Council of Medical Research, which provided financial support
Vijesh S. KuttiattComments to Author , Sanughosh Kalpathodi, Sindhu T. Gangadharan, Lalitha Kailas, Easwaran Sreekumar, Suja M. Sukumaran, and Radhakrishnan R. Nair
Author affiliations: Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India (V.S. Kuttiatt, S. Kalpathodi, E. Sreekumar, S.M. Sukumaran, R.R. Nair)Sree Avittom Thirunal Hospital, Government Medical College,Thiruvananthapuram (S.T. Gangadharan, L. Kailas)


We thank Sunil Vaidya, for providing the Vero/human-SLAM cell line and laboratory-adapted measles viral strains D4 and D8. We thank Iype Joseph for useful discussions; Manoj P for help with DNA sequencing; Sara Jones and Rachy Abraham for assistance with cell culture; and Pradip Fulmali, Sanjai D, and technical personnel of Rajiv Gandhi Centre for Biotechnology for diagnostic support.


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Technical Appendix

Suggested citation for this article: Kuttiatt VS, Kalpathodi S, Gangadharan ST, Kailas L, Sreekumar E, Sukumaran SM, et al. Detection of measles virus genotype B3, India [letter]. Emerg Infect Dis. 2014 Oct [date cited]. http://dx.doi.org/10.3201/eid2010.130742

DOI: 10.3201/eid2010.130742

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