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 ::  Abstract
 :: Introduction
 ::  Materials and Me...
 :: Results
 :: Discussion
 ::  References
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BRIEF REPORT
Year : 2016  |  Volume : 62  |  Issue : 2  |  Page : 105-108

Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India


Department of Virology and Immunology, Haffkine Institute for Training, Research and Testing, Mumbai, Maharashtra, India

Date of Submission23-Apr-2016
Date of Decision30-Sep-2015
Date of Acceptance06-Jan-2016
Date of Web Publication15-Apr-2016

Correspondence Address:
P Charan
Department of Virology and Immunology, Haffkine Institute for Training, Research and Testing, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0022-3859.175006

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 :: Abstract 

Context: Rabies poses a serious public health concern in developing countries such as India. Aims: The study focuses on molecular diagnosis of street rabies virus (RABV) from human clinical specimens received from Maharashtra, India. Materials and Methods: Nucleoprotein gene from eight (of total 20 suspected samples) rabies cases that tested positive for rabies antigen using reverse transcriptase-polymerase chain reaction (RT-PCR) were sequenced. Results: Sequence analysis using basic local alignment search tool (BLAST) and multiple sequence alignment (MSA) and phylogenetic analysis showed similarity to previously reported sequences from India and those of Arctic lineages. Conclusions: The circulating RABV strains in Maharashtra, India show genetic relatedness to RABV strains reported from Indo-Arctic lineages and India-South and Japan.


Keywords: Epidemiology, nucleoprotein gene, phylogenetic analysis, rabies


How to cite this article:
Mehta S, Charan P, Dahake R, Mukherjee S, Chowdhary A. Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India. J Postgrad Med 2016;62:105-8

How to cite this URL:
Mehta S, Charan P, Dahake R, Mukherjee S, Chowdhary A. Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India. J Postgrad Med [serial online] 2016 [cited 2019 Aug 19];62:105-8. Available from: http://www.jpgmonline.com/text.asp?2016/62/2/105/175006



 :: Introduction Top


Rabies is a zoonotic disease that leads to fatal encephalitis in humans and other mammalian species. Rabies virus (RABV) belongs to the Rhabdoviridae family and Lyssavirus genera. Rabies is a major problem in Asia. India is reported to have the highest incidences of rabies globally. [1],[2] A large number of dog bite cases are reported annually from Mumbai city, Maharashtra, India; however, dog bite deaths are more often seen in rural Maharashtra, India.

Arctic-like RABV strains are the most predominantly circulating strain in Asian countries. [3],[4],[5],[6] In the Indian subcontinent, only Andaman and Nicobar islands, India and Lakshadweep group of islands, India are declared rabies-free. [7] While Japan as well is rabies-free now, [8] there have been intermittent cases of imported rabies reported in Japan through travelers from rabies-prevalent countries. [8],[9] Apart from crossing boundaries, species-jump from foxes to dogs has also been observed. [10],[11] Phylogenetic studies allow for identifying the lineage, origin, and type of circulating viral strains into new geographic areas. [12],[13]

For routine diagnosis, N gene is the preferred target as it is highly conserved, strongly expressed, allows for antigenic classification of Lyssavirus, and can be employed to determine genetic differences within circulating strains. [14],[15],[16] This study focuses on molecular characterization of street RABV isolated from human clinical specimens from Maharashtra, India.


 :: Materials and Methods Top


Specimens

A total of 29 specimens from 20 (eight postmortem, 12 antemortem) patients suspected of rabies were received for rabies testing between January 2011 and February 2013 that included four corneal impression smears (CIS), seven nuchal biopsies, two saliva cultures, nine cerebrospinal fluid (CSF), and seven brain biopsies. In some cases, more than one type of specimen was received from the same suspect. CIS were tested using fluorescent antibody test (FAT) [17] while nuchal biopsy, CSF, and brain biopsy were tested using FAT and reverse transcriptase-polymerase chain reaction (RT-PCR). Rabies-positive cases, where an alternate specimen to corneal impression smear was available, were taken up for sequencing study. Standard laboratory strains and isolates from canine brains (street virus) were included as reference strains.

Nested reverse transcriptase-polymerase chain reaction

Brain/nuchal biopsy were homogenized and centrifuged to separate supernatant while CSF was directly used for RNA extraction as per the manufacturer's instructions QIAamp ® Viral RNA mini kit (Qiagen, India) and used for detection of RABV N gene using a protocol by Nagaraj et al. with minor modifications. [18] Primers were as follows: Outer Forward: 5'-GCTCTAGAACACCTCTACAATGGATGCCGACAA-3', Outer Reverse: 5'- GGATTGAC(AG)AAGATCTTGCTCAT-3', Inner Forward: 5' TTGT(AG)GA(TC)CAATATGAGTACAA-3', and Inner Reverse: 5' CCGGCTCAAACATTCTTCTTA-3'.

Nucleotide sequencing

The purified samples were sent to M/s. Ocimum Biosolutions Ltd. (Hyderabad, India) for sequencing. Standard reference RABV nucleotide sequence (GenBank Accession # NC_001542) and other sequences submitted to GenBank were used for multiple sequence alignment (MSA). The sequences were aligned using Clustal W algorithm in Molecular Evolutionary Genetics Analysis (MEGA) v5.10, [19] and BioEdit Sequence alignment editor software [20] was used for phylogenetic analysis.


 :: Results Top


Reverse transcriptase-polymerase chain reaction and sequencing

Of the total 20 patients suspected of rabies, eight tested positive by RT-PCR. Their gene sequences and amino acid sequences were determined and compared with sequences of RABV isolates from GenBank.

Phylogenetic analysis

MSA of all the sequences showed similarity-based grouping leading to the formation of four different clusters [Table 1] and [Figure 1]a. On comparison with Asian RABV isolates, the phylogenetic tree exhibited closeness of Cluster 1 to India-South and Arctic lineages while Cluster 2 grouped closer to sequences from Japan and Korea. Similarly, Cluster 3 showed grouping with India-South isolates and Cluster 4 with sequences from Japan [Figure 1]b.
Figure 1: (a) Phylogenetic analysis of N gene sequences of clinical rabies isolates from the present study showing cluster formation in reference to standard sequence from GenBank (b) Phylogenetic tree of N gene sequences of clinical rabies isolates in comparison with Asian isolates from GenBank. The filled circles () are standard laboratory sequences. The tree is rooted on the Rabies N gene reference sequence

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Table 1: Clinical samples and reference strains included in the study


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Identity plot

Analysis of the deduced amino acid sequences with other reported sequences revealed substitutions as shown in [Figure 2].
Figure 2: Identity plot of rabies nucleoprotein amino acid sequence showing polymorphisms

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 :: Discussion Top


A multicentric survey revealed that among the total rabies-related deaths reported, 38.3% were children from rural areas and an appalling 74.5% were adults in urban areas. [7] Such reports emphasize the need for ensuring vaccine-preventable deaths and regular surveillance data and characterization of the circulating strains in endemic areas. In this study, we have performed molecular diagnosis of rabies-positive clinical samples received from various parts of Maharashtra, India in order to assess the type of RABV circulating strains. Predominant circulation of RABV strains with Arctic lineages in Asian countries has been reported earlier. [12] Our study shows results coherent with these observations. Interestingly, Cluster 2 and Cluster 4 showed similarities to sequences from Japan which has now been declared rabies-free. [21] This may be a classic example of cross-border spread of the disease. Based on the amino acid identity plot, probable polymorphisms were observed at positions I84T/S, S135P, and V179I. Geographic distribution of the clusters indicates that prevalence of rabies may be due to proximity of these countries. Surveillance studies and epidemiological data will prove vital that include registration and vaccination of domestic dogs, [21] regular awareness programs, better reporting network, and facilities for suspected cases. The government may strengthen the networking among anti-RABV clinics, veterinary hospitals, animal control organizations, medical institutions, and diagnostic centers to deal with people and animals that may have come in contact with confirmed rabies cases. To our knowledge, this is the first ever report on molecular characterization of circulating RABV strains in Maharashtra, India. Complete genome sequencing can provide insight into the variations in original lineage and highlight the gene pool of circulating street RABV strains.

Sequence data

GenBank accession numbers for the sequences reported in our study are KJ201891-KJ201902.

Financial support and sponsorship

Nil.

Conflicts of interest

We hereby declare that none of the authors of this paper have any conflict of interest.

 
 :: References Top

1.
World Health Organization. WHO Expert Consultation on Rabies. Second report. World Health Organ Tech Rep Ser 2013:1-139.  Back to cited text no. 1
    
2.
Kole AK, Roy R, Kole DC. Human rabies in India: A problem needing more attention. Bull World Health Organ 2014;92:230.  Back to cited text no. 2
    
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Lang SL, Tao XY, Guo ZY, Tang Q, Li H, Yin CP, et al. Molecular characterization of viral G gene in emerging and re-emerging areas of rabies in China, 2007 to 2011. Virol Sin 2012; 27:194-203.  Back to cited text no. 3
    
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Pant GR, Lavenir R, Wong FY, Certoma A, Larrous F, Bhatta DR, et al. Recent emergence and spread of an arctic-related phylogenetic lineage of rabies virus in Nepal. PLoS Negl Trop Dis 2013;7:e2560.  Back to cited text no. 4
    
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Yang DK, Park YN, Hong GS, Kang HK, Oh YI, Cho SD, et al. Molecular characterization of Korean rabies virus isolates. J Vet Sci 2011;12:57-63.  Back to cited text no. 5
    
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Nadin-Davis SA, Sheen M, Wandeler AI. Recent emergence of the Arctic rabies virus lineage. Virus Res 2012;163:352-62.  Back to cited text no. 6
    
7.
Sudarshan MK, Madhusudana SN, Mahendra BJ, Rao NS, Ashwath Narayana DH, Abdul Rahman S, et al. Assessing the burden of human rabies in India: Results of a national multi-center epidemiological survey. Int J Infect Dis 2007;11:29-35.  Back to cited text no. 7
    
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Yamamoto S, Iwasaki C, Oono H, Ninomiya K, Matsumura T. The first imported case of rabies into Japan in 36 years: A forgotten life-threatening disease. J Travel Med 2008;15:372-4.  Back to cited text no. 8
    
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Tobiume M, Sato Y, Katano H, Nakajima N, Tanaka K, Noguchi A, et al. Rabies virus dissemination in neural tissues of autopsy cases due to rabies imported into Japan from the Philippines: Immunohistochemistry. Pathol Int 2009;59:555-66.  Back to cited text no. 9
    
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Madhusudana SN, Mani R, Ashwin YB, Desai A. Rabid fox bites and human rabies in a village community in southern India: Epidemiological and laboratory investigations, management and follow-up. Vector Borne Zoonotic Dis 2013;13:324-9.  Back to cited text no. 10
    
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David D, Yakobson B, Smith J, Stram Y. Molecular epidemiology of rabies virus isolates from Israel and other middle-and Near-Eastern countries. J Clin Microbiol 2000;38:755-62.  Back to cited text no. 11
    
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Nadin-Davis SA, Turner G, Paul JP, Madhusudana SN, Wandeler AI. Emergence of Arctic-like rabies lineage in India. Emerg Infect Dis 2007;13:111-6.  Back to cited text no. 12
    
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Nagarajan T, Mohanasubramanian B, Seshagiri EV, Nagendrakumar SB, Saseendranath MR, Satyanarayana ML, et al. Molecular epidemiology of rabies virus isolates in India. J Clin Microbiol 2006; 44:3218-24.  Back to cited text no. 13
    
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Heinemann MB, Fernandes-Matioli FM, Cortez A, Soares RM, Sakamoto SM, Bernardi F, et al. Genealogical analyses of rabies virus strains from Brazil based on N gene alleles. Epidemiol Infect 2002;128:503-11.  Back to cited text no. 14
    
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Jayakumar R, Tirumurugaan KG, Ganga G, Kumanan K, Mahalinga Nainar A. Characterization of nucleoprotein gene sequence of an Indian isolate of rabies virus. Acta Virol 2004;48:47-50.  Back to cited text no. 15
    
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Zienius D, Sajute K, Zilinskas H, Stankevicius A. Phylogenetic analysis of the rabies virus n-coding region in lithuanian rabies isolates. Acta Vet Brno 2009;78:273-80.  Back to cited text no. 16
    
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CONTROL CFD, PREVENTION. Protocol for postmortem diagnosis of rabies in animals by direct fluorescent antibody testing: A minimum standard for rabies diagnosis in the United States. 2010.   Back to cited text no. 17
    
18.
Nagaraj T, Vasanth JP, Desai A, Kamat A, Madhusudana SN, Ravi V. Ante mortem diagnosis of human rabies using saliva samples: Comparison of real time and conventional RT-PCR techniques. J Clin Virol 2006;36:17-23.  Back to cited text no. 18
    
19.
Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-9.  Back to cited text no. 19
    
20.
Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series; 1999.  Back to cited text no. 20
    
21.
Takahashi-Omoe H, Omoe K, Okabe N. Regulatory systems for prevention and control of rabies, Japan. Emerg Infect Dis 2008;14:1368-74.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2]
 
 
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