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

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.

Introduction

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.[12] 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.[3456] 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.[89] Apart from crossing boundaries, species-jump from foxes to dogs has also been observed.[1011] Phylogenetic studies allow for identifying the lineage, origin, and type of circulating viral strains into new geographic areas.[1213] 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.[141516] This study focuses on molecular characterization of street RABV isolated from human clinical specimens from Maharashtra, India.

Materials and Methods

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

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 1a]. 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 1b].

Table 1

Clinical samples and reference strains included in the study

Cluster no.	Laboratory ID	Age/sex	Post mortem/ante mortem	GenBank accession no.
1	HICVYR1812*	60/F	PM	KJ201895
	HICVYR1912*	15/F	PM	KJ201896
2	HICVYR2012*	6/M	PM	KJ201897
	HICVYR0113*	55/M	PM	KJ201891
3	HICVYRSI**	—	—	KJ201899
	HICVYRSII**	—	—	KJ201900
4	HICVYR3411*	4/M	PM	KJ201898
	HICVYR0212*	56/M	PM	KJ201892
	HICVYR0412***	6/M	AM	KJ201893
	HICVYR0512#	6/M	AM	KJ201894
	HICVYRBHK##	—	—	KJ201901
	HICVYRMNTCVS###	—	—	KJ201902

*Human brain, **Canine brain, ***Human CSF, #Human nuchal biopsy, ##BHK21-adapted CVS, ###Mouse brain-adapted CVS

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

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

Discussion

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.