Characterization of nucleoprotein gene sequence of an Indian isolate of rabies virus

Rabies occurs in all parts of Indian sub-continent except Andaman and Nicobar and Lakshadweep group of islands. The full-length nucleoprotein (N) gene sequence of a rabies virus isolate from India is reported for the first time and the same has been compared with available N gene sequences from the database. A central domain of 230 amino acids (aa) from aa 141 to aa 370 exhibited more than 95% similarity. There were 8 amino acid positions (aa 29, 32, 38, 84, 119, 379, 438, and 439) at which substitution was unique for Indian isolates but common for laboratory strains. In antigenic epitopes, except for a single amino acid difference at the antigenic site IV, the amino acids were conserved. The Indian isolate also possessed two Bam HI sites (aa 247 and 278), while the other Asian isolates had only one site at aa 278 or were not digested with Bam HI at all. Phylogenetic analysis also demonstrated that the Indian isolate was closely related to the Sri Lankan isolate and grouped in the cluster that comprised of the isolates from other Asian countries namely China and Pakistan.     

Genetic Characterization of Rabies Virus Isolates in Korea

In investigation of the genetic characteristics of rabies viruses in Korea, the nucleotide and deduced amino acid sequences of the nucleoprotein (N) gene were determined in four Korean rabies virus strains obtained from dogs and raccoons, and were compared with published sequences for non-Korean rabies viruses. Three Korean rabies virus isolates had identical nucleotide sequences, and the fourth differed at only one nucleotide position. The Korean virus isolates had 84.5–92.0% nucleotide sequence similarity and 94.0–99.2% amino acid sequence similarity with non-Korean rabies virus isolates. In a phylogenetic tree based on partial nucleotide sequences of the N gene, the Korean rabies viruses formed a single cluster closely related to Arctic rabies viruses (FXCAN, 9141RUS, and 94260NEP). However, they were divergent from other Asian rabies viruses (94256SRL, 8677MAL, ChiNo.7, Phil 12301, and Mdn1278).*The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY730595, AY730596 and AY730597.     

Isolation of bluetongue virus serotype 1 (BTV-1) from goats and its phylogenetic relationship to other BTV-1 isolates worldwide based on full-length sequence of genome segment-2

Eight bluetongue viruses (BTV) were isolated in BHK-21 cell culture from blood of goats suffering from peste des petits ruminants. These viruses were identified as BTV serotype 1 (BTV-1) by RT-PCR using VP2-gene-based primers coupled with sequencing of the PCR products. All of the isolates showed similar genome migration profile in 8% polyacrylamide gel electrophoresis. The genome segment-2 (seg-2) of one isolate (MKD18/India/2008) was amplified piecemeal by overlapping PCR, and the products were sequenced to obtain full-length seg-2. Phylogenetic analysis based on the seg-2 sequence revealed that MKD18 is closely related to Australian BTV-1 isolates, with 86.3–86.8% nucleotide identity. Phylogenetic analysis based on the partial sequence of seg-2 (541 bp, nucleotides 1,304–1,844) showed that the Indian BTV-1 isolates, namely, MKD18, Avikanagar, Sirsa-3 and Chennai, are very closely related to each other, with more than 99.6% nucleotide identity. Although a high degree of similarity exists, the Indian BTV-1 isolates collected over the past 25 years should be studied to demonstrate the co-existence of different VP2 antigenic profiles.     

Molecular epidemiology of rabies virus isolates from Israel and other middle- and Near-Eastern countries

A total of 226 isolates of rabies virus from different areas of Israel, including three human isolates and one sample from South Lebanon were identified between 1993 and 1998 by direct immunofluorescence using monoclonal antibodies to the viral nucleoprotein (N). An epidemiological survey based on nucleotide sequence analysis of 328 bp from the C terminus of the N coding region and the noncoding region between the nucleoprotein and the phosphoprotein (NS gene) was performed. Phylogenetic analysis of the isolates from Israel showed that they were related geographically, but not according to host species. Five variants, related groups distributed among four geographical regions, were identified. In each region, rabies virus was isolated from more than one animal species. A comparison of the sequence analysis of rabies virus samples from the rest of world revealed a 2-nucleotide change that distinguished the Middle East variants from the rest.     

Molecular characterization of KGH, the first human isolate of rabies virus in Korea

The complete genome sequence of the KGH strain of the first human rabies virus, which was isolated from a skin biopsy of a patient with rabies, whose symptoms developed due to bites from a raccoon dog in 2001. The size of the KGH strain genome was determined to be 11,928 nucleotides (nt) with a leader sequence of 58 nt, nucleoprotein gene of 1,353 nt, phosphoprotein gene of 894 nt, matrix protein gene of 609 nt, glycoprotein gene of 1,575 nt, RNA-dependent RNA polymerase gene of 6,384 nt, and trailer region of 69 nt. Sequence similarity was compared with 39 fully sequenced rabies virus genomes currently available, and the result showed 70.6–91.6 % at the nucleotide level, and 82.8–97.9 % at the amino acid level. The deduced amino acids in the viral protein were compared with those of other rabies viruses, and various functional regions were investigated. As a result, we found that the KGH strain only had a unique amino acid substitution that was identified to be associated either with host immune response and pathogenicity in the N protein, or with a related region regulating STAT1 in the P protein, and related to pathogenicity in G protein. Based on phylogenetic analyses using the complete genome of 39 rabies viruses, the KGH strain was determined to be closely related with the NNV-RAB-H strain and transplant rabies virus serotype 1, which are Indian isolates, and was confirmed to belong to the Arctic-like 2 clade. The KGH strain was most closely related to the SKRRD0204HC and SKRRD0205HC strain when compared with Korean animal isolates, which was separated around the same time and place, and belonged to the Gangwon III subgroup.     

Molecular epidemiology of rabies in Guangxi Province, south of China.

BACKGROUND: Surveillance data for rabies in Guangxi Province in China showed that human rabies cases have gradually increased since 1996. OBJECTIVE: To evaluate the epidemiology of rabies at the molecular level and provide suggestions for effective prevention of rabies in Guangxi. STUDY DESIGN: Since 2000, 1569 brains from suspected rabid animals were collected from different areas of Guangxi. Rabies virus was isolated from 42 samples. RT-PCR was used to amplify a 455 nucleotide segment of the 3'-terminal of the N gene. The sequencing data from that segment was used for phylogenetic analysis. RESULTS: Nucleotide homology comparisons and phylogenetic tree analysis based on this sequence indicated that all the rabies virus isolates from Guangxi belonged to genotype 1 and could be divided into four groups. Groups I, II and IV included 23, 10 and 8 isolates, respectively. These had nucleotide homologies of 97.1-100%, 98.2-100% and 99.1-99.6%, respectively. Only the GXN119 strain belonged to group III. Group I had two group-specific mutations: T90N and E110D. Group II had one group-specific mutation of T42S. CONCLUSIONS: This study showed that rabies virus isolates from Guangxi have a close genetic relationship and topographical distribution.     

Molecular characterization of the full-length genome of a rabies virus isolate from India

Rabies is an important public health problem in South East Asia, with cases in this part of the world contributing to about 70% of the global burden. A large number of rabies cases occur in India, however, there is no organized system of surveillance and hence there is a lack of reliable data. Moreover, comprehensive molecular epidemiological studies have not been performed on Indian virus isolates. In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus obtained from the brain of an infected patient. Comparison of the genomic sequence with those of the ten fully sequenced rabies strains available in GenBank showed nucleotide homology ranging from 97% with AY956319 to 81% with AY705373. Amino acid homology of nucleoprotein ranged from 99.7% with AY352493 to 92% with DQ875051. In case of the glycoprotein gene, the homology ranged from 98.8% with AY956319 to 87.2 % with AY705373. An extensive nucleoprotein, glycoprotein, and full-length genome-based phylogenetic analysis was performed along with sequences available from the GenBank. Phylogenetic analysis of the complete genome sequence indicated that this isolate exhibited close homology with the ex Indian strain AY956319. Primer sequences and the scheme of amplification can be availed from the authors on request.     

Sequence analysis of the VP2 gene hypervariable region of infectious bursal disease viruses from India

Attempts have been made to characterize infectious bursal disease virus (IBDV) isolates collected from different parts of India during 1993 to 1999. Phylogenetic analysis was performed on a sequence generated by cycle sequencing comprising the variable region of the VP2 gene of 14 isolates. Indian IBDV isolates had divergence of 0.2 to 4.3% at nucleotide and 0 to 2.2% at amino acid levels among themselves. Nine nucleotide changes were found in Indian IBDV field isolates, resulting in the four specific amino acid changes at 222P-A, 256V-I, 294L-I and 299N-S, reported regularly in very virulent isolates. One of the Indian IBDV isolates, UP1/99, had change D to N at position 212 in the first hydrophilic region. The serine-rich heptapeptide sequence 'SWSASGS' was conserved in all the isolates. Phylogenetically, all Indian field isolates were found to be closely related to very virulent IBDV isolates from Europe, Japan, China and Israel.     

Molecular epidemiology of rabies virus isolates in India

In India, rabies is enzootic and is a serious public health and economic problem. India has a large population of stray dogs which, together with a lack of effective control strategies, might have led to the persistence of rabies virus (RV) in the canine population. Our objective was to study the molecular epidemiology of RV isolates in India based on nucleotide sequence analysis of 29 RV isolates originating from different species of animals in four states. Here we have analyzed two sets of sequence data based upon a 132-nucleotide region of the cytoplasmic domain (CD) of the G gene (G-CD) and a 549-nucleotide region (Psi-L) that combines the noncoding G-L intergenic region (Psi) and a fragment of the polymerase gene (L). Phylogenetic analysis revealed that the RV isolates belong to genotype 1 and that they were related geographically but were not related according to host species. Five different genetic clusters distributed among three geographical regions were identified. Comparison of the deduced amino acid sequences of G-CD between RV isolates revealed three amino acid changes (amino acid 462G [aa462G], aa465H, and aa468K) that distinguished the Indian RVs from RV isolates in other parts of the world. Analysis of the data indicated that the dog rabies virus variants are the major circulating viruses in India that transmit the disease to other domestic animals and humans as well.     

A new outbreak of rabies in rare Ethiopian wolves (Canis simensis)

Between October 2008 and May 2009, five brain samples from the carcasses of the rare Ethiopian wolf (Canis simenensis) were submitted for rabies virus testing. Rabies virus was detected in all five samples, and this confirmed that a further outbreak of rabies had occurred within the wolf population in the Bale Mountains of Ethiopia. Sequence comparison of a partial fragment of the nucleoprotein-coding gene demonstrated that all viruses showed 100% sequence identity, suggesting a single introduction of rabies virus.     

Molecular characterization,isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.     

Complete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV) from India

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus (PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan (AY355307), and two isolates (AY362547 and AY102034) from Thailand.     

中国狂犬病毒疫苗株（5aG株）糖蛋白基因的克隆与序列分析

用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法从中国狂犬疫苗株（５ａＧ株）病毒感染细胞中扩增得到该株病毒糖蛋白基因，并进行序列测定。结果表明该基因开放阅读框架全长１５７５ｂｐ，编码５０５个氨基酸的成熟糖蛋白及Ｎ－端１９个氨基酸的信号肽，该基因与其他株系相应基因比较，核酸序列同源性为８８％～９１％，氨基酸序列同源性为８７％～９０％，其中膜外区同源性高于膜内区及跨膜区同源性。糖蛋白中重要功能区段嗜乙酰胆碱受体区段、抗原位点３等在个株系间高度保守。     

Recent emergence of the Arctic rabies virus lineage

The rabies viruses that circulate in Arctic countries and in much of northern and central Asia are phylogenetically closely related and collectively referred to as the Arctic/Arctic-like (AL) lineage. The emergence and spread of this lineage is of significant interest given that rabies remains a serious zoonotic disease in many parts of Asia, especially in India where the prevalence of dog rabies leads to frequent human exposures and deaths. Previous molecular epidemiological studies of rabies viruses in India identified the AL lineage as the type circulating across much of the country. To further explore the relationship of Indian and Arctic rabies viruses, a collection of samples recovered from Rajasthan state in northern India was characterised at the N gene locus. Combination of these data with a larger collection of samples from India, central/northern Asia and the Arctic has permitted detailed phylogenetic analysis of this viral lineage and estimation of its time-frame of emergence. These analyses suggest that most current Indian viruses emerged from a common progenitor within the last 40 years and that the entire Arctic/AL lineage emerged within the last 200 years, a time-frame in accord with historical records of the invasion of Canada by the Arctic clade.     

Complete nucleotide sequence of cherry virus A (CVA) infecting sweet cherry in India

Cherry virus A (CVA) is a graft-transmissible member of the genus Capillovirus that infects different stone fruits. Sweet cherry (Prunus avium L; family Rosaceae) is an important deciduous temperate fruit crop in the Western Himalayan region of India. In order to determine the health status of cherry plantations and the incidence of the virus in India, cherry orchards in the states of Jammu and Kashmir (J&K) and Himachal Pradesh (H.P.) were surveyed during the months of May and September 2009. The incidence of CVA was found to be 28 and 13% from J&K and H.P., respectively, by RT-PCR. In order to characterize the virus at the molecular level, the complete genome was amplified by RT-PCR using specific primers. The amplicon of about 7.4 kb was sequenced and was found to be 7,379 bp long, with sequence specificity to CVA. The genome organization was similar to that of isolates characterized earlier, coding for two ORFs, in which ORF 2 is nested in ORF1. The complete sequence was 81 and 84% similar to that of the type isolate at the nucleotide and amino acid level, respectively, with 5′ and 3′ UTRs of 54 and 299 nucleotides, respectively. This is the first report of the complete nucleotide sequence of cherry virus A infecting sweet cherry in India.     

Further support of genetic conservation in Indian isolates of Rice tungro bacilliform virus by sequence analysis of an isolate from North–Western India

The genomic sequence of an isolate of Rice tungro bacilliform virus (RTBV), collected from the state of Punjab (Pb), a non-endemic tungro region from North–Western India was determined. In silico comparison of the 7931-bp sequence with isolates from Southeast Asia and the three previously characterized Indian isolates, revealed not only similar genome size to other Indian isolates but also high degree of homology both at nucleotide (>93 %) and amino acid (>96 %) levels among them. On the other hand, like the other Indian isolates, RTBV-Pb showed much lower nucleotide (<87 %) and amino acid (<90 % in most of the open reading frames) identities with the Southeast Asian isolates owing to several nucleotide substitutions and indels. In-depth annotation comparisons reinforce the hypothesis that Indian isolates of RTBV have diverged sufficiently from the Southeast Asian ones to form a separate group.     

Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5–100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.     

Antigenic and molecular characterization of rabies virus in Argentina

The nucleoprotein genes of 54 human, domestic and wild animals rabies isolates obtained in Argentina between 1995 and 2002 were characterized using monoclonal antibodies and partial gene sequence analysis. The antigenic and genetic diversities of rabies virus in samples from bat and bat-related cases were studied, leading to the identification of five distinct genetic variants. Rabies viruses isolated from vampire bat related cases were very similar to each other, showing 98.9% overall similarity. Specific antigenic variants (AgV) were detected associated with different insectivorous bats species, in samples from Tadarida brasiliensis and Eumops patagonicus bats. In contrast, isolates from Myotis sp. and Histiotus sp. bats could not be matched to any antigenic type. Additionally, bat rabies cases were also detected in southern provinces previously considered rabies-free. Finally, two independent antigenic and genetic variants co-circulating in northern Argentina were found in isolates obtained from dogs and dog-related cases, suggesting two independent cycles of virus transmission. This is the first national coordinated study of antigenic as well as molecular epidemiology of rabies in Argentina. The information presented here will improve our knowledge about rabies epidemiology and therefore, will assist preventing fatal human cases.