During investigations into the outbreak of encephalitis in 1996 in the Kerala state in India, an arbovirus was isolated from a Culex tritaeniorhynchus mosquito pool. It was characterized as a Japanese encephalitis and West Nile virus cross-reactive arbovirus by complement fixation test. A plaque reduction-neutralization test was performed using hyperimmune sera raised against the plaque-purified arbovirus isolate. The sera did not show reactivity with Japanese encephalitis virus and were weakly reactive with West Nile virus. Complete open reading frame sequence analysis characterized the arbovirus as Bagaza virus (BAGV), with 94.80 % nucleotide identity with African BAGV strain DakAr B209. Sera collected from the encephalitic patients during the acute phase of illness showed 15 % (8/53) positivity for anti-BAGV neutralizing antibodies. This is the first report of the isolation of BAGV from India. The presence of anti-BAGV neutralizing antibodies suggests that the human population has been exposed to BAGV.An outbreak of Japanese encephalitis (JE) was reported from the Allapuzza, Thiruvanthapuram and Kottayam districts of Kerala state, India, during 1996. Only 33 % (50/ 150) of the sera collected from hospitalized cases were confirmed as JE by immunoglobulin M (IgM) ELISA. Other clinical specimens were not available for further investigations. Entomological investigations during the outbreak were carried out and 184 mosquito pools collected from the affected area were processed for isolation in 2-day-old Swiss mice by the intra-cranial route (Rodrigues et al., 1980;George et al., 1984). One pool from Culex tritaeniorhynchus showed sickness in inoculated mice. Brains from sick mice were harvested and suspended in 10 % bovalbumin phosphate saline. The suspensions were stored at 270 u C and designated as the arbovirus isolate (96363). The isolate showed cross-reactivity with anti-JE virus (JEV) and anti-West Nile virus (WNV) immune sera in a complement fixation (CF) test (Pavri & Ghosh, 1969; Rodrigues et al., 1980;Damle et al., 1998).The isolate did not react with immune sera raised against other circulating arboviruses, including Chandipura (Rhabdoviridae), Sindbis (Togaviridae), Chikungunya (Togaviridae), Kyasanur forest disease (Flaviviridae), Batai (Bunyaviridae) and Dengue (Flaviviridae) viruses (Paul et al., 1970; Rodrigues et al., 1980;George et al., 1984).In this study, we present the genetic characterization of the arbovirus isolate and serological analysis of available sera collected from encephalitis patients during 1996. The Institutional Animal Ethical Committee approved this work and ethical guidelines were strictly followed according to their recommendations. The arbovirus isolate was plaque-purified to rule out the possibility of isolation of both JEV and WNV from the mosquito pool. The mouse brain stock of the arbovirus isolate was passaged twice in porcine stable kidney (PS) cells to amplify the virus. A single plaque was selected from the first PS cell passage and then subjected to two sequential ...
Background & objectives:Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome.Methods:Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains.Results:Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries.Interpretation & conclusions:Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.
A c c e p t e d M a n u s c r i p t 2 SummaryThe spread and establishment of Chandipura virus (CHPV) infection in India has raised serious epidemiological concerns. Virus interface with the vertebrate hosts, humans and vector competence are the important parameters of disease prevalence. Interestingly, in the present study a highly zoophilic species of sandfly Sergentomyia is emerging as a potential vector of CHPV in Gujarat. It is probably the first report from India that male sandflies have been found to be positive for CHPV by RT-PCR. These finding signifies the vertical transmission of the virus among sandflies and has epidemiological significance.Health Officers, Gujarat, referred nine pools comprising 277 adult sandflies from the disease affected and non affected areas to National Institute of Virology, Pune. Pools were subjected to RT-PCR and sequencing. Of the nine, two female and one male pool was positive for Gujarat and Maharashtra and recently in Orissa and Bihar. All these outbreaks were characterized by very high mortality rates ranging from 28% to 78.3% (2-5 and NIV Unpublished data). In India the first isolation of CHPV was reported from sandflies belonging to the genus Phlebotomus species (6). In the recent past the role of Sergentomyia has been established in transmission of CHPV from endemic region of AP and Maharashtra (7-8). Transovarial transmission of CHPV has also been reported in the laboratory (9). This has raised an alarm of the role of variety of vectors capable of transmitting this virus.This study presents the molecular evidence for the detection and the characterization of CHPV from sandflies obtained from three districts of Gujarat in 2015. Human CHPV cases were also reported during the same period. To our knowledge this is perhaps the first report on detection of CHPV from sandflies from this state.The study was approved by Institutional Biosafety Committee. A small CHPV outbreak was reported during June-August 2015 from Gujarat where four districts were involved (10). During the same period NIV received nine pools comprising 277 adult sandflies from Health Officers of Gujarat ( Figure. 1.). Of them 100 male and 114 female sandflies could be recovered and processed. Table 1 shows details of the sandflies received.The sandfly specimens were chilled and identified on wet ice using the taxonomic key by Lewis (11). Individual pools of the sandflies were prepared based on genus identification and gender. Immediately after segregation, pools of male individual (2-32) and female individual (5-27) sandflies were ground separately using Knotes pestles (Sigma), centrifuged, filtered
Agroforests are increasingly seen as ancillary conservation landscapes that effectively integrate production needs while sustaining biodiversity goals. The conservation potential of these land uses can be significantly improved by using evidence‐based management practices. In this study, we examine the community assembly of anuran amphibians and identify vulnerable species based on their life‐history traits across tea and coffee agroforests and non‐agricultural forest fragments in the Anamalai Hills of India. We conducted visual and auditory encounter surveys for amphibians along streams and terrestrial habitats. A modified joint species distribution model was used to examine the drivers of species richness, community composition and species co‐occurrence patterns. At the community level, mean species richness was greatest in forest fragments followed by coffee and least in tea agroforests. Community composition was associated with both land use (tea/coffee/forest) and habitat (stream/terrestrial), with the greatest composition difference between coffee and forest. Life‐history traits were significant drivers of species occupancies, particularly in forests. Fast‐flowing water breeding amphibians and smaller body sizes were positively associated with forest streams over coffee and tea streams. Elevation was a strong predictor of amphibian occupancy with nine species showing negative association and 14 species showing positive association. Twelve species also showed a significant positive association with the wetter year, seven of which belonged to direct‐developing frogs. This suggests that even single‐year declines in rainfall could have detrimental effects on populations and make these species vulnerable to climate change. The results of the study have important conservation consequences for agroforests in the Western Ghats. Stream restoration efforts across elevation gradients could significantly improve habitats for different amphibian assemblages in agroforests.
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