Diversity of sandflies in Vidarbha region of Maharashtra, India, a region endemic to Chandipura virus encephalitis

Background & objectives: Sandflies are implicated as vectors of Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae). The virus is prevalent in central India including Vidarbha region of Maharashtra. CHPV causes encephalitis in children below 15 yr of age with case fatality rates ranging from 56 to 78 per cent. The present study was undertaken to determine the sandfly fauna in the CHPV endemic Vidharba region. Methods: A year round survey of sandflies was conducted at 25 sites in three districts of Vidarbha region. Sandflies were collected from their resting sites using handheld aspirators and identified using taxonomical keys. Results: A total of 6568 sandflies were collected during the study. Approximately 99 per cent of the collection belonged to genus Sergentomyia, which was represented by Ser. babu, Ser. bailyi and Ser. punjabensis. Genus Phlebotomus was represented by Ph. argentipes and Ph. papatasi. Ser. babu was the predominant species (70.7%) collected during the study. Ph. argentipes was detected in four villages with 0.89 per cent, whereas Ph. papatasi was detected in only one village with 0.32 per cent of the total collection. CHPV could not be isolated despite processing all the sandflies for virus isolation in cell culture. Interpretation & conclusions: The present study showed influence of higher temperature and relative humidity on sandfly population dynamics. An important observation during the study was the absence or decline in the population of Ph. papatasi and Ph. argentipes in the study area. Surge in Sergentomyia population and their breeding/resting in close vicinity to humans pose a concern as they are known to harbour CHPV and other viruses of public health importance

Investigating the role of Ebp1 in Chandipura virus infection

ErbB-3 binding protein 1 (Ebp1) is a host protein which binds ErbB-3 receptor to induce signalling events for cell growthregulation. In addition, Ebp1 also interacts with ribonucleoprotein complexes. In recent times, Ebp1 was found to play anantagonistic role in viral infections caused by Influenza and Rinderpest viruses. In our present work we have tried tounderstand the role of Ebp1 in Chandipura virus (CHPV) infection. We have observed an induction in Ebp1 expressionupon CHPV infection similar to other viruses. However, unlike other viruses an overexpressed Ebp1 only reduces viralprotein expression, but does not affect its progeny formation. Additionally, this effect is being carried out in an indirectmanner, as there is no interaction between Ebp1 and viral proteins. This is despite Ebp1’s presence in viral inclusion bodies.

Changing clinical scenario in Chandipura virus infection.

Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae) garnered global attention as an emerging neurotropic pathogen inflicting high mortality in children within 24 h of commencement of symptoms. The 2003-2004 outbreaks in Central India witnessed case fatality rates ranging from 56-75 per cent in Andhra Pradesh and Gujarat with typical encephalitic symptoms. Due to the acute sickness and rapid deterioration, the precise mechanism of action of the virus is still unknown. Recent studies have shown increased expression of CHPV phosphoprotein upto 6 h post infection (PI) demonstrating CHPV replication in neuronal cells and the rapid destruction of the cells by apoptosis shed light on the probable mechanism of rapid death in children. Phlebotomine sandflies are implicated as vectors due to their predominance in endemic areas, repeated virus isolations and their ability to transmit the virus by transovarial and venereal routes. Significant contributions have been made in the development of diagnostics and prophylactics, vaccines and antivirals. Two candidate vaccines, viz. a recombinant vaccine and a killed vaccine and siRNAs targeting P and M proteins have been developed and are awaiting clinical trials. Rhabdomyosarcoma and Phlebotomus papatasi cell lines as well as embryonated chicken eggs have been found useful in virus isolation and propagation. Despite these advancements, CHPV has been a major concern in Central India and warrants immediate attention from virologists, neurologists, paediatricians and the government for containing the virus.

Isolation of Chandipura virus (Vesiculovirus: Rhabdoviridae) from Sergentomyia species of sandflies from Nagpur, Maharashtra, India.

Background & objectives: An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra state, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. Methods: Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE). The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. Results: CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. Interpretation & conclusions: This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission.

Chandipura virus encephalitis outbreak among children in Nagpur division, Maharashtra, 2007.

Background & objectives: An outbreak of acute encephalitis syndrome (AES) among children from Nagpur division, Maharashtra was investigated to confirm the aetiology and to describe clinico-epidemiological features. Methods: AES cases among children <15 yr, from Nagpur division, hospitalized between June-September 2007, were investigated. Serum and cerebrospinal fluid (CSF) were tested for IgM antibodies against Chandipura virus (CHPV) and Japanese encephalitis virus (JEV) and for CHPV RNA by RT-PCR. Partial N gene sequences were used for phylogenetic analysis. Virus isolations were attempted in rhabdomyosarcoma (RD) cell line. Sandflies were collected, pooled and tested for CHPV RNA by RT-PCR. Results: A total of 78 AES cases were recorded in children <15 yr of age. Case fatality ratio was 43.6 per cent. Male to female ratio was 1:1.2. Chandipura (CHP) was confirmed in 39 cases. CHPV RNA was detected in both CSF and serum specimens of 2 cases and in serum of 22 cases. Phylogenetic analysis showed 99.98 – 100 per cent nucleotide identity in the sequences studied. Anti-CHPV IgM antibodies were detected in CSF of 2 cases and in serum of 8 cases. Seroconversion to anti-CHPV IgM antibodies was observed in 5 cases. Clinical manifestations of CHP cases (n=38) were fever (100%), convulsion (76.3%), altered sensorium (34.2%), headache (23.7%), vomiting (44.7%) and diarrhoea (23.7%). CHPV RNA was detected in one of two pools of sandflies from affected locality. Interpretation & conclusions: Chandipura virus was confirmed as the aetiological agent of this acute encephalitis outbreak with high case-fatality among children.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

Background & objectives: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Methods: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID50) and indirect immunofluorescence assay (IFA). Results: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Interpretation & conclusions: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.