Vijay P. Bondre

Enteroviruses in Patients with Acute Encephalitis, Uttar Pradesh, India

An outbreak of viral encephalitis occurred in northern India in 2006. Attempts to identify an etiologic agent in cerebrospinal fluid by using reverse transcription–PCR showed positivity to enterovirus (EV) in 66 (21.6%) of 306 patients. Sequencing and phylogenetic analyses of PCR products from 59 (89.3%) of 66 specimens showed similarity with EV-89 and EV-76 sequences.

Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients

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.

De novo identification of viral pathogens from cell culture hologenomes

BackgroundFast, specific identification and surveillance of pathogens is the cornerstone of any outbreak response system, especially in the case of emerging infectious diseases and viral epidemics. This process is generally tedious and time-consuming thus making it ineffective in traditional settings. The added complexity in these situations is the non-availability of pure isolates of pathogens as they are present as mixed genomes or hologenomes. Next-generation sequencing approaches offer an attractive solution in this scenario as it provides adequate depth of sequencing at fast and affordable costs, apart from making it possible to decipher complex interactions between genomes at a scale that was not possible before. The widespread application of next-generation sequencing in this field has been limited by the non-availability of an efficient computational pipeline to systematically analyze data to delineate pathogen genomes from mixed population of genomes or hologenomes.FindingsWe applied next-generation sequencing on a sample containing mixed population of genomes from an epidemic with appropriate processing and enrichment. The data was analyzed using an extensive computational pipeline involving mapping to reference genome sets and de-novo assembly. In depth analysis of the data generated revealed the presence of sequences corresponding to Japanese encephalitis virus. The genome of the virus was also independently de-novo assembled. The presence of the virus was in addition, verified using standard molecular biology techniques.ConclusionsOur approach can accurately identify causative pathogens from cell culture hologenome samples containing mixed population of genomes and in principle can be applied to patient hologenome samples without any background information. This methodology could be widely applied to identify and isolate pathogen genomes and understand their genomic variability during outbreaks.

Neutralization escape variant of West Nile virus associated with altered peripheral pathogenicity and differential cytokine profile

In order to understand the factors influencing pathogenicity of a virus, two neutralization escape (NE) variants were selected from wild type lineage 1 West Nile virus (WNV) 68856 strain pathogenic by intra-peritoneal (i.p.) route using monoclonal antibodies (MAbs) against envelope (E) protein. Both NE IF1A7 1.1 and NE IVC3F10 1.2 were resistant to neutralization and were neurovirulent by intra-cranial (i.c.) inoculation. Growth kinetics in porcine stable (PS) kidney and baby hamster kidney (BHK) cells was unchanged. In contrast to parent WNV only NE IF1A7 1.1 failed to cause lethal encephalitis on i.p. inoculation and was non pathogenic. NE IF1A7 1.1 variant showed delayed replication kinetics in murine peritoneal exudate cells (PEC) and Neuro 346 cells in vitro. In comparison with parent WNV and NE IVC3F10 1.2 variant, non pathogenic variant exhibited significantly reduced tumour necrosis factor α (TNF-α) induction in infected animals and PEC. Other cytokines like Interleukin (IL)-10, IL-6 and Interferon (IFN)-β remained unchanged. However, IL-1β did not follow the pattern and was higher only in parent WNV-infected PEC. The E gene sequences of these NE variants showed three common amino acid substitutions at residues E50, E89 and E242. A unique E156 (ser→pro) substitution in NE IF1A7 1.1, was absent in NE IVC3F10 1.2 variant suggested probable virulence marker. Our data indicates possible role of WNV E protein in induction of TNF-α and IL-1β and its association with WNV pathogenesis.

Scrub Typhus as a Cause of Acute Encephalitis Syndrome, Gorakhpur, Uttar Pradesh, India

Outbreaks of acute encephalitis syndrome (AES) have been occurring in Gorakhpur Division, Uttar Pradesh, India, for several years. In 2016, we conducted a case–control study. Our findings revealed a high proportion of AES cases with Orientia tsutsugamushi IgM and IgG, indicating that scrub typhus is a cause of AES.

A large outbreak of Japanese encephalitis predominantly among adults in northern region of West Bengal, India

Unusual rise of acute encephalitis syndrome cases (AES) were reported in July 2014 in the northern region of West Bengal, India. Investigations were carried out to characterize the outbreak and to identify the associated virus etiology. This observational study is based on 398 line listed AES cases, mostly (70.8%, 282/398) adults, with case fatality ratio of 28.9% (115/398). Japanese encephalitis virus infection was detected in 134 (49.4%) among 271 AES cases tested and most of them (79.1%, 106/134) were adults. The study reports a large outbreak of genotype III Japanese encephalitis among adults in northern region of West Bengal, India. J. Med. Virol. 88:2004–2011, 2016.

Bagaza virus inhibits Japanese encephalitis & West Nile virus replication in Culex tritaeniorhynchus & Cx. quinquefasciatus mosquitoes.

Background & objectives: Studies have shown that certain flaviviruses influence susceptibility of mosquitoes by inhibiting/enhancing replication of important flaviviruses. Hence, a study was designed to determine whether Bagaza virus (BAGV), a flavivirus isolated from Culex tritaeniorhynchus mosquitoes in India, alters susceptibility of Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes to Japanese encephalitis (JEV) and West Nile viruses (WNV). Methods: JEV and WNV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes in the presence of BAGV was carried out by intrathoracic (IT) inoculation and oral feeding methods. Mosquitoes were infected with BAGV and WNV/JEV either simultaneously or in a phased manner, in which mosquitoes were infected with BAGV by IT inoculation followed by super-infection with JEV/WNV after eight days post-infection (PI). JEV and WNV yield on 7th and 14th day PI after super-infection was determined by 50 per cent tissue culture infective dose (TCID50) method. Results: In Cx. tritaeniorhynchus mosquitoes, prior infection with BAGV significantly reduced JEV and WNV replication while in Cx. quinquefasciatus, BAGV influence was only seen with WNV. Reduction in virus titre was observed in IT inoculated and oral fed mosquitoes irrespective of the infection mode. JEV replication was also found reduced in Cx. tritaeniorhynchus mosquitoes persistently infected with BAGV at passage four. Interpretation & conclusions: BAGV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes altered their susceptibility to JEV and WNV producing low virus yield. However, the role of BAGV in inhibiting JEV/WNV replication in field mosquitoes needs further investigations.

Isolation and characterization of Oya virus a member of Simbu serogroup, family Bunyaviridae, isolated from Karnataka, India.

During a study on Japanese encephalitis (JE) from Kolar district of Karnataka state, India in 1986; two virus isolates were obtained in infant Swiss albino mouse from a pig and a human serum sample. For characterization of these virus isolates, they were propagated in Vero CCL-81 cells. These virus isolates were screened for flaviviruses (Japanese encephalitis, West Nile, Dengue, Kyasanur forest disease) and Alphavirus (Chikungunya) by RT-PCR and found to be negative. Further these they were screened for bunyaviruses using genus-specific primers. A virus isolate from a human sample was sequenced using next generation sequencing; which identified it as Oya virus, Simbu group of the genus Orthobunyavirus of the family Bunyaviridae. Phylogenetic analysis of L, M, S (N and NSs) revealed its close association with Chinese strain of Oya virus in Simbu serogroup with the distance of 6.5>4.2>3.2% for nucleotides and 2.4>0.8>0.0% for the amino acid of L>M>S segments respectively. Based on the PCR results; an isolate from pig sample was also confirmed as Oya virus. This study was strengthened by findings of IgG antibody positivity against Oya virus in retrospective serum samples of suspected febrile illness cases from this area by an indigenously developed ELISA. Oya virus positivity was also recorded in human samples collected from Karnataka using nested RT-PCR. This is the first report of the presence of Oya virus in human samples. Further studies are needed to determine disease-causing potential in humans.

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.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

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.