Publications by authors named "Vijay P Bondre"

23 Publications

  • Page 1 of 1

Infectious causes of acute encephalitis syndrome hospitalizations in Central India, 2018-20.

J Clin Virol 2022 Aug 28;153:105194. Epub 2022 May 28.

Kakatiya Medical College, Warangal, Telangana, India.

Background: We enhanced surveillance of hospitalizations of all ages for acute encephalitis syndrome (AES) along with infectious aetiologies, including the Japanese encephalitis virus (JEV).

Methods: From October 2018 to September 2020, we screened neurological patients for AES in all age groups in Maharashtra and Telangana States. AES cases were enrolled at study hospitals along with other referrals and sampled with cerebrospinal fluid, acute and convalescent sera. We tested specimens for non-viral aetiologies viz. leptospirosis, typhoid, scrub typhus, malaria and acute bacterial meningitis, along with viruses - JEV, Dengue virus (DENV), Chikungunya virus (CHIKV), Chandipura virus (CHPV) and Herpes simplex virus (HSV).

Results: Among 4977 neurological hospitalizations at three study site hospitals over two years period, 857 (17.2%) were AES. However, only 287 (33.5%) AES cases were eligible. Among 278 (96.9%) enrolled AES cases, infectious aetiologies were identified in 115 (41.4%) cases, including non-viral in 17 (6.1%) cases - leptospirosis (8), scrub-typhus (3) and typhoid (6); and viral in 98 (35.3%) cases - JEV (58, 20.9%), HSV (22, 7.9%), DENV (15, 5.4%) and CHPV (3, 1.1%). JEV confirmation was significantly higher in enrolled cases than referred cases (10.2%) (p < 0.05). However, the contribution of JEV in AES cases was similar in both children and adults. JE was reported year-round and from adjacent non-endemic districts.

Conclusions: The Japanese encephalitis virus continues to be the leading cause of acute encephalitis syndrome in central India despite vaccination among children. Surveillance needs to be strengthened along with advanced diagnostic testing for assessing the impact of vaccination.
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http://dx.doi.org/10.1016/j.jcv.2022.105194DOI Listing
August 2022

The Outbreaks of Acute Encephalitis Syndrome in Uttar Pradesh, India (1978-2020) and Its Effective Management: A Remarkable Public Health Success Story.

Front Public Health 2021 9;9:793268. Epub 2022 Feb 9.

Encephalitis Group, National Institute of Virology, Pune, India.

Introduction: Acute encephalitis syndrome (AES) is a major public health enigma in India and the world. Uttar Pradesh (UP) is witnessing recurrent and extensive seasonal AES outbreaks since 1978. Government of India and UP state government have devised various mitigation measures to reduce AES burden and AES associated mortality, morbidity and disability in Uttar Pradesh. The aim of this study was to describe the public health measures taken in order to control seasonal outbreaks of AES in UP between 1978 and 2020.

Methods: We used literature review as a method of analysis, including the Indian government policy documents. This review utilized search engines such as PubMed, Google Scholar, Research Gate, Cochrane, Medline to retrieve articles and information using strategic keywords related to Acute Encephalitis Syndrome. Data was also collected from progress reports of government schemes and websites of Indian Council of Medical Research (ICMR), National Vector Borne Disease Control Programme (NVBDCP) and Integrated Disease Surveillance Programmes (IDSP).

Results: The incidence of AES cases in UP have declined from 18.2 per million population during 2005-2009 to 15 per million population during 2015-2019 [CI 12.6-20.6, -value < 0.001] and case fatality rate (CFR) reduced from 33% during 1980-1984 to 12.6% during 2015-2019 [CI 17.4-30.98, -value < 0.001]. AES incidence was 9 (2019) and 7 (2020) cases per million populations respectively and CFR was 5.8% (2019) and 5% (2020). This decline was likely due to active surveillance programs identifying aetiological agents and risk factors of AES cases. The identified etiologies of AES include Japanese encephalitis virus (5-20%), Enterovirus (0.1-33%), Orientia tsutsugamushi (45-60%) and other viral (0.2-4.2%), bacterial (0-5%) and Rickettsial (0.5-2%) causes. The aggressive immunization programs against Japanese encephalitis with vaccination coverage of 72.3% in UP helped in declining of JE cases in the region. The presumptive treatment of febrile cases with empirical Doxycycline and Azithromycin (EDA) caused decline in Scrub Typhus-AES cases. Decrease in incidence of vector borne diseases (Malaria, Dengue, Japanese Encephalitis and Kala Azar) i.e., 39.6/100,000 population in 2010 to 18/100,000 population in 2017 is highlighting the impact of vector control interventions. Strengthening healthcare infrastructure in BRD medical college and establishment of Encephalitis Treatment Centre (ETC) at peripheral health centres and emergency ambulance services (Dial 108) reduced the referral time and helped in early treatment and management of AES cases. The AES admissions increased at ETC centres to 60% and overall case fatality rate of AES declined to 3%. Under clean India mission and Jal Jeevan mission, proportion of population with clean drinking water increased from 74.3% in 1992 to 98.7% in 2020. The proportion of household having toilet facilities increased from 22.9% in 1992 to 67.4% in 2020. Provisions for better nutritional status under state and national nutrition mission helped in reducing the burden of stunting (52%) and wasting (53.4%) among under five children in 1992 to 38.8% (stunting) and 36.8% (wasting) in year 2018. These factors have all likely contributed to steady AES decline observed in UP.

Conclusion: There is a recent steady decline in AES incidence and CFR since implementation of intensive AES surveillance system and JE immunization campaigns which is highlighting the success of interventions made by central and state government to control seasonal AES outbreaks in UP. Currently, AES incidence is 9 cases per million population (in year 2019) and mortality is 5.8%.
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http://dx.doi.org/10.3389/fpubh.2021.793268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8863615PMC
April 2022

Childhood encephalitis hospitalizations associated with virus agents in medium-endemic states in India.

J Clin Virol 2021 11 14;144:104970. Epub 2021 Sep 14.

ICMR - National Institute of Virology, Pune, Maharashtra, India; Kakatiya Medical College, Warangal, Telangana, India; Government Medical College, Nagpur, Maharashtra, India; Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, Maharashtra, India.

Background: Japanese encephalitis (JE) is the leading cause of childhood acute encephalitis syndrome (AES) in India. We enhanced the AES surveillance in sentinel hospitals to determine trends and virus etiologies in central India.

Methods: The neurological hospitalizations among children ≤15 years were tracked by using the AES case definition implemented by the national program. Acute and convalescent sera along with cerebrospinal fluid (CSF) specimens were collected and tested at the strengthened site hospital laboratories for anti-JE, anti-Dengue and anti-Chikungunya virus by IgM ELISA; along with Chandipura virus RT-PCR. Herpes simplex and enterovirus testing was undertaken at the reference laboratory.

Results: Among 1619 pediatric neurological hospitalizations reported during 2015-16, AES case definition was fulfilled in 332 (20.5%) cases. After excluding 52 non-AES cases, 280 AES cases resident from study districts were considered eligible for study. The treating physicians diagnosed non-viral causes in 90 cases, therefore 190 (67.9%) of 280 AES cases were suspected with viral etiologies. We enrolled 140 (73.7%) of 190 eligible AES cases. Viral etiologies were confirmed in 31 (22.1%) of 140 enrolled AES cases. JE (n = 22) was the leading cause. Additional non-JE viral agents included Chikungunya (5), Dengue (2) and Chandipura (2). However, only 21 (9.4%) of 222 additional AES cases referred from peripheral hospitals were confirmed as JE.

Conclusions: Japanese encephalitis virus continues to be the leading cause of childhood acute encephalitis syndrome in central India despite vaccination program. Surveillance needs to be intensified for assessing the true disease burden of Japanese encephalitis following vaccination program implementation.
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http://dx.doi.org/10.1016/j.jcv.2021.104970DOI Listing
November 2021

Association of single nucleotide polymorphisms in the CD209, MMP9, TNFA and IFNG genes with susceptibility to Japanese encephalitis in children from North India.

Gene 2022 Jan 14;808:145962. Epub 2021 Sep 14.

ICMR-Regional Research Medical Centre, Gorakhpur, Uttar Pradesh, India.

Japanese encephalitis (JE), an acute encephalitis syndrome disease caused by infection with JE virus (JEV), is an important mosquito borne disease in developing countries. The clinical outcomes of JEV infection show inter individual differences. Only in a minor percent of the infected subjects, the disease progresses into acute encephalitis syndrome. Single nucleotide polymorphisms in the host immune response related genes are known to affect susceptibility to JE. In the present study, 238 JE cases and 405 healthy controls (HCs) without any known history of encephalitis were investigated for SNPs in the CD209 MX1, TLR3, MMP9, TNFA and IFNG genes which are important in the immune response against JEV by PCR based methods. The results revealed higher frequencies of heterozygous genotypes of CD209 rs4804803, MMP9 rs17576, TNFA rs1800629 and IFNG rs2430561 in JE cases compared to HCs. These SNPs were associated with JE in an over-dominant genetic model (Odds ratio with 95% CI 1.51 (1.09-2.10) for CD209 rs4804803, 1.52 (1.09-2.11) for MMP9 rs17576, and 1.55 (1.12-2.15) for IFNG rs2430561). The association of G/A genotype of TNFA rs1800629 with JE was confirmed in a larger sample size. The results suggest the association of CD209 rs4804803, MMP9 rs17576, IFNG rs2430561 and TNFA rs1800629 polymorphisms with susceptibility to JE.
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http://dx.doi.org/10.1016/j.gene.2021.145962DOI Listing
January 2022

Molecular Detection and Genetic Characterization of from Hospitalized Acute Encephalitis Syndrome Cases During Two Consecutive Outbreaks in Eastern Uttar Pradesh, India.

Vector Borne Zoonotic Dis 2021 10 29;21(10):747-752. Epub 2021 Jun 29.

ICMR-RMRC, Gorakhpur, India.

Seasonal outbreaks of acute encephalitis syndrome (AES) have been reported especially in the pediatric population with a high case fatality rate in Eastern Uttar Pradesh, India. (OT) is a causative agent of scrub typhus that has been recently identified as a major cause of AES. However, the specific genotypes of OT responsible for AES cases of this region are not known. Therefore, the present study was undertaken to understand the molecular epidemiology of OT prevailing in the AES endemic Eastern Uttar Pradesh region of India. The study was conducted on 2529 hospitalized AES cases from August 2016 to December 2017. The presence of antibodies against OT from cerebrospinal fluid (CSF) and serum samples were tested using OT IgM enzyme-linked immunosorbent assay (ELISA), whereas OT DNA was tested from whole blood and CSF specimens targeting the partial gene of 56 kDa using nested PCR. Phylogenetic analysis was conducted with sequences ( = 241) generated in this study. Among the studied AES cases, 50% were found positive for antibodies against OT, whereas 37% of cases were positive for OT DNA. The genetic analysis study revealed that Gilliam (93.8%) is the prevailing genotype of OT followed by Karp (6.16%) genotype in AES cases. Furthermore, the Gilliam strains of this study showed they were >99% identical to earlier reported Gilliam strains from AES cases. We observed the presence of two main OT genotypes in AES cases, among which the majority of OT genotypes fall under the Gilliam clade. The understanding of predominant genotype will be beneficial for its future implications in vaccine development strategies and the development of rapid diagnostic tests.
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http://dx.doi.org/10.1089/vbz.2021.0003DOI Listing
October 2021

Abundance of Ticks (Acari: Ixodidae) and Presence of Rickettsia and Anaplasma in Ticks Infesting Domestic Animals From Northern India.

J Med Entomol 2021 05;58(3):1370-1375

ICMR-National Institute of Virology, Pune, Maharashtra, India.

Rickettsia and Anaplasma are bacteria that can be transmitted by hematophagous arthropods such as ticks infesting animals in close proximity to humans. The main objective of the present study was to investigate abundance of common tick species infesting domestic animals and presence of Rickettsia and Anaplasma in tick populations. Adult ticks were collected from domestic animals in rural areas and screened by molecular detection of bacterial DNA for these two genera of bacteria. A total of 1,778 adult ixodid tick specimens were collected from 200 cattle, 200 buffaloes, 200 goats, and 40 dogs. The collection consisted of four species of ixodid ticks, Rhipicephalus (Boophilus) microplus (Canestrini) (83.8%), Hyalomma kumari (Sharif) (7.1%), Rhipicephalus sanguineus (Latreille) (6.4%), and Dermacentor auratus (Supino) (2.7%) infesting the domestic animals. The prevalence of all the collected tick species was highest in the month of October. Anaplasma spp. was the most frequently identified bacteria (3.3%) in tested ticks. Of 17 positive tick pools for Anaplasma spp., 14 pools were from ticks infesting cattle, 2 pools of ticks collected from buffalo, and the remaining pool were ticks infesting a goat at the time of collection. Although 1.6% tick pools of R. microplus collected from cattle tested positive for Rickettsia spp., present investigation provides evidence of the most prevalent ixodid ticks infesting domestic animals and the presence of obligate intracellular bacteria, Rickettsia and Anaplasma, in these ticks collected in the Gorakhpur division of Northern India.
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http://dx.doi.org/10.1093/jme/tjaa296DOI Listing
May 2021

Primary varicella zoster virus infection-related hemiparesis and fatal neurological complications in an immunocompetent girl.

Natl Med J India 2019 Nov-Dec;32(6):381-382

Department of Paediatrics, Baba Raghav Das Medical College, Gorakhpur, Uttar Pradesh, India.

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http://dx.doi.org/10.4103/0970-258X.303630DOI Listing
September 2021

Genetic characterization of dengue virus serotype 2 isolated from dengue fever outbreaks in eastern Uttar Pradesh and western Bihar, India.

J Med Virol 2021 06 17;93(6):3322-3329. Epub 2020 Jul 17.

Encephalitis Group, ICMR-National Institute of Virology, Microbial Containment Complex, Pune, Maharashtra, India.

Dengue (DEN) is the most common cause of mosquito-borne endemic viral diseases in the tropical and subtropical countries. DEN outbreaks associated with multiple dengue virus (DV) serotypes have been regularly reported in different parts of India. This study was done during DEN outbreaks in 2015 to 2016 in UP and Bihar where DEN-2 was found as the only prevalent serotype. DV-2 was the only serotype amplified in serotype-specific reverse-transcription polymerase chain reaction from sera of 210 (65.21%) out of 322 DV NS1 antigen-positive patients. Further genetic analysis based on full-length envelope (E) protein sequence derived from patient's sera as well as DV isolate showed the circulation of lineages I and III of DV-2 cosmopolitan genotype during 2015 and lineage II during 2016. Finally, the phylogenetic analysis using the E gene sequence revealed that these DV-2 strains have a close genetic relationship with the recently reported DV-2 genotypes from DEN outbreaks reported from different parts of north India. These results showed the circulation of cosmopolitan genotype of DV-2 in eastern Uttar Pradesh and western Bihar, India. The genetic database generated on circulating DV strains in this study will be useful as reference for disease surveillance and strengthening laboratory diagnosis protocols.
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http://dx.doi.org/10.1002/jmv.26239DOI Listing
June 2021

Association of single nucleotide polymorphisms in TNFA and CCR5 genes with Japanese Encephalitis: A study from an endemic region of North India.

J Neuroimmunol 2019 11 26;336:577043. Epub 2019 Aug 26.

ICMR-National Institute of Virology, Pune, Maharashtra, India.

TNFA, IL1B, HMGB1, IL10, CXCL8, CCL2 and CCR5 gene polymorphisms were investigated in 183 Japanese Encephalitis (JE) cases and 361 healthy controls from North India. Higher frequency of TNFA rs1800629 G/A, CCR5 rs1799987 genotypes with A allele and lower frequency of combination lacking TNFA rs1800629 A, CCR5 rs333 Δ32, andCCR5 rs1799987 A alleles and CCL2 rs1024611 G/G genotype was observed in JE cases. TNFA rs1800629 A and CCR5 rs1799987 A alleles were associated with susceptibility while combination lacking TNFA rs1800629 A, CCR5 rs333 Δ32, and rs1799987 A alleles and CCL2 rs1024611 G/G genotype was associated with protection to JE.
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http://dx.doi.org/10.1016/j.jneuroim.2019.577043DOI Listing
November 2019

Chickenpox and measles clusters among college students in Pune, Maharashtra.

Virusdisease 2017 Sep 20;28(3):337-340. Epub 2017 Sep 20.

National Institute of Virology [NIV], Indian Council of Medical Research, 130/1, Sus Road, Pashan, Pune, 411021 India.

Chickenpox and measles, both vaccine preventable febrile rash illnesses, present in a comparatively severe form among young adults/adults than among children. Immunity levels against chickenpox are not known in India and those against measles have been found variable across the country. Places where students or adults/young adults from various parts of the country come together pose a peculiar challenge in preventive policy making regarding these diseases. In this article, we present findings from parallel outbreaks of the two diseases in a graduate/postgraduate institute in the city of Pune. A team from National Institute of Virology [Pune] investigated outbreak of febrile rash illness in a premier graduate institute and found that it was a case of two parallel outbreaks of chickenpox and measles. In this outbreak chickenpox cases did not present with greater severity but measles cases were severe. The concerned institute hosts more than 800 students and 300 staff including faculty. These outbreaks were contained because of the alert physician in the institute; but it also highlights a need for uniform policies across such educational institutions in the country.
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http://dx.doi.org/10.1007/s13337-017-0395-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685002PMC
September 2017

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

Indian J Med Res 2016 Nov;144(5):750-760

National Institute of Virology (ICMR), Pune, 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.
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http://dx.doi.org/10.4103/ijmr.IJMR_747_14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393087PMC
November 2016

Monocytes and B cells support active replication of Chandipura virus.

BMC Infect Dis 2016 09 14;16:487. Epub 2016 Sep 14.

National Institute of Virology, Sus Road, Pashan, Pune, 411021, India.

Background: Interaction between immune system and Chandipura virus (CHPV) during different stages of its life cycle remain poorly understood. The exact route of virus entry into the blood and CNS invasion has not been clearly defined. The present study was undertaken to assess the population in PBMC that supports the growth of virus and to detect active virus replication in PBMC as well as its subsets.

Methods: PBMC subsets viz.: CD3(+), CD14(+), CD19(+), CD56(+)cells were separated and infected with CHPV. The infected cells were then assessed for transcription (N gene primer) and replication (NP gene primer) of CHPV by PCR. The supernatant collected from infected cells were titrated in Baby Hamster Kidney (BHK) cells to assess virus release. The cytokine and chemokine expression was quantified by flow cytometry.

Results: Amplification of N and NP gene was detected in CD14(+) (monocyte) and CD19(+) (B cell), significant increase in virus titre was also observed in these subsets. It was observed that, although the levels of IL-6 and IL-10 were elevated in CD14(+) cells as compared to CD19(+)cells, the differences were not significant. However the levels of TNFα and IL-8 were significantly elevated in CD14(+) cells than in CD19(+)cells. The levels of chemokine (CXCL9, CCL5, CCL2, CXCL10) were significantly elevated in CHPV infected PBMC as compared to uninfected cells. CCL2 and CXCL9 were significantly increased in CHPV infected CD14(+)cells as compared to CD19(+) cells.

Conclusion: CD14(+)and CD19(+)cells support active replication of CHPV. High viral load was detected in CD14(+) cells infected with CHPV hence it might be the primary target cells for active replication of CHPV. An elevated levels of cytokines and chemokines observed in CD14(+) cells may help in predicting the pathogenecity of CHPV and possible entry into the central nervous system.
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http://dx.doi.org/10.1186/s12879-016-1794-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024506PMC
September 2016

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

J Med Virol 2016 11 2;88(11):2004-11. Epub 2016 May 2.

Department of Paediatric, North Bengal Medical College, Siliguri, Darjeeling, 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. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jmv.24556DOI Listing
November 2016

Molecular characterization of IFN-T expressed in buffalo embryonic trophoblasts and expression of recombinant BuIFN-T1a2 and BuIFN-T8 isoforms in E. coli.

Protein Expr Purif 2016 06 11;122:8-14. Epub 2016 Feb 11.

Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India. Electronic address:

Interferon tau (IFN-T) acts as a signaling molecule for maternal recognition of pregnancy (MRP) in ruminants. Aim of the present study was to identify various Buffalo Interferon tau (BuIFN-T) transcripts in buffalo trophoblast, phylogenetic comparison of these sequences with known mRNA sequences of buffalo, bovine, caprine and ovine and to express and purify the recombinant BuIFN-T (rBuIFN-T) isoforms. Following RNA extraction from trophectodermal cells, RT-PCR was performed using Ifn-t gene specific primers. 13 distinct cDNA variants encoding eight different BuIFN-T proteins were identified. BuIFN-T1a2 and BuIFN-T8 were expressed in prokaryotic expression system at 37 °C, 25 °C and 16 °C with 1 mM IPTG for 12 h and the recombinant proteins expressed at 16 °C were partially purified by Immobilised Metal Affinity Chromatography (IMAC). BuIFN-T isoforms have greater nucleotide and amino acid homology with caprine (98-100%, 96-100%), ovine (94-97%, 90-95%) and bovine (89.6-90.6%, 82-86%). These novel BuIFN-T isoforms contained pronounced nucleotide and amino acid sequence identity with one another (99.1-99.8%, 98-99%) but moderate sequence identity with previously identified buffalo IFN-T (90-92%, 82-86%). Solubility of expressed recombinant isoforms (rBuIFN-T1a2 and rBuIFN-T8) was highest at 16 °C. In conclusion, 13 distinct Ifn-t gene variants exist in trophectoderm of in vitro developed buffalo blastocysts that encode eight different proteins. rBuIFN-T1a2 and rBuIFN-T8 were successfully expressed in soluble form in Escherichia coli expression system at 16 °C with 1 mM IPTG and the resulting recombinant proteins were partially purified by IMAC.
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http://dx.doi.org/10.1016/j.pep.2016.02.005DOI Listing
June 2016

Development and characterization of reverse genetics system for the Indian West Nile virus lineage 1 strain 68856.

J Virol Methods 2015 Dec 24;226:31-9. Epub 2015 Sep 24.

Encephalitis Group, National Institute of Virology, Microbial Containment Complex, Pashan, Pune 411021, India. Electronic address:

Our previous studies on West Nile virus (WNV) strains isolated from human patients in India suggested substantial variation at the genetic level reflecting their variable pathogenesis. This study describes the development of reverse genetics system for a neurovirulent WNV isolate 68856 and its characterization. Full length viral cDNA was cloned into bacterial artificial chromosome (BAC) under the transcription control of T7 promoter. The RNA transcripts obtained by in vitro transcription were infectious in mammalian cells upon transfection. Cytopathic effect caused by synthetic RNA transcripts in mammalian cells, detection of cell associated viral protein after transfection and recovery of genetic markers in the progeny virus genome marked the successful development of reverse genetics system for WNV. Replication potential and plaque morphology of newly expressed virus along with its antigenic cross reactivity with the parental virus suggests synthesis of biologically identical replicative virus. Comparative neuropathogenesis studies in murine model indicated that the three genetic changes occurred in the recombinant virus during in vitro transcription has no impact on viral pathogenesis. The stable infectious cDNA clone generated from the neurovirulent Indian WNV strain will serve as a valuable experimental tool to study the viral factors contributing towards pathogenesis, host-virus interaction and immune evasion.
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http://dx.doi.org/10.1016/j.jviromet.2015.09.008DOI Listing
December 2015

Temporal changes of Japanese encephalitits virus in different brain regions of rat.

Indian J Med Res 2013 ;138:219-23

Department of Neurology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.

Background & Objectives: Japanese encephalitis virus (JEV) infection results in acute encephalitic illness. The affinity of JEV to different regions of brain and temporal changes in viral load have not been studied. This study was conducted to describe localization of JEV to different regions of the brain at different stages of disease in a rat model of Japanese encephalitis (JE).

Methods: Twelve days old Wistar rats were inoculated intracerebrally with a dose of 3 x 10⁶ pfu/ml of JEV. After 3, 6, 10 and 20 days post-inoculation, brains were dissected out and different regions of brain (cortex, striatum, thalamus and mid brain) were taken. Motor deficit was assessed by the rota rod and JEV RNA copies were evaluated using real-time PCR assay.

Results: There was a significant increase in motor deficit in rats inoculated with JEV compared to the controls. JEV RNA copies were present in all studied regions of the brain on days 3, 6 and 10 post-inoculation. Maximum number of JEV RNA copies were present in the mid brain on days 3 and 10 post-inoculation. JEV RNA copies were not detected in any of the brain regions on day 20.

Interpretation & Conclusions: This study reports JEV RNA load in different brain regions of rat with higher affinity of JEV virus to thalamus and mid brain compared to other regions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788207PMC
April 2014

De novo identification of viral pathogens from cell culture hologenomes.

BMC Res Notes 2012 Jan 6;5:11. Epub 2012 Jan 6.

CSIR Institute of Genomics and Integrative Biology (CSIR-IGIB), Mall Road, Delhi 110007, India.

Background: Fast, 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.

Findings: We 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.

Conclusions: Our 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.
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http://dx.doi.org/10.1186/1756-0500-5-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284880PMC
January 2012

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

J Gen Virol 2009 Nov 1;90(Pt 11):2644-2649. Epub 2009 Jul 1.

National Institute of Virology, Pashan, Pune 411 021, India.

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.
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http://dx.doi.org/10.1099/vir.0.012336-0DOI Listing
November 2009

Enteroviruses in patients with acute encephalitis, uttar pradesh, India.

Emerg Infect Dis 2009 Feb;15(2):295-8

National Institute of Virology, Pune, 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.
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http://dx.doi.org/10.3201/eid1502.080865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657625PMC
February 2009

Detection and isolation of Japanese encephalitis virus from blood clots collected during the acute phase of infection.

Am J Trop Med Hyg 2007 Dec;77(6):1139-45

National Institute of Virology, Sus Road, Pashan, Pune, Maharashtra 411021 India.

Clinical specimens from an encephalitis outbreak in the Lakhimpur area of Uttar Pradesh, India, were investigated for identification and characterization of the etiologic agent. IgM capture ELISA showed recent Japanese encephalitis virus (JEV) infection. JEV isolation was attempted from white blood cells (WBCs) separated from blood clots of 12 patients (9 IgM positive and 3 negative) by serial co-culturing with phytohemagglutinin P-stimulated peripheral blood mononuclear leukocytes (PBMCs) obtained from pre-screened JEV sero-negative healthy individuals. JEV was isolated from two IgM-positive blood clots. Isolate 014178 was detected in WBCs and in the first passage of PBMCs by ELISA and reverse transcriptase-polymerase chain reaction. Isolate 014173 was detectable only after a second passage in PBMC co-culture. Sequence analysis of 346 nt of the C-prM region showed homology with JEV strain GP78. This is the first report on isolation of JEV from patient blood clots. Our study shows that the co-cultures of PBMCs separated from patient blood clots provide an additional source for JEV isolation.
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December 2007

West Nile virus isolates from India: evidence for a distinct genetic lineage.

J Gen Virol 2007 Mar;88(Pt 3):875-884

Microbial Containment Complex Unit, National Institute of Virology (ICMR), Sus Road, Pashan, Pune 411 021, India.

The complete genomic sequence of one human isolate of West Nile virus (WNV) and the partial genomic sequences of 14 other strains from India isolated in the period 1955-1982 from different hosts and geographical areas were determined. Phylogenetic analyses based on complete and partial genomic sequences (921 nt of the C-prM-E region) revealed that WNV could be classified into five distinct groups that differed from each other by 20-25% at the complete genome level and by 20-26% using partial sequences. Of the Indian isolates, 13 formed a distinct genetic lineage, lineage 5, whereas two isolates, one from a human patient (1967) and another from a bat (1968), were related closely to lineage 1 strains. The complete genomic sequence of the Indian isolate, 804994, showed 20-22% genetic divergence from the previously proposed lineage 1 and 2 strains and 24-25% divergence from isolates of the newly proposed lineages 3 (Rabensburg isolate 97-103 of 1997) and 4 (Russian isolate LEIV-Krnd88-190 of 1998). Similarly, the partial genomic sequences of the Indian isolates showed 21-26% divergence from lineage 1 and 2 strains and from the Rabensburg (97-103) and Russian (LEIV-Krnd88-190) isolates. Cross-neutralization using strain-specific polyclonal antibodies against lineage 1 strain Eg-101 and representative Indian strains suggests substantial antigenic variation. This study documents circulation of WNV strains typical to India for 27 years and the introduction of lineage 1 strains during 1967-1968. These results indicate strongly that WNV should be classified into five genetic lineages, with Indian viruses constituting the distinct genetic lineage 5.
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http://dx.doi.org/10.1099/vir.0.82403-0DOI Listing
March 2007

Vibrio cholerae persistence in aquatic environments and colonization of intestinal cells: involvement of a common adhesion mechanism.

FEMS Microbiol Lett 2005 Mar;244(2):267-73

Istituto di Microbiologia e Scienze Biomediche, Università Politecnica delle Marche, Ancona 60131, Italy.

Forty-one Tnpho A mutants of Vibrio cholerae O1 classical strain CD81 were analyzed for their ability to interact with chitin particles, Tigriopus fulvus copepods and the Intestine 407 cell line compared to the parent strain. Thirteen mutants were less adhesive than CD81; in particular, T21, T33 and T87 were less adhesive towards all substrates and insensitive to inhibition by N-acetyl glucosamine (GlcNAc). By SDS-PAGE analysis of sarkosyl-insoluble membrane proteins (siMPs) isolated from mutants and parent, it was found that a 53 kDa siMP is missing in T21, T33 and T87 mutants. It is hypothesized that this protein might have the function to mediate adherence to GlcNAc-containing substrates both in the aquatic environment and in human intestine.
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http://dx.doi.org/10.1016/j.femsle.2005.01.052DOI Listing
March 2005
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