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► Chandipura virus (Rhabdoviridae: Vesiculovirus) is an encephalitis causing virus of the pediatric group with high case fatality rate. ► A candidate vaccine against the virus is developed and evaluated. ► Beta propio lactone—the vaccine produces 100% sero-conversion in Swiss albino mice. ► Neutralizing antibody after the third dose ranged from 1:80 to 1:320. ► Mice with antibody titer >20 survived intra cranial virus challenge. A Vero cell based vaccine candidate against Chandipura (CHP) virus (Rhabdoviridae: Vesiculovirus), was developed and evaluated for immunogenicity in mice. Virus was purified by ultracentrifugation on 30% glycerol cushion followed by differential centrifugation on 10–60% sucrose gradient and inactivated with β-propio lactone at a concentration of 1:3500. The inactivated product was blended with aluminium phosphate (3%) and immunized 4-week-old Swiss albino mice. Neutralizing antibodies in the range of 1:10 to 160 and 1:80 to 1:320 was detected with 85% and 100% sero-conversion after 2nd and 3rd dose, respectively. All the immunized mice with antibody titer above 1:20 survived live virus challenge. The vaccine candidate has potential to be an efficient vaccine against CHP virus.

Dengue (DEN) is a rapidly spreading arboviral disease transmitted by Aedes mosquitoes. Although it is endemic in India, dengue virus (DENV) infection has not been reported from tribal areas of Madhya Pradesh. Investigations were conducted to establish the aetiology of sudden upsurge of cases with febrile illness in June 2013 from tribal villages of Mandla district of Madhya Pradesh, India. The rapid response team of the National Institute for Research in Tribal Health, Jabalpur, conducted clinical investigations and field surveys to collect the samples from suspected cases. Samples were tested using molecular and serological tools. Collected mosquitoes were identified and tested for the presence of virus using semi nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The sequences were analysed to identify serotype and genotype of the virus. Of the 648 samples collected from 18 villages of Mandla, 321 (49.53%) were found to be positive for dengue. The nRT-PCR and sequencing confirmed the aetiology as dengue virus type 2. Eighteen per cent of patients needed hospitalization and five deaths were attributed to dengue. The virus was also detected from Aedes aegypti mosquito, which was incriminated as a vector. Phylogenetic analysis revealed that the dengue virus 2 detected belonged to cosmopolitan genotype of the virus. Dengue virus serotype 2 was detected as the aetiological agent in the outbreak in tribal villages of Mandla district of Madhya Pradesh. Conducive man-made environment favouring mosquitogenic conditions and seeding of virus could be the probable reasons for this outbreak. Urgent attention is needed to control this new threat to tribal population, which is already overburdened with other vector borne diseases.

Influenza A(H1N1) viruses of the 2009 pandemic (A(H1N1)pdm09) continue to cause outbreaks in the post-pandemic period. During January to May 2015, an upsurge of influenza was recorded that resulted in high fatality in central India. Genetic lineage, mutations in the hemagglutinin (HA) gene and infection by quasi-species are reported to affect disease severity. The objective of this study is to present the molecular and epidemiological trends during the 2015 influenza outbreak in central India. All the referred samples were subjected to qRT–PCR for diagnosis. HA gene sequencing (23 survivors and 24 non-survivors) and cloning were performed and analyzed using Molecular Evolutionary Genomic Analyzer (MEGA 5·05). Of the 3625 tested samples, 1607 (44·3%) were positive for influenza A(H1N1)pdm09, of which 228 (14·2%) individuals succumbed to death. A significant trend was observed in positivity (P = 0·003) and mortality (P < 0·0001) with increasing age. The circulating A(H1N1)pdm09 virus was characterized as belonging to clade-6B. Clinically significant mutations were detected. Patients infected with the quasi-species of the virus had a greater risk of death (P = 0·009). This study proposes a robust molecular and clinical surveillance program for the detection and characterization of the virus, along with prompt treatment protocols to prevent outbreaks.

Hepatitis A virus (HAV) infection, a major cause of childhood hepatitis is transmitted by orofaecal route. Children mostly suffer with subclinical infection but may have serious clinical implications leading to hospitalization and mortality. IgM ELISA and nRT PCR were conducted on the blood samples collected from HAV suspected paediatric cases referred to the viral diagnostic laboratory in the Regional Medical Research Centre for Tribals at Jabalpur, Central India. The nRT PCR products were sequenced and phylogenetic analysis was done. Of the 195 samples tested, 41 (21%) were positive for HAV antibodies, among which 38 (92%) belonged to paediatric age group and 32 per cent of these were hospitalized. nRT PCR and sequencing confirmed the presence of HAV. Phylogenic analysis revealed circulation of genotype III A in central India. Regular serological and molecular monitoring would aid in understanding epidemiology of HAV and plan intervention strategies.

Dengue is an important arboviral disease. All four dengue virus serotypes are reported to be circulating in India. It is also known that different serotypes, genotypes and clades of genotype determine outbreak severity. Dengue affected children are known to have serious disease outcome. We carried out this study to give reliable diagnosis of dengue infection in children and to detect circulating serotype in central India. Samples collected from paediatric patients suspected to have dengue fever were subjected to IgM and IgG ELISA to determine dengue virus infection. Samples collected within 0-5 days of onset of illness and positive by IgM ELISA were tested by nested reverse transcription polymerase chain reaction (nRT-PCR). The PCR products were sequenced and analyzed. Of the 89 samples tested, 18 and 7 were positive for dengue IgM and IgG, respectively. Dengue activity was observed in both Jabalpur city and adjoining rural settings. One sample found positive by nRT-PCR was further sequenced to confirm dengue virus 4 as aetiological agent. Our findings demonstrated dengue virus infection in children and adolescent in central India. Because of continuous changing epidemiology, it is important to monitor dengue virus activity at both serological and molecular level in this part of the country for better patient care and management.

Dengue is regarded as the most important arboviral disease. Although sporadic cases have been reported, serotypes responsible for outbreaks have not been identified from central India over the last 20 years. We investigated two outbreaks of febrile illness, in August and November 2012, from Korea district (Chhattisgarh) and Narsinghpur district (Madhya Pradesh), respectively. Fever and entomological surveys were conducted in the affected regions. Molecular and serological tests were conducted on collected serum samples. Dengue-specific amplicons were sequenced and phylogenetic analyses were performed. In Korea and Narsinghpur districts 37·3% and 59% of cases were positive, respectively, for dengue infection, with adults being the worst affected. RT–PCR confirmed dengue virus serotype 1 genotype III as the aetiology. Ninety-six percent of infections were primary. This is the first time that dengue virus 1 outbreaks have been documented from central India. Introduction of the virus into the population and a conducive mosquitogenic environment favouring increased vector density caused the outbreak. Timely diagnosis and strengthening vector control measures are essential to avoid future outbreaks.

Two major factors, higher temperatures and the application of insecticides, can drastically alter the genetic structure of a vector mosquito population. Due to these two stresses, the majority of the population gets wiped out, but the ones that withstand the stress and survive are likely to pass on survivability, and have an altered physiology. Our study shows that exposures to higher temperatures and DDT during the larval stage affects their susceptibility as adult mosquitoes to the DEN-2 virus. The overall transcription and translation status of heat shock protein (Hsp70) in virus high- and low-susceptible was the same as that in other batches. In the case of a DDT-resistant (R-7) strain two bands were obtained during RT-PCRs after heat shock. These two alleles were obtained only with HY-1 in which R-7 males were used for the crosses, suggesting that the second allele is probably male sex linked. The higher expression of Hsp70 may provide DDT-resistant strains a better chance of survival high temperature environments, particularly in homozygotes and hybrids. It was also interesting to note that these strains have a significantly lower susceptibility to the virus. Wide-spread DDT-resistance and a rise in temperature above the average temperature during summer may result in a population with a low susceptibility to the virus. Several families of heat shock proteins are known to be expressed in mosquitoes, and may have a cumulative role in determining susceptibility to the virus, which itself is governed by several genes.

Objectives: Mosquito densonucleosis viruses (DNVs) are known to persistently infect the insect cell line and mosquito population in nature, causing mortality in mosquitoes. Here we report the isolation and characterization of a DNV from Aedes aegypti and its distribution among different Ae. aegypti populations from India. Methods: We screened Ae. aegypti mosquito populations from different states of India by PCR. Virus isolation and purification was performed using a cesium chloride gradient from a positive mosquito colony. Characterization of this isolate was carried out by electron microscopy, Western blot and sequencing. Results: Electron microscopy showed the presence of parvovirus-like particles, and Western blot showed the presence of 2 viral proteins of 40 and 41 kDa. A total of 3,776 bases of genome were sequenced, which included a 3′UTR of 128 bases, a coding region of 3,507 bases and a 5′UTR of 141 bases. Three open reading frames (ORFs) were identified and characterized. The NIVDNV genome showed 95% similarity with Culex pipiens pallens DNV and 93% similarity with Ae. aegypti DNV. Conclusion: Phylogenetic analysis of all 3 ORFs showed that this new isolate falls in the lineage of Brevidensovirus along with other mosquito DNVs.

Chikungunya (CHIK) virus has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic which occurred in 1971. For active surveillance, it is necessary to have a test, which can detect the virus from a large number of field-collected mosquitoes. The present study describes the standardization of monoclonal antibody (MAb) based antigen capture ELISA to detect chikungunya virus antigen from the mosquitoes. CHIK virus antigen from suspension of experimentally infected mosquitoes and their progeny was captured on mouse polyclonal antibody, while biotinylated CHIK Mab was used as a probing antibody. CHIK virus antigen in the head squashes of virus inoculated mosquitoes was detected using indirect immunofluorescence antibody (IFA) test for confirmation of ELISA results. The ELISA test was sensitive enough to detect antigen even if a small fraction of a single infected mosquito homogenate was incorporated in the test. The IFA test failed to detect CHIK antigen in 10 and 25 microliters of suspension whereas with ELISA it was detected in all the samples. Progeny of Aedes aegypti and Ae. albopictus mosquitoes infected with chikungunya virus did not show the possibility of existence of transovarial transmission. This test is rapid and simple since it can be completed in two days as compared to the conventional mosquito inoculation and IFA techniques, which require at least 10 days. There is an additional advantage with this test that a large number of samples can be processed, and the remaining homogenate of the mosquitoes can be used for screening other viruses. Experimental data raised using this test showed that transovarial transmission of this virus does not occur in these vector species.

Effect of Bacillus thuringiensis H-14 toxin was studied on the adults and larvae of Aedes aegypti mosquitoes. Incorporation of 1% commercial formulation of toxin in the adult blood meal caused hyper-peristaltic movement in the gut. As a result mosquitoes defecated ingested blood within 15–30 min. Similar effect of toxin was seen when it was fed to mosquitoes along with 10% glucose. The toxin in the defecated blood was active and could kill fresh larvae. It seems that this is a kind of defence response against the toxin. Atropine sulphate, which is known to reduce involuntary muscle movements, was incorporated in the blood meal. Higher amount of atropine sulphate in the blood meal of adult mosquitoes caused low mortality, but its combination with toxin caused significantly higher mortality. It is suggested that due to arrest of peristaltic movement by atropine sulphate, adults could not excrete the toxin from the gut. Experiments were also conducted to determine if the toxin causes a similar effect in the larvae. Larval bioassays were conducted with and without atropine sulphate. Addition of atropine sulphate did not show any noticeable increase in mortality in the larvae.