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Since the first reported case of COVID-19 in December 2019, several SARS-CoV-2 variants have evolved, and some of them have shown higher transmissibility, becoming the prevalent strains. Genomic epidemiological investigations into strains from different time points, including the early stages of the pandemic, are very crucial for understanding the evolution and transmission patterns. Using whole-genome sequences, our study describes the early landscape of SARS-CoV-2 variants in central India retrospectively (including the first known occurrence of SARS-CoV-2 in Madhya Pradesh). We performed amplicon-based whole-genome sequencing of randomly selected SARS-CoV-2 isolates ( n = 38) collected between 2020 and 2022 at state level VRDL, ICMR-NIRTH, Jabalpur, from 11899 RT-qPCR-positive samples. We observed the presence of five lineages, namely B.1, B.1.1, B.1.36.8, B.1.195, and B.6, in 19 genomes from the first wave cases and variants of concern (VOCs) lineages, i.e., B.1.617.2 (Delta) and BA.2.10 (Omicron) in the second wave cases. There was a shift in mutational pattern in the spike protein coding region of SRAS-CoV-2 strains from the second wave in contrast to the first wave. In the first wave of infections, we observed variations in the ORF1Ab region, and with the emergence of Delta lineages, the D614G mutation associated with an increase in infectivity became a prominent change. We have identified five immune escape variants in the S gene, P681R, P681H, L452R, Q57H, and N501Y, in the isolates collected during the second wave. Furthermore, these genomes were compared with 2160 complete genome sequences reported from central India that encompass 109 different SARS-CoV-2 lineages. Among them, VOC lineages Delta (28.93%) and Omicron (56.11%) were circulating predominantly in this region. This study provides useful insights into the genetic diversity of SARS-CoV-2 strains over the initial course of the COVID-19 pandemic in central India.

Leptospirosis, an underdiagnosed zoonotic disease in India, was studied retrospectively in Madhya Pradesh, Central India. Between 2018 and 2019, 2617 samples from patients with hepatitis-related symptoms were collected. Of these, 518 tested negative for hepatitis and other tropical viral diseases under the VRDL project were analyzed for leptospira IgM using ELISA. 68 (13.12%) were positive for leptospirosis. Common symptoms included fever (97.45%) and jaundice (42.27%), with renal involvement in 30.88% of cases. Higher incidence was observed in the 31-60 age group, especially during monsoon and post-monsoon seasons. The study highlights the need for increased clinician awareness and inclusion of leptospirosis in screening panels to differentiate tropical illnesses in India., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the research work reported here., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

The rise of Crimean-Congo Hemorrhagic Fever (CCHF), poses a significant global health challenge, urging immediate action and continuous surveillance. With no available vaccines, monitoring pathogen presence is critical to identify at-risk areas promptly. A study was designed to assess the incidence of CCHF virus in goats and cattle using commercial ELISA IgG kits in tribal-dominated regions. Overall, 16% of the samples (n = 63/393) were positive for CCHF virus-specific IgG antibodies, whereas sero-prevalence detected in cattle 11.6% [95% CI:7-17.7] and in goats 18.9% [95% CI: 13.76-24.01], respectively. Statistically, Animal gender and age didn't significantly affect prevalence (p-value >0.05). Our finding indicates unnoticed CCHF virus circulation. Notably, lack of public awareness about zoonotic diseases in the study region was recorded. To combat this emerging tick-borne disease effectively, it's crucial to screen individuals with hemorrhagic manifestations in healthcare settings and active surveillance of ticks to prevent unwarranted public health outbreaks and design preventive interventions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the research. Declaration of Generative AI and AI-assisted technologies in the writing process. The authors declare that generative artificial intelligence (AI) and AI-assisted technologies were used to improve readability and language. The author(s) reviewed and edited the content as needed and take full responsibility for the content of the publication., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background and Objectives: Exploring the disease profile in a defined area helps policymakers to understand local health issues. It is essential to collect such information in countries, like India, which have a wide range of socioeconomic, geographic, and cultural diversity. Madhya Pradesh is the second largest state of India and has diversified populations living in urban, rural, and tribal areas. In this study, we performed a hospital record-based analysis to find out the status of different diseases in various outpatient departments (OPDs) of the District Hospital where patients from both rural and urban areas are treated., Materials and Methods: The retrospective data was collected from medical records available for the period September 2018 to September 2020. These datasets were analyzed to determine the most common presentations among patients., Results and Conclusions: A total of 138,756 records of patients were available for this study, whose department-wise analysis revealed that most records were related to respiratory tract infection, superficial dermatophytosis, anemia among women, suppurative otitis media, and pneumonia. This study provides a broad overview of the overall health issues of people living in rural and urban areas of Datia. However, a major limitation of the study was that other correlations with age and gender were not established due to the absence of such information. Nonetheless, these findings will help policymakers and researchers to set the agenda for interventions and set goals for achieving better health for all people including those living in rural and tribal regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mishra, Barde, Awasthi, Kumawat, Gaur, Singh, Srivastava and Das.)

Background & Objectives: Infection by hepatitis B virus (HBV) results in acute or chronic hepatitis. Based on sequence differences of eight per cent or more, HBV is divided into 10 genotypes (A to J) and 35 sub-genotypes. Molecular characterization of the circulating HBV genome has helped in understanding the epidemiology and its clinical importance. Spiti valley in Himachal Pradesh, which shares its border with Tibet, is one of the most HBV prevalent areas in India. Since information about the circulating genotype/s of HBV in this area is limited, this study was conducted to identify the circulating HBV genotypes., Methods: The surface and partial reverse transcriptase gene regions were sequenced using 14 hepatitis B surface antigen-positive samples., Results: Out of the 14 hepatitis B surface antigen-positive samples 11 sample gave quality sequence for further analysis. All the 11 samples belonged to subtype ayw2. The phylogenetic and recombination analysis revealed that five out of 11 samples were of genotype CD1 and the rest six were of genotype D3., Interpretation & Conclusions: The CD1 recombinant sub-genotype might have immigrated during past or present transcontinental migration between the adjacent countries. Further studies using full-genome sequencing and high sample size will be helpful to understand this epidemiology and to combat the high prevalence of HBV in the area.

Background: COVID-19 has inundated the entire world disrupting the lives of millions of people. The pandemic has stressed the healthcare system of India impacting the psychological status and functioning of health care workers. The aim of this study is to determine the burnout levels and factors associated with the risk of psychological distress among healthcare workers (HCW) engaged in the management of COVID 19 in India., Methods: A cross-sectional study was conducted from 1 September 2020 to 30 November 2020 by telephonic interviews using a web-based Google form. Health facilities and community centres from 12 cities located in 10 states were selected for data collection. Data on socio-demographic and occupation-related variables like age, sex, type of family, income, type of occupation, hours of work and income were obtained was obtained from 967 participants, including doctors, nurses, ambulance drivers, emergency response teams, lab personnel, and others directly involved in COVID 19 patient care. Levels of psychological distress was assessed by the General health Questionnaire -GHQ-5 and levels of burnout was assessed using the ICMR-NIOH Burnout questionnaire. Multivariable logistic regression analysis was performed to identify factors associated with the risk of psychological distress. The third quartile values of the three subscales of burnout viz EE, DP and PA were used to identify burnout profiles of the healthcare workers., Results: Overall, 52.9% of the participants had the risk of psychological distress that needed further evaluation. Risk of psychological distress was significantly associated with longer hours of work (≥ 8 hours a day) (AOR = 2.38, 95% CI(1.66-3.41), income≥20000(AOR = 1.74, 95% CI, (1.16-2.6); screening of COVID-19 patients (AOR = 1.63 95% CI (1.09-2.46), contact tracing (AOR = 2.05, 95% CI (1.1-3.81), High Emotional exhaustion score (EE ≥16) (AOR = 4.41 95% CI (3.14-6.28) and High Depersonalisation score (DP≥7) (AOR = 1.79, 95% CI (1.28-2.51)). About 4.7% of the HCWs were overextended (EE>18); 6.5% were disengaged (DP>8) and 9.7% HCWs were showing signs of burnout (high on all three dimensions)., Conclusion: The study has identified key factors that could have been likely triggers for psychological distress among healthcare workers who were engaged in management of COVID cases in India. The study also demonstrates the use of GHQ-5 and ICMR-NIOH Burnout questionnaire as important tools to identify persons at risk of psychological distress and occurrence of burnout symptoms respectively. The findings provide useful guide to planning interventions to mitigate mental health problems among HCW in future epidemic/pandemic scenarios in the country., Competing Interests: The authors have declared that no competing interests exist.

Hepatitis Delta virus (HDV) infection amongst Hepatitis B virus (HBV) infected patients increases morbidity and mortality. The prevalence varies temporally and spatially. The present study aimed to evaluate the HDV prevalence in central India. Samples received from January 2018 to December 2019 were tested for viral hepatitis markers. Randomly picked 372 HBsAg positive samples were tested for the presence of HDV total antibodies using ELISA, of these 8 were found positive. This study for the first-time documents presence of HDV with 2.1% prevalence from central India. We recommend screening for better patient management and bringing down the disease burden., Competing Interests: Declaration of competing interest The author declare no conflicts of interest., (Copyright © 2021 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

We conducted a nationally representative population-based survey in 60 districts from 15 Indian states covering all five geographic regions during 2017-2018 to estimate the age specific seroprevalence of dengue. Of the 12,300 sera collected, 4,955 were positive for IgG antibodies against dengue virus using IgG Indirect ELISA indicating past dengue infection. We tested 4,948 sera (seven had inadequate volume) positive for IgG antibodies on indirect ELISA using anti-dengue IgG capture ELISA to estimate the proportion of dengue infections with high antibody titers, suggestive of acute or recent secondary infection. Of the 4,948 sera tested, 529 (10.7%; 95% CI: 9.4-12.1) were seropositive on IgG capture ELISA. The proportions of dengue infections with high titers were 1.1% in the northeastern, 1.5% in the eastern, 6.2% in the western, 12.2% in the southern, and 16.7% in the northern region. The distribution of dengue infections varied across geographic regions, with a higher proportion of infections with high antibody titer in the northern and southern regions of India. The study findings could be useful for planning facilities for clinical management of dengue infections.

Background: Diphtheria is re-emerging as a public health problem in several Indian states. Most diphtheria cases are among children older than 5 years. In this study, we aimed to estimate age-specific immunity against diphtheria in children aged 5-17 years in India., Methods: We used residual serum samples from a cross-sectional, population-based serosurvey for dengue infection done between June 19, 2017, and April 12, 2018, to estimate the age-group-specific seroprevalence of antibodies to diphtheria in children aged 5-17 years in India. 8309 serum samples collected from 240 clusters (122 urban and 118 rural) in 60 selected districts of 15 Indian states spread across all five geographical regions (north, northeast, east, west, and south) of India were tested for the presence of IgG antibodies against diphtheria toxoid using an ELISA. We considered children with antibody concentrations of 0·1 IU/mL or greater as immune, those with levels less than 0·01 IU/mL as non-immune (and hence susceptible to diphtheria), and those with levels in the range of 0·01 to less than 0·1 IU/mL as partially immune. We calculated the weighted proportion of children who were immune, partially immune, and non-immune, with 95% CIs, for each geographical region by age group, sex, and area of residence (urban vs rural)., Findings: 29·7% (95% CI 26·3-33·4) of 8309 children aged 5-17 years were immune to diphtheria, 10·5% (8·6-12·8) were non-immune, and 59·8% (56·3-63·1) were partially immune. The proportion of children aged 5-17 years who were non-immune to diphtheria ranged from 6·0% (4·2-8·3) in the south to 16·8% (11·2-24·4) in the northeast. Overall, 9·9% (7·7-12·5) of children residing in rural areas and 13·1% (10·2-16·6) residing in urban areas were non-immune to diphtheria. A higher proportion of girls than boys were non-immune to diphtheria in the northern (17·7% [12·6-24·2] vs 7·1% [4·1-11·9]; p=0·0007) and northeastern regions (20·0% [12·9-29·8] vs 12·9% [8·6-19·0]; p=0·0035)., Interpretation: The findings of our serosurvey indicate that a substantial proportion of children aged 5-17 years were non-immune or partially immune to diphtheria. Transmission of diphtheria is likely to continue in India until the immunity gap is bridged through adequate coverage of primary and booster doses of diphtheria vaccine., Funding: Indian Council of Medical Research., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background & Objectives: Infections caused by arboviruses and transmitted by Aedes species mosquitoes are a serious health concern. India is endemic for diseases like Dengue, Chikungunya and recently Zika has been reported from few states. Vector control is the only way to contain these diseases, however, data regarding vectors from central India is lacking; to fulfill the lacuna we conducted this study., Methods: Entomological surveys were conducted from November 2017 to December 2018 for Aedes species in Dengue endemic areas of central India. The mosquitoes were identified, pooled and tested for the presence of Dengue, Chikungunya and Zika viruses by RT-PCR. The PCR products were sequenced to identify serotypes and genotypes of viruses., Results: A total of 2991 adults of Aedes specimens were collected and tested. Ae. aegypti (94.6%) was found to be the most abundant species. Highest mosquito density was recorded in the monsoon periods. Dengue (n=5) and Chikungunya (n=4) virus were detected from pools of female Ae. aegypti. One pool of male Ae. aegypti was positive for Dengue virus-3 and Chikungunya virus. Zika virus was not detected from any pool., Interpretation & Conclusion: The findings suggest that Ae. aegypti is the principal vector of Dengue and Chikungunya, which is capable to transmit these viruses vertically. The findings have epidemiological importance and will be helpful to program managers., Competing Interests: None

Background: Since its re-emergence in 2005, chikungunya virus (CHIKV) transmission has been documented in most Indian states. Information is scarce regarding the seroprevalence of CHIKV in India. We aimed to estimate the age-specific seroprevalence, force of infection (FOI), and proportion of the population susceptible to CHIKV infection., Methods: We did a nationally representative, cross-sectional serosurvey, in which we randomly selected individuals in three age groups (5-8, 9-17, and 18-45 years), covering 240 clusters from 60 selected districts of 15 Indian states spread across all five geographical regions of India (north, northeast, east, south, and west). Age was the only inclusion criterion. We tested serum samples for IgG antibodies against CHIKV. We estimated the weighted age-group-specific seroprevalence of CHIKV infection for each region using the design weight (ie, the inverse of the overall probability of selection of state, district, village or ward, census enumeration block, and individual), adjusting for non-response. We constructed catalytic models to estimate the FOI and the proportion of the population susceptible to CHIKV in each region., Findings: From June 19, 2017, to April 12, 2018, we enumerated 117 675 individuals, of whom 77 640 were in the age group of 5-45 years. Of 17 930 randomly selected individuals, 12 300 individuals participated and their samples were used for estimation of CHIKV seroprevalence. The overall prevalence of IgG antibodies against CHIKV in the study population was 18·1% (95% CI 14·2-22·6). The overall seroprevalence was 9·2% (5·4-15·1) among individuals aged 5-8 years, 14·0% (8·8-21·4) among individuals aged 9-17 years, and 21·6% (15·9-28·5) among individuals aged 18-45 years. The seroprevalence was lowest in the northeast region (0·3% [95% CI 0·1-0·8]) and highest in the southern region (43·1% [34·3-52·3]). There was a significant difference in seroprevalence between rural (11·5% [8·8-15·0]) and urban (40·2% [31·7-49·3]) areas (p<0·0001). The seroprevalence did not differ by sex (male 18·8% [95% CI 15·2-23·0] vs female 17·6% [13·2-23·1]; p=0·50). Heterogeneous FOI models suggested that the FOI was higher during 2003-07 in the southern and western region and 2013-17 in the northern region. FOI was lowest in the eastern and northeastern regions. The estimated proportion of the population susceptible to CHIKV in 2017 was lowest in the southern region (56·3%) and highest in the northeastern region (98·0%)., Interpretation: CHIKV transmission was higher in the southern, western, and northern regions of India than in the eastern and northeastern regions. However, a higher proportion of the population susceptible to CHIKV in the eastern and northeastern regions suggests a susceptibility of these regions to outbreaks in the future. Our survey findings will be useful in identifying appropriate target age groups and sites for setting up surveillance and for future CHIKV vaccine trials., Funding: Indian Council of Medical Research., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Serology remains the mainstay for diagnosis of scrub typhus. In central India, diagnosis of this neglected emerging zoonotic disease suffers due to lack of standardized region-specific cutoff value and diagnostic test. Henceforth, standardized region-specific cutoff value for diagnostic ELISA play a significant role in initial treatment of patients and to differentiate other febrile diseases in endemic setup. A total of 144 patients of all age groups with acute undifferentiated febrile illness patients, forty healthy controls, dengue and chikungunya positive thirty-five samples in each category, respectively were enrolled in the study and subjected to IgM ELISA (InBioS, International, Inc.). Samples showing OD value more than 0.5 in IgM ELISA, were subjected to nested PCR. Both, receiver operating characteristic (ROC) curve and healthy volunteer samples mean with +3 SD were considered to generate region specific cutoff OD value. A total of 48 patients were diagnosed as cases of scrub typhus through IgM ELISA. Out of 48 samples, 30 were positive by nested PCR. The ROC curve analysis revealed a diagnostic ELISA cutoff value of 0.73 with sensitivity and specificity of 95% and 100%, respectively. The cut off arrived from healthy volunteer is mean OD + 3 SD is 0.72. Considering the significance of scrub typhus diagnosis for treatment and to understand disease dynamics in region wise, the cutoff value of >0.72 for diagnostic ELISA for Madhya Pradesh in central India can be used., Competing Interests: None

Purpose: Hepatitis B virus (HBV) is one of the leading causes of morbidity and mortality across the globe. The pathogenesis, clinical outcomes, disease progression and response to antiviral treatment of HBV depend on infecting genotypes and mutations across HBV genome. There is a lack of such information from central India. The present study was planned to identify genotype/subgenotype and epidemiologically important mutation in HBV circulating in the area., Methods: Samples positive for HBsAg by ELISA from 2012 to 2016 were included and analysed in this retrospective study. The amplification of partial S gene (n = 25) and full genome (n = 10) was carried out to determine the genotype/subgenotype and genome wide mutations of HBV. The sequencing data was analysed using bioinformatics tools., Results: All 25 sequences belonged to genotype D; subgenotypes D1, D2, D3 and D5 with dominance of D1 were detected in the study subjects. Mutational profiling revealed the presence of nucleotide substitutions in promoter/regulatory/precore region associated with liver disease progressions. The amino acid (aa) changes associated with vaccine escape, immune escape, antiviral resistance and progression to liver cirrhosis (LC) or hepatocellular carcinoma (HCC) were detected., Conclusions: This maiden molecular study on HBV from central India indicates that the genotype D with subgenotypes D1, D2, D3 and D5 harbouring mutations of clinical and epidemiological importance are in circulation. This study will serve as a baseline for future. Studies with larger sample size may aid in identifying the circulation of more genotypes., (Copyright © 2021 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Introduction: The TORCH infections during pregnancy are associated with adverse congenital abnormalities, poor foetal outcome and subsequent reproductive failures. The absence of baseline data on status of TORCH infections and associated foetal outcomes prompted us to conduct the current study in Central India., Materials and Methods: : A total of 144 high-risk pregnant women attending tertiary care unit, suspected for TORCH infections were enrolled from August 2017 to December 2018. All the participants were tested for the presence of IgM and IgG antibodies and followed up to record the foetal outcome., Results: The overall TORCH infection (IgM positivity) positivity rate was 61.1% (88/144). Rubella was the most prevalent infection (46.5%) followed by herpes simplex virus (HSV) 1 and 2 (41%) and cytomegalovirus (CMV) (34.7%). The highest IgG sero-positivity was recorded against CMV (88.6%), followed by Rubella (86.8%), HSV 1 and 2 (28.4%), and toxoplasmosis (15.2%). Follow-up of IgM TORCH positive pregnant women revealed that majority of the neonates/infants are having congenital cardiac abnormalities (39.2%), followed by microcephaly/hydrocephaly (25%), low birth weight (10.7%), and deafness (3.6%). Thirty-two percent of neonatal mortality was associated to multiple TORCH infections., Conclusion: A high prevalence of IgM seropositivity of TORCH infection was noted in the present study with the increased rate of poor foetal outcome warrants the need of proper prenatal counselling, universal immunisation and nutritional supplements during pregnancy., Competing Interests: None

Background & Objectives: Chikungunya (CHIK) is a neglected, re-emerging arboviral disease. Limited information on CHIK-confirmed cases during interepidemic period is available from India. This surveillance study was conducted in Madhya Pradesh (MP), India, during the years 2016-2017, to provide information about CHIK cases., Methods: Blood samples collected from patients suspected having CHIK were tested by immunoglobulin (Ig) IgM ELISA or real time reverse transcription-polymerase chain reaction (rRT-PCR) for the detection of CHIK virus (CHIKV)-specific IgM antibodies or viral RNA, respectively. Partial envelope-1 gene sequencing was done. Clinical and demographic data were collected and analyzed., Results: Of the 4019 samples tested, 494 (12.2%) were found positive for CHIKV infection. The positivity was detected in both rural and urban areas. The mean age of CHIK-positive cases was 33.12±18.25 yr. Headache and joint pain were the most prominent symptoms, 34.6 per cent (171/494) of the CHIK cases required hospitalization and six patients with CHIKV infection died. The East/Central/South African genotype of CHIKV was found to be circulating in the study area., Interpretation & Conclusions: Our study recorded a higher CHIK positivity during 2016-2017 in comparison to earlier reports from MP, India. A high proportion of CHIK cases required hospitalization and deaths were also reported, which indicated the severity of the disease in the study area. In-depth molecular analysis of the virus and other risk factors is essential to understand the trends in disease severity., Competing Interests: None

Background & Objectives: Dengue virus (DENV) causes outbreaks and sporadic cases in tropical and subtropical countries. Documenting intricacies of DEN outbreaks is important for future interventions. The objective of this study was to report clinical, laboratory and epidemiological features of DEN outbreaks reported in different districts of Central India in 2016., Methods: In 2016, outbreaks (n=4) suspected of DEN were investigated by rapid response team. Door-to-door fever and entomological surveys were conducted. Blood samples were collected and tested using NS1 or IgM ELISA; real-time reverse transcription-polymerase chain reaction was done to identify serotypes of DEN virus (DENV). NS1-positive samples were tested for the presence of IgG by ELISA. Clinical and demographic data were collected and analyzed., Results: Outbreaks occurred in both urban and rural areas in monsoon season and Aedes aegypti was identified as the vector. Fever, chills, headache and myalgia were the major symptoms; no fatality was recorded. Of the 268 DEN suspects, 135 (50.4%) were found serologically positive. DEN positivity was higher (n=75; 55.56%) among males and in the age group of 16-45 yr (n=78; 57.8%). DENV 3 followed by DENV 2 were detected as the major responsible serotypes. High attack rates (up to 38/1000) and low cumulative IgG prevalence (14.9%) were recorded in rural areas., Interpretation & Conclusions: Our study showed that DENV 3 was the major serotype responsible for outbreaks that occurred in monsoon. High attack rates and lower number of secondary infections in rural areas indicated that DENV is emerging in rural parts of Central India. Early diagnosis at local level and timely intervention by mosquito control activities are needed to avoid such outbreaks in future., Competing Interests: None

Viral hepatitis is a considerable public health burden affecting millions of people throughout the world. The incidence of viral hepatitis varies greatly depending upon geographic locations, age and gender. Exploring the etiological spectrum and clinic-epidemiological profile of acute viral hepatitis (AVH) becomes essential for strategizing the preventive measures to control the diseases. An epidemiological data depicting AVH situation and its etiologies is missing from central India. With the aim of fulfilling this lacuna, the present analysis was done on samples tested over a period of 2 years from July 2015 to June 2017. Of the 1901 hepatitis cases, 597 individuals (31.4%) were positive for AVH infection and HEV was the predominant cause followed by HBV, HAV and HCV. Co-infections of hepatitis viruses were detected in 42 cases. Co-infection of HEV with HBV was the commonest pattern. Male preponderance was observed among AVH positive cases and the age group of 26-45 years was the most susceptible to the viral hepatitis infections, except hepatitis A, which was the most frequent among children. Two hundred patients (33.45%) required hospitalization and 51 deaths were attributed to AVH infections. The analysis for the first time reports intricacies and viral etiologies of AVH in central India. Regular diagnosis of AVH etiology and monitoring of cases will help in patient management and assist disease control programs to take policy decisions., Competing Interests: Conflicts of interestAuthors declare that there is no conflict of interest., (© Indian Virological Society 2019.)

Hepatitis B virus (HBV) infection is an important health issue across the world. With 4% to 7% prevalence of HBV, India is designated as an intermediate endemic country. However, HBV prevalence is significantly high in some pockets of the country, especially among tribal populations. HBV prevalence and associated risk factors in residents of far-flung two Himalayan valleys, Lahaul and Spiti of Himachal Pradesh (HP), were estimated. This was a community-based cross-sectional study. Blood samples were collected and tested for the presence of hepatitis B surface antigen (HBsAg) using ELISA. Data was collected in a predesigned semi-structured format. Univariate and multivariate analyses of risk factors were done using software SPSS 25. Samples from 1,327 individuals residing in 32 villages were tested, of these 141 (10.6%) were found positive for HBsAg. High prevalence (17.2%) of HBV was recorded in Spiti valley but not in Lahaul valley (3.1%). Both sexes were equally affected. Positivity was higher in adults than in children. High risk sexual behavior (OR = 2.0; 95% CI: 1.1-3.6), having an HBV positive person in the family (OR = 2.4; 95% CI: 1.4-4.3), being a student (OR: 11.2; 95% CI 3.9-32.1) and preacher (OR = 9.0; 95% CI: 3.6-22.4) were the most prominent risk factors associated with HBsAg positivity. Mass immunization for HBV along with information, education, communication and behavior change communication for curtailing risk behavior for avoiding risk factors is essential in the area.

Background & Objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India., Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases., Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups., Interpretation & Conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme., Competing Interests: None

Purpose: Dengue viruses (DENVs), the causative agents of dengue (DEN), are classified into four serotypes and several genotypes. Identifying circulating serotypes and genotypes has clinical and epidemiological importance; however, limited information in this regard is available from Central India. This laboratory-based study was done to fill this lacuna., Materials and Methods: The samples collected in the acute phase of illness were subjected to DEN NS1 ELISA, and NS1-positive samples (n = 80) were subjected to serotyping; representative samples from each serotype were sequenced to identify genotypes., Results: Seventy-one (88.75%) samples could be serotyped. All the four DENV serotypes with dominance of DENV-3 (n = 33; 47%) were detected. DENV-4 was detected after a gap of 3 years. Cases with multiple DENV serotype infection were identified. Genotyping showed that DENV-1 belonging to genotype III, DENV-2 cosmopolitan (IV), DENV-3 genotype III lineage C and DENV-4 genotype I were in circulation in the year 2016., Conclusion: Our study documents the molecular characteristics of DENV circulating in the area. Detection of heterologous DENV serotype with dominance of DENV-3 emphasises the need for regular molecular monitoring., Competing Interests: None

Background: Dengue is most important arboviral disease spreading to newer areas. Timely diagnosis and mosquito control are only ways to prevent outbreaks; however there are very few studies demonstrating this hypothesis., Methods: During this study laboratory diagnosis for Dengue was provided to patient and the programme manager on the same day of receipt of the sample. The mobile number of the patient/kin of patient was used to contact patient and reach their address to conduct vector control within 24h of case detection., Results: Eighty three present cases were diagnosed within 24h and 82% houses were surveyed within 24h of reporting of the positive cases. The prompt diagnosis proactive vector control model helped keeping dengue outbreaks at bay., Conclusion: Timely diagnosis and prompt vector control activities can prevent dengue outbreaks. It is suggested to enhance diagnostic capabilities at local level and use new technologies such as mobile phones and internet to strengthen health systems to curb DEN., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Influenza A(H1N1)pdm09 virus pandemic struck India in 2009 and continues to cause outbreaks in its post-pandemic phase. Diminutive information is available about influenza A(H1N1)pdm09 from central India. This observational study presents epidemiological and molecular findings for the period of 6 years. Throat swab samples referred from districts of Madhya Pradesh were subjected to diagnosis of influenza A(H1N1)pdm09 following WHO guidelines. Clinical and epidemiological data were recorded and analyzed. Hemagglutinin (HA) gene sequencing and phylogenetic analysis were performed. The H275Y mutation responsible for antiviral resistance was tested using allelic real-time RT-PCR. Out of 7365 tested samples, 2406 (32.7%) were positive for influenza A(H1N1)pdm09, of which 363 (15.08%) succumbed to infection. Significant trends were observed in positivity (χ 2  = 50.8; P < 0.001) and mortality (χ 2  = 24.4; P < 0.001) with increasing age. Mutations having clinical and epidemiological importance were detected. Phylogenetic analysis of HA gene sequences revealed that clade 7, 6A, and 6B viruses were in circulation. Oseltamivir resistance was detected in three fatal cases. Influenza A(H1N1)pdm09 viruses having genetic diversity were detected from central India and continues to be a concern for public health. This study highlights the need of year-round monitoring by establishment of strong molecular and clinical surveillance program., (© 2017 Wiley Periodicals, Inc.)

Human Respiratory syncytial virus (hRSV) is the major cause of respiratory tract infection in both children and adults, virtually all children acquire infection with hRSV by the age of 3 years. Two subgroups of the virus, hRSV-A and hRSV-B based on sequence variability of G protein gene are divided into 11 and 17 genotypes, respectively. Very limited data regarding circulating genotypes is available from India. This study aimed to detect and characterize the circulating genotype of hRSV from central India. Throat swabs collected from patient's having influenza like illness (ILI) were subjected to RT-PCR for diagnosis, further sequencing and phylogenetic analysis was performed using primers specific for C-terminal end of G gene. Out of 526 tested samples 62 (12%) were found positive, 90% cases were from children under 3-year age children. Both hRSV-A and hRSV-B were detected in equal proportions. Sequence analysis of 15 samples revealed circulation of genotypes NA1, ON1 of hRSV-A, and BA9 of hRSV-B. We advocate molecular surveillance of hRSV for better patient management and epidemiological monitoring., (© 2017 Wiley Periodicals, Inc.)

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.

The objective of this study was to demonstrate the utility of dengue virus (DENV) non structural protein 1 (NS1) based rapid diagnostic test (RDT) for use in tribal and difficult to reach areas for early dengue (DEN) diagnosis in acute phase patients and evaluate its sensitivity and specificity against DENV NS1 enzyme linked immune sorbent assay (ELISA) and real time reverse transcriptase polymerase chain reaction (qRT-PCR). The DENV NS1 RDT was used for preliminary diagnosis during outbreaks in difficult to reach rural and tribal areas. The diagnosis was confirmed by DENV NS1 ELISA in the laboratory. The samples were also tested and serotyped by qRT-PCR. The results were evaluated using statistical tests. The DENV NS1 RDT showed 99.2% sensitivity and 96.0% specificity when analyzed using DENV NS1 ELISA as standard. The specificity and sensitivity of the RDT when compared with qRT-PCR was 93.6% and 91.1%, respectively. The serotype specific evaluation showed more than 90% sensitivity and specificity for DENV-1, 2, and 3. The RDT proved a good diagnostic tool in difficult to reach rural and tribal areas. Further evaluation studies with different commercially available RDTs in different field conditions are essential, that will help clinicians and patients for treatment and programme managers for timely intervention., (© 2017 Wiley Periodicals, Inc.)

The purpose of this study was to evaluate rubella susceptibility of pregnant women from central India as rubella infection can be devastating for the newborn if it occurs in the mother in the first trimester of pregnancy, which may lead to congenital rubella syndrome (CRS). There are very few studies about seroprevalence of rubella from India and none from central India. The study was conducted among women attending the obstetric department of a tertiary care hospital, in which 369 antenatal cases were tested for the presence of immunoglobulin G antibody for rubella and its titer. Data were analyzed using statistical tests. A total of 141 (38.2%) women were found susceptible to rubella. There was no significant difference in rubella susceptibility among different socioeconomic classes, ages, and gravidity. A large proportion of pregnant women were found to be rubella susceptible, posing immense threat of CRS to their newborns. A robust program for rubella immunization targeting young adult women is needed to avoid CRS.

Background and Aims: An acute hepatitis, caused by hepatitis E virus (HEV), is a significant global health concern, especially in developing countries. HEV has 1 serotype and 8 genotypes, which are further divided into subtypes. Despite the availability of sporadic cases and outbreak data from India, specific information regarding HEV epidemiological data is lacking from central India. This study was conducted to understand epidemiological and molecular features of HEV in central India., Methods: In this 3-year study conducted from July 2012 to June 2015, IgM ELISA was used for the diagnosis of suspected HEV cases. For identifying the genotype, nRT PCR was conducted and the PCR products were sequenced and analysed. Clinical and demographic data were analysed using statistical tools to highlight the trends., Result: Out of 1,369 suspected cases, 341 (24.9%) were positive for HEV. The positivity was significantly higher in males (69.2%) and in the age group of > 15-45 years (72.5%). The HEV cases peaked during the summer. Subtypes 1a and 1f of genotype 1 were detected in the area during the study period., Conclusion: HEV is a major aetiological agent of viral hepatitis in central India with adults and males at higher risk of infection. Two subtypes of the virus were detected in the region. Continuous serological surveillance and molecular monitoring will help to understand the epidemiology of HEV infection, outbreak mitigation and aid in providing treatment., (© 2018 S. Karger AG, Basel.)

Background & Objectives: Various conventional methods such as gaseous, vapour and misting systems, fogging, manual spray and wipe techniques employing a number of chemical agents are used for decontamination of enclosed spaces. Among all these methods, use of aerosolized formaldehyde is the most preferred method due to cost-effectiveness and practical aspects. However, being extremely corrosive in nature generating very irritating fumes and difficulty in maintaining a high level of gas concentration, many laboratories prefer the vaporization of hydrogen peroxide (H 2 O 2 ) as an alternative. We present here the results of using H 2 O 2 vapour in combination with plasma irradiation for quick decontamination of closed chambers., Methods: The present study describes a decontamination method, using plasma irradiation in combination with H 2 O 2 (5%). Effect of plasma irradiation and H 2 O 2 on the viability of bacterial spores (Bacillus subtilis), Chikungunya and Kyasanur Forest Disease viruses was assessed., Results: Data suggest that with the combination of H 2 O 2 vapour and plasma irradiation, within short time (three minutes), decontamination of surfaces and space volume could be achieved. Although it showed damage of spores present on the strips, it did not show any penetration power., Interpretation & Conclusions: The results were encouraging, and this method was found to be efficient for achieving surface sterilization in a short time. This application may be useful in laboratories and industries particularly, those working on clean facility concept following good laboratory and manufacturing practices., Competing Interests: None.

Background & Objectives: 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., Methods: 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., Results: 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., Interpretation & Conclusions: 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.

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.

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.

Background: Dengue and chikungunya present with very similar signs and symptoms in the initial stage of illness and so it is difficult to distinguish them clinically. Both are transmitted by Aedes aegypti and Aedes albopictus mosquitoes. This study was conducted with the aim to explore the co-circulation of dengue and chikungunya viruses in central India., Materials and Methods: Samples from suspected dengue cases were subjected to dengue immunoglobulin M (lgM) enzyme-linked immunosorbent assay (ELISA) and dengue-negative samples were tested with chikungunya-specific IgM ELISA. The samples collected in acute phase of illness were tested by nested reverse transcription polymerase chain reaction (nRT-PCR). Chikungunya virus (CHIKV) sequences were analysed to determine their genotype., Results: Of 138 samples screened for dengue, 21 (15.2%) were positive, and of 119samples screened for chikungunya, 13 (10.9%) were positive. Dengue viruses 1 and 4 were found co-circulating with chikungunya virus in Jabalpur, central India. The chikungunya virus detected belonged to the East Central South African genotype., Conclusion: Accurate and timely diagnosis would help in patient management and use of resources. It is advocated to simultaneously test samples for these two diseases in endemic areas. This will also aid in understanding the epidemiology of chikungunya.

Background: Vector-borne diseases (VBDs) caused by parasites and viruses are a major cause of morbidity and mortality in Madhya Pradesh (MP), central India. These diseases are malaria, lymphatic filariasis, dengue and chikungunya. Epidemiological information is lacking on different VBDs that are commonly prevalent in rural-tribal areas of MP, except on malaria., Methods: The studies were carried out at the request of Government of Madhya Pradesh, in three locations where many VBDs are endemic. Data on malaria/filaria prevalence were collected by repeatedly undertaking cross-sectional parasitological surveys in the same areas for 3 years. For dengue and chikungunya, suspected cases were referred to the research centre., Results: Monitoring of results revealed that all the diseases are commonly prevalent in the region, and show year-to-year variation. Malaria slide positivity (the number of malaria parasitaemic cases, divided by the total number of blood smears made) was 18.7% (190/1018), 16.4% (372/2266) and 20.4% (104/509) respectively in the years 2011, 2012 and 2013. There was a strong age pattern in both Plasmodium vivax and P. falciparum. The slide vivax rate was highest among infants, at 5% (odds ratio [OR] = 3.8; 95% confidence interval [CI] - 1.5 to 9.4; P<0.05) and the highest slide falciparum rate was 20% in children aged 1-4 years (OR = 2.0; 95%CI 1.5 to 2.7; p<0.0001). This age-related pattern was not seen in other VBDs. The microfilaria rate was 7.5%, 7.6% and 7.8% in the years 2010, 2012 and 2013, respectively. Overall, microfilaria rates were higher in males (8.7%) as compared to females 6.4% (OR = 1.5; 95% CI = 1.1 to 2.0; P < 0.01). The prevalence of dengue was 48% (dengue viruses 1 and 4 - DENV-1 and DENV-4), 59% (DENV-1) and 34% (DENV-3) respectively, in the years 2011, 2012 and 2013 among referred samples, while for chikungunya very few samples were found to be positive., Conclusion: Despite recent advances in potential vaccines and new therapeutic schemes, the control of VBDs remains difficult. Therefore, interruption of transmission still relies on vector-control measures. A coordinated, consistent, integrated vector-management approach is needed to control malaria, filaria, dengue and chikungunya.

Background: Lymphatic filariasis is a major vector-borne disease and India bears 81% of the filariasis burden in South East Asia. Screening and mass drug administration is carried out in 11 known endemic districts of Madhya Pradesh. However, regular monitoring is not carried out in non-endemic areas., Methods: Cross sectional surveys were carried out to examine blood samples for filariasis. Entomological surveys were conducted for infective mosquitoes., Results: Altogether, 24 of 252 samples were positive for filariasis with a mean microfilaria count of 0.8. The entomological investigations showed 7.4% infection and 1.4 % infectivity rates in vectors., Conclusion: This study reveals the emergence of new foci of lymphatic filariasis in Madhya Pradesh. We conclude that filariasis surveys should be carried out in non-endemic areas of India to achieve the goal of elimination.

Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background & Objectives: 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., Methods: 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., Results: 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., Interpretation & Conclusions: 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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Dengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The current lack of an approved tetravalent vaccine and ineffective insecticide control measures warrant a search for alternatives to effectively combat DENV. The trans-splicing variant of the Tetrahymena thermophila group I intron catalytic RNA, or ribozyme, is a powerful tool for post-transcriptional RNA modification. The nature of the ribozyme and the predictability with which it can be directed makes it a powerful tool for modifying RNA in nearly any cell type without the need for genome-altering gene therapy techniques or dependence on native cofactors., Results: Several anti-DENV Group I trans-splicing introns (αDENV-GrpIs) were designed and tested for their ability to target DENV-2 NGC genomes in situ. We have successfully targeted two different uracil bases on the positive sense genomic strand within the highly conserved 5'-3' cyclization sequence (CS) region common to all serotypes of DENV with our αDENV-GrpIs. Our ribozymes have demonstrated ability to specifically trans-splice a new RNA sequence downstream of the targeted site in vitro and in transfected insect cells as analyzed by firefly luciferase and RT-PCR assays. The effectiveness of these αDENV-GrpIs to target infecting DENV genomes is also validated in transfected or transformed Aedes mosquito cell lines upon infection with unattenuated DENV-2 NGC., Conclusions: Analysis shows that our αDENV-GrpIs have the ability to effectively trans-splice the DENV genome in situ. Notably, these results show that the αDENV-GrpI 9v1, designed to be active against all forms of Dengue virus, effectively targeted the DENV-2 NGC genome in a sequence specific manner. These novel αDENV-GrpI introns provide a striking alternative to other RNA based approaches for the transgenic suppression of DENV in transformed mosquito cells and tissues.

Background: Densonucleosis viruses are the etiological agents of insect's disease. We have reported the isolation of densovirus from India and its distribution among the natural populations of Aedes aegypti mosquitoes across the country. Since densonucleosis virus persistently infects mosquito populations, and is demonstrated to negatively affect multiplication of dengue virus in Aedes albopictus, it would be interesting to study if this virus has a role in determining the susceptibility of the vector mosquito Ae. aegypti to chikugunya virus., Methods: Mosquito cell lines and adult Ae. aegypti mosquitoes infected with densovirus were superinfected with Chikungunya virus and both the viruses were quantitated by determining their genomic copy number by real time amplification. Comparison was made between the log of genomic copy numbers of the viruses in the presence and absence of each other., Results: The log of copy number of the viruses did not vary due to co-infection. Even though the RNA copy number of chikungunya virus increased over the period of time, no change was observed in the RNA copy number between the control and the co-infected group on any given day. Similarly, DNA copy number of densovirus also remained unchanged between the control and the co-infected groups., Conclusion: Chikungunya virus neither stimulates the replication of densovirus nor is its own replication suppressed due to co-infection. Ae. aegypti mosquitoes with densovirus infection were as susceptible to infection by chikungunya virus as the uninfected mosquitoes.

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analyses of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost 50 years (1956-2005). Analysis of complete envelope (E) gene sequences of 37 strains of DENV-2 from India, together with globally representative strains, revealed that the American genotype, which circulated predominantly in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, known to be involved functionally in the cellular tropism of the virus, were shown to be under positive selection pressure. The rate of nucleotide substitution estimated for DENV-2 was 6.5x10(-4) substitutions per site year(-1), which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 73 and 100 years (1905-1932), which correlates with the historical record of traffic between India and South America and suggests transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the Cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian Cosmopolitan strains was about 47 years, with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean between 1955 and 1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.

Kyasanur Forest disease virus (KFDV) is enzootic to India and maintained in ticks, mammals, and birds. It causes severe febrile illness in humans and was first recognized in 1957 associated with a high number of deaths among monkeys in Kyasanur Forest. Genetic analysis of 48 viruses isolated in India during 1957-2006 showed low diversity (1.2%). Bayesian coalescence analysis of these sequences and those of KFDVs from Saudi Arabia and the People's Republic of China estimated that KFDVs have evolved at a mean rate of approximately 6.4 x 10(-4) substitutions/site/year, which is similar to rates estimated for mosquito-borne flaviviruses. KFDVs were estimated to have shared a common ancestor in approximately 1942, fifteen years before identification of the disease in India. These data are consistent with the view that KFD represented a newly emerged disease when first recognized. Recent common ancestry of KFDVs from India and Saudi Arabia, despite their large geographic separation, indicates long-range movement of virus, possibly by birds.

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., (Copyright 2009 S. Karger AG, Basel.)

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.

Aim: Intestinal ischemia (Ii) is an abdominal emergency due to blockade of the superior mesenteric artery resulting in 60-100% mortality if diagnosed late. Changes in several biochemical parameters such as D (-)-lactate, Creatinine kinase isoenzymes and lactate dehydrogenase suggested for early diagnosis, lack specificity and sensitivity. Therefore a biochemical parameter with greater sensitivity needs to be identified., Methods: Wistar male rats were randomly assigned into two groups; control sham operated (n = 24) and ischemic test (n = 24) group. Superior mesenteric arterial occlusion was performed in the ischemic test group for 1 h. Alcohol dehydrogenase (ADH) was estimated in blood from portal vein, right ventricle of heart, dorsal aorta (DA) and inferior vena cava (IVC). The Serum glutamic acid pyruvate transaminase (SGPT) was also estimated in blood from portal vein and right ventricle of heart., Results: A significant increase (P<0.001) in the levels of ADH in both portal blood as well as heart blood of the test group (232.72+/-99.45 EU and 250.85+/-95.14 EU, respectively) as compared to the control group (46.39+/-21.69 EU and 65.38+/-30.55 EU, respectively) were observed. Similarly, increased levels of ADH were observed in blood samples withdrawn from DA and IVC in test animals (319.52+/-80.14 EU and 363.90+/-120.68 EU, respectively) as compared to the control group (67.68+/-63.22 EU and 72.50+/-58.45 EU, respectively). However, in test animals there was significant increase in SGPT in portal blood (P = 0.054) without much increase in heart blood., Conclusion: Significant increase in the levels of ADH in portal and heart blood within 1 h of SMA occlusion without increase in SGPT in heart blood, suggests that the origin of ADH is from ischemic intestine and not from liver. Similarly, raised ADH levels were found in DA and IVC as well. IVC blood does represent peripheral blood sample. A raised level of ADH in test animals confirms it to be a potential marker in the early diagnosis of Ii.