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Glycoprotein C gene based molecular subtyping of a bovine herpesvirus -1 isolate from uttar pradesh, India.

ABSTRACT: Bovine herpesvirus -1 (BHV-1) is the etiological agent of many clinical syndromes in cattle which causes huge economic losses to the animal husbandry sector annually. Since the first report of its presence in India in 1976, the disease is considered to be endemic in the country. In the present study, a case of keratoconjunctivitis in a cow was investigated to find out the underlying cause of the condition. The clinical material (ocular swab) was tested by BHV-1 glycoprotein D gene specific PCR using in house designed primers and found to be positive by the presence of a 212 bp DNA product in agarose gel electrophoresis. The virus was isolated in MDBK cell line in the third passage and the serum from the animal, was positive for antibodies against BHV-1 by ELISA. A 575 bp segment of the glycoprotein C gene of the isolate was amplified by PCR, cloned and sequenced. On phylogenetic analysis, it was seen that the sequence matched with published BHV-1.1 sequences from USA and Uruguay whereas it was divergent from Brazilian BHV-1.1 isolates. This study highlights the isolation, rapid and sensitive detection of BHV-1 virus from clinical cases and its subtyping by nucleotide sequencing and subsequent phylogenetic analysis which gives invaluable information about the molecular epidemiology of BHV-1 subtypes prevalent in the country.

SUBMITTER: Ravishankar C

PROVIDER: S-EPMC3550786 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Project description:Abstract Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Cryptococcosis affects more than one million people per year worldwide. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. The ecological niche of the etiological agents of C. neoformans and C. gattii are not well established yet in various parts of India. Methodology The present study was performed on the C. neoformans isolated from the Eucalyptus terreticornis at Varanasi, India. A total of 265 samples including flowers, leaves, bark, and nearby soil of E. terreticornis. The fungal pathogens were identified by both the conventional and molecular methods. The isolates were also grown in Staib's and tobacco agar medium. In addition, all isolates were identified on the basis of different biochemical tests such as urease test and canavanine-glycine-bromothymol blue (CGB). Further, molecular characterization was carried out by using PCR and DNA sequence analyses. Results Of all the 265 samples, 11 were positive for C. neoformans. 8% (4/50) for E. tereticornis bark, 5% (3/60) for nearby soil, 6.66% (2/30) for debris, and 3.07% (2/65) for decayed wood from tree hollows. C. neoformans was not recovered from the leaves and flowers of E. tereticornis. The presence of brown colonies on Staib's and tobacco agar media confirmed that the isolated pathogen is Cryptococcus. Further the isolates of Cryptococcus were tested for urease production using Christensen urea citrate agar medium in which all isolates were found to hydrolyze urea rapidly and reddish pink color was obtained confirming it to be C. neoformans. All isolated yeast were negative for Canavanine glycinebromothymol blue (CGB) as they fail to produce a color change in the medium. Five strains were identified as C. neoformans var. grubii and one of the isolates was identified as C. neoformans var. neoformans. Molecular typing grouped the isolates into two major genotypes. Five were molecular type VNI (serotype A, var. grubii), one of the isolates was molecular type VNIV (serotype D, var. neoformans). Figure 3 shows the Gel electrophoresis image of ITS1- ITS4 amplicons. The nucleotide sequences were submitted in NCBI GenBank with accession numbers MZ824412, MZ848145, MZ823611, MZ882370, MZ882372, and MZ817986. Conclusion In spite of a major Eucalyptus growing region of India, there is no report of C. neoformans/C. gattii-Eucalyptus association from Eastern Uttar Pradesh. Therefore, the current study would certainly be helpful in the establishment of molecular epidemiology of Cryptococcus in this area.

Project description:The emergence and rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a global crisis that required a detailed characterization of the dynamics of mutational pattern of the viral genome for comprehending its epidemiology, pathogenesis and containment. We investigated the molecular evolution of the SASR-CoV-2 genome during the first, second and third waves of COVID-19 in Uttar Pradesh, India. Nanopore sequencing of the SARS-CoV-2 genome was undertaken in 544 confirmed cases of COVID-19, which included vaccinated and unvaccinated individuals. In the first wave (unvaccinated population), the 20A clade (56.32%) was superior that was replaced by 21A Delta in the second wave, which was more often seen in vaccinated individuals in comparison to unvaccinated (75.84% versus 16.17%, respectively). Subsequently, 21A delta got outcompeted by Omicron (71.8%), especially the 21L variant, in the third wave. We noticed that Q677H appeared in 20A Alpha and stayed up to Delta, D614G appeared in 20A Alpha and stayed in Delta and Omicron variants (got fixed), and several other mutations appeared in Delta and stayed in Omicron. A cross-sectional analysis of the vaccinated and unvaccinated individuals during the second wave revealed signature combinations of E156G, F157Del, L452R, T478K, D614G mutations in the Spike protein that might have facilitated vaccination breach in India. Interestingly, some of these mutation combinations were carried forward from Delta to Omicron. In silico protein docking showed that Omicron had a higher binding affinity with the host ACE2 receptor, resulting in enhanced infectivity of Omicron over the Delta variant. This work has identified the combinations of key mutations causing vaccination breach in India and provided insights into the change of [virus's] binding affinity with evolution, resulting in more virulence in Delta and more infectivity in Omicron variants of SARS-CoV-2. Our findings will help in understanding the COVID-19 disease biology and guide further surveillance of the SARS-CoV-2 genome to facilitate the development of vaccines with better efficacies.

Project description:BackgroundThe sustainable development goals (SDG) aim at satisfying three-fourths of family planning needs through modern contraceptive methods by 2030. However, the traditional methods (TM) of family planning use are on the rise, along with modern contraception in Uttar Pradesh (UP), the most populous Indian state. This study attempts to explore the dynamics of rising TM use in the state.MethodsWe used a state representative cross-sectional survey conducted among 12,200 Currently Married Women (CMW) aged 15-49 years during December 2020-February 2021 in UP. Using a multistage sampling technique, 508 primary sampling units (PSU) were selected. These PSU were ASHA areas in rural settings and Census Enumeration Blocks in urban settings. About 27 households from each PSU were randomly selected. All the eligible women within the selected households were interviewed. The survey also included the nearest public health facilities to understand the availability of family planning methods. Univariate and bivariate analyses were conducted. Appropriate sampling weights were applied.ResultsOverall, 33.9% of CMW were using any modern methods and 23.7% any TM (Rhythm and withdrawal) at the time of survey. The results show that while the modern method use has increased by 2.2 percentage points, the TM use increased by 9.9 percentage points compared to NFHS-4 (2015-16). The use of TM was almost same across women of different socio-demographic characteristics. Of 2921 current TM users, 80.7% started with TM and 78.3% expressed to continue with the same in future. No side effects (56.9%), easy to use (41.7%) and no cost incurred (38.0%) were the main reasons for the continuation of TM. TM use increased despite a significant increase (66.1 to 81.3%) in the availability of modern reversible methods and consistent availability of limiting methods (84.0%) in the nearest public health facilities.ConclusionInitial contraceptive method was found to have significant implications for current contraceptive method choice and future preferences. Program should reach young and zero-parity women with modern method choices by leveraging front-line workers in rural UP. Community and facility platforms can also be engaged in providing modern method choices to women of other parities to increase modern contraceptive use further to achieve the SDG goals.

Project description:IntroductionAbortion is legal in India and medication abortion (MA) using a combined regimen of mifepristone and misoprostol is the preferred method. Users increasingly purchase MA kits directly from pharmacies, in some cases experiencing perceived complications and approaching a facility for care. We present findings of a qualitative research tracing the decision-making pathway(s) of MA users in Uttar Pradesh, India, to help understand knowledge and behaviour gaps, and recommend ways to improve the overall quality of care at these service delivery points.MethodsForty in-depth interviews were conducted with recent MA users (20 each of clinic and pharmacy clients) across three districts. Providers were purposively selected in collaboration with an international organization selling MA kits, using their list of pharmacies and clinics. MA users were identified from the clients of the selected providers, and additionally through the snow ball method. Interviews were conducted in Hindi with verbal informed consent in a private place convenient to the respondent. Transcripts were translated to English and analysed thematically.ResultsUsers first sought MA kits at pharmacies out of convenience, low cost and customer anonymity. Men often purchased kits for their partners and trusted the chemist for guidance on dosage, progression and side effects. For side effects or other concerns after using an MA kit, users first visited their neighbourhood doctor or traditional practitioner. These providers either attempted to treat the issue and failed, or directly advised her to consult a gynaecologist. The final point of care was gynaecologists, preferably female private practitioners with their own clinics. They diagnosed most abortion-related cases as incomplete abortions, emptying the uterus using the dilation and curettage method. Comparatively low cost and convenience made users inclined towards repeat use of MA.ConclusionThere are information gaps at various stages in the MA pathway that need to be addressed. Large scale public information programmes are required on safe abortion care- when is it legal, where to obtain MA, dosage, side effects and signs of possible complications. Pharmacists could be trained or incentivized to improve their quality of care to facilitate adequate exchange of information on MA. Since, for most couples, the male partner purchases MA, information approaches or tools are needed that pharmacists can give men to share directly with the MA user.

Project description:BackgroundSalinity is known to affect almost half of the world's irrigated lands, especially rice fields. Furthermore, cyanobacteria, one of the critical inhabitants of rice fields have been characterized at molecular level from many different geographical locations. This study, for the first time, has examined the molecular diversity of cyanobacteria inhabiting Indian rice fields which experience various levels of salinity.ResultsTen physicochemical parameters were analyzed for samples collected from twenty experimental sites. Electrical conductivity data were used to classify the soils and to investigate relationship between soil salinity and cyanobacterial diversity. The cyanobacterial communities were analyzed using semi-nested 16S rRNA gene PCR and denaturing gradient gel electrophoresis. Out of 51 DGGE bands selected for sequencing only 31 which showed difference in sequences were subjected to further analysis. BLAST analysis revealed highest similarity for twenty nine of the sequences with cyanobacteria, and the other two to plant plastids. Clusters obtained based on morphological and molecular attributes of cyanobacteria were correlated to soil salinity. Among six different clades, clades 1, 2, 4 and 6 contained cyanobacteria inhabiting normal or low saline (having EC < 4.0 ds m(-1)) to (high) saline soils (having EC > 4.0 ds m(-1)), however, clade 5 represented the cyanobacteria inhabiting only saline soils. Whilst, clade 3 contained cyanobacteria from normal soils. The presence of DGGE band corresponding to Aulosira strains were present in large number of soil indicating its wide distribution over a range of salinities, as were Nostoc, Anabaena, and Hapalosiphon although to a lesser extent in the sites studied.ConclusionLow salinity favored the presence of heterocystous cyanobacteria, while very high salinity mainly supported the growth of non-heterocystous genera. High nitrogen content in the low salt soils is proposed to be a result of reduced ammonia volatilization compared to the high salt soils. Although many environmental factors could potentially determine the microbial community present in these multidimensional ecosystems, changes in the diversity of cyanobacteria in rice fields was correlated to salinity.

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Glycoprotein C gene based molecular subtyping of a bovine herpesvirus -1 isolate from uttar pradesh, India.

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