Determination of G and P genotypes of bovine group A rotavirus with emergence of unusual G- and P-type combinations from neonatal calf diarrhea in Kashmir, India.

A total of 490 diarrhoeic samples from calves aged between 0 and 6 months were screened for the presence of different G- and P-genotypes of rotavirus circulating in bovines in the Kashmir Valley. Of the 490 diarrhoeic samples, Group A rotavirus was detected in 68 (13.87%) samples by polymerase chain reaction (PCR) followed by RNA-PAGE. Genotyping analysis revealed G10, G6, G3, P[11] and P[5] to be the predominant types. The most common types of combinations detected were G10P[11] (27.90%) and G6P[11] (20.60%). The prevalence rate of G10 and P[11] decreased from 60% to 36.76% and 100%-69.11%, respectively. Genotypes G6, G3, P[1] and P[5], which were not previously reported, were detected and unusual combinations such as G6P[11], G3P[11], G10P[5], G3P[5], G6P[1], G6P[5], G6+G8P[11] were also observed for the first time. Fluctuations in the predominant types, emergence of new types and possible genetic reassortment events suggest an unstable epidemiological situation and the need for continuous surveillance of the circulating types to ensure the suitability of the vaccination programme. The present data suggests G10, G6, P[11] and P[5] genotypes could be incorporated in the polyvalent vaccine to offer increased protection against bovine rotavirus infection in India.

Seasonal pattern in occurrence of rotavirus infection (RV) in diarrheic children, calves and piglets from Bareilly, India.

Rapid and reliable diagnosis for diarrhoeal disease is critically important for the differentiation of etiological agents and subsequent suitable treatment modalities. The objective of the study is to reveal the seasonal pattern in the occurrence of rotavirus in diarrheic children, calves and piglets from Bareilly, Uttar Pradesh, India. A total of 115 diarrhoeal samples were collected, out of which 51 were collected during post-monsoon/autumn (September 2018-November 2018) and 64 during the winter season (December 2018-February 2019). The samples were collected from children <5 years (n = 50), piglets <3 months (n = 35) and calves <6 months of age (n = 30). These samples were screened by ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) by targeting the VP6 gene of rotavirus A (RVA) and the two were compared. In RNA-PAGE 29.4% (5/17), 6.3% (1/16) and 0% (0/18) samples collected from children, calves and piglets, respectively were rotavirus positive during the autumn season while 45.5% (15/33), 21.4% (3/14) and 17.7% (3/17) samples in the winter season. In RT-PCR, 41.2% (7/17), 12.5% (2/16) and 0% (0/18) samples were rotavirus positive in the autumn season while 51.5% (17/33), 28.6% (4/14) and 29.4% (5/17) samples in winter season collected from children, calves and piglets, respectively. On statistical analysis, no significant difference between the season and number of positives in children and calves (p > 0.05) was observed, however in piglets significantly higher number of RVA positives were detected in the winter season than autumn (p < 0.01). The diagnostic test comparison of RNA-PAGE and RT-PCR showed no statistically significant difference in detecting the RVA positives (p > 0.05). Overall the percent positivity showed a seasonal pattern with higher positivity in winter as compared to autumn season.

Molecular characterization of porcine rotavirus A from India revealing zooanthroponotic transmission.

Rotaviruses A (RVA) are leading causes of diarrhea and dehydration in piglets and imply great economic loss to the pig farming community. In this study, the porcine RVA genotypes circulating in western and northern parts of India were determined by screening 214 fecal samples from diarrheic (n = 144) and non-diarrheic (n = 70) pigs. Subsequently, the structural (VP4 and VP7) and nonstructural (NSP3, and NSP4) genes were amplified, sequenced, and genetically characterized. The RVA positivity percentage was 7.94% (17/214) by RNA-PAGE and 10.28% (22/214) by RT-PCR. Higher RVA positivity was observed in samples from Uttar Pradesh (24.07%) followed by Maharashtra (6.77%) and Goa (2.38%). The sequence and automated genotyping software analysis confirmed the circulation of G4P[6] and G9P[13] RVA strains in porcine population. To note, the sequence similarity of the VP7 gene of Porcine/INDIA/RVA/PK-13 IVRI/Maharashtra/G4 and Porcine/INDIA/RVA/P-8/IVRI/U.P./G9 strain showed a relationship of 96.83 and 98.89% at the nucleotide level with human RVA strains indicating inter-species transmission. Additionally, the NSP3 (T1) and NSP4 (E1) genes (genotypes) also showed genetic relatedness with human RVA strains. Overall, the nucleotide sequences of VP7, NSP3, and NSP4 genes of porcine RVA indicate zooanthroponotic transmission. Further, we report the detection of G9P[13] RVA strain in porcine for the first time from India.HIGHLIGHTSRVA positivity was 7.94% (17/214) by RNA-PAGE and 10.28% (22/214) by RT-PCRThe RVA strain G9P[13] reported for the first time in Indian pigletsVP7, NSP3 and NSP4 genes analysis of porcine RVA showed genetic relatedness with human strains indicating evidence of zooanthroponotic transmission.

Investigation of a large waterborne acute gastroenteritis outbreak caused by group B rotavirus in Maharashtra state, India.

An acute gastroenteritis outbreak at Devli Karad village, Maharashtra, India with an attack rate of 22.6% affected mainly adolescent and adult population. The viral investigations conducted on fecal specimens of patients hospitalized indicated the presence of rotavirus B (RVB) using RNA polyacrylamide gel electrophoresis and reverse transcription polymerase chain reaction. The samples collected from the source of drinking water also showed the presence of the only RVB. Absence of other viral agents and identification of RVB of genotype G2 as the etiological agent of the acute gastroenteritis outbreak highlights, the necessity of monitoring RVB, the viral agent known for its large outbreak potential.

Identification of group B rotavirus as an etiological agent in the gastroenteritis outbreak in Maharashtra, India.

Acute gastroenteritis outbreak occurred at Pargaon, Maharashtra, India in 1789 cases with an attack rate of 32.5% between November to December 2015. The stool specimens (n = 32) were investigated for different enteric viral agents using conventional methods. Transmission electron microscopy and RNA polyacrylamide gel electrophoresis respectively identified morphologically distinct rotavirus particles in 28% and RNA migration pattern of Group B Rotavirus (GBR) in 72% of the specimens. Reverse transcription polymerase chain reaction and nucleotide sequencing confirmed presence of GBR in 97% of the samples analyzed. The predominance of GBR infections and absence or insignificant presence of other agents confirmed GBR as an etiological agent of the gastroenteritis outbreak occurred in Maharashtra, India.

Zooanthroponotic transmission of rotavirus in Haryana State of Northern India.

Rotaviruses are the major cause of severe gastroenteritis and mortality in young children and animals. Due to segmented nature of dsRNA genome and wide host range, vast genetic and antigenic diversity exists amongst different isolates of rotaviruses. A total of 230 fecal ovine and caprine samples collected from organized farms and villages in Haryana were screened for rotavirus detection. Samples were screened by latex agglutination test and RNA-PAGE followed by RT-PCR and nucleic acid sequencing. The latex agglutination test showed 25 newborn lamb and 4 kid fecal samples positive for rotavirus. However, RNA-PAGE showed only 9 lamb fecal samples positive for rotavirus. All the samples were subjected to RT-PCR employing vp4 and vp7 gene specific primers of group A rotavirus of ovine, bovine and human origin. Only two samples from lamb (Sheep18/Hisar/2013 and Sheep22/Hisar/2013) showed vp4 and vp7 gene specific amplification with human group A rotavirus (GAR) specific primer. However, they did not show any amplification with ovine and bovine rotavirus specific primers. The nucleotide as well as deduced amino acid sequence analysis of vp4 gene of these isolates showed >98/97% and vp7 gene >95/94% nt/aa identity with human GAR from different regions of the world. Based on nucleotide similarity search, Sheep18/Hisar/2013 and Sheep22/Hisar/2013 isolates were genotyped as G1P[8] and G1P[4]. Phylogenetic analysis also confirmed that these isolates were clustered closely with human rotaviruses from different regions of the world. Earlier, higher prevalence of human rotaviruses was reported from the sample collecting area. The amplification of ovine samples with human rotavirus gene specific primers, sequence identity and phylogenetic analysis strongly suggests the zoonotic transmission of human GAR to sheep.

Detection of rotavirus from hospitalized diarrheic children in uttar pradesh, India.

In the present study 220 stool samples collected from diarrheic children admitted to different hospitals and nursing homes of Uttar Pradesh and Uttarakhand were screened for rotavirus. Of 220 diarrheic samples screened 46 samples were found to be positive for rotavirus by RNA PAGE. All the isolates exhibited 4-2-3-2 migration pattern suggesting group A rotavirus. Both long and short electropherotypes were prevalent in these regions. Six different electropherotypes were detected in this study period. Male diarrheic children were found to be more susceptible to rotavirus infection (22.96 %) than that of the female ones (17.64 %). Viral RNA isolated from stool samples again subjected to VP4 gene amplification by RT-PCR using con2 and con3 primer which resulted 876 bp product suggesting group A rotavirus. Besides virus isolation was successfully done using MA104 cell line.

Genotypic determination of human group A rotaviruses from Goa and Meghalaya states, India.

INTRODUCTION: Rotavirus is the leading cause of diarrhoea in young children in India, responsible for an estimated 21357 mean numbers of deaths in 2010. Various genotypes of rotaviruses evolved due to mutational changes have been recognized. In this study, we determined the genotypes of rotaviruses involved in diarrhea in Goa and Meghalaya states of India. METHODS: The dsRNA of rotaviruses was extracted from stool samples and detected by Ribonucleic Acid-Polyacrylamide gel electrophoresis (RNA PAGE) and Reverse transcription-polymerase Chain Reaction (RT-PCR) targeting the partial VP7 gene. The full length VP7 and partial VP4 genes of rotavirus strains were amplified by RT-PCR followed by nucleotide sequencing. The RotaC classification tool was used to determine the genotypes. RESULTS: The positivity of rotavirus by PAGE and RT-PCR was observed to be 43.10% and 39.65% in Goa and 38% and 36% in Meghalaya, respectively. Though long electrophoretic profile was appeared to be the most predominant rotavirus type in circulation in these two states, 96% of long and 84.61% short electropherotype profiles could be detected by RT-PCR. The dsRNA of rotavirus extracted from 36 samples could be transcribed and amplified by beg9end9 primers for G genotyping, while, 41 by con3con2 primers for P genotyping. G1P[8] and G1P[6] genotypes were commonly circulated in Goa and G1P[8] and G1P[4] genotypes in Meghalaya. On nucleotide analysis, 6 samples from Goa showed G1 genotype specificity, while, 3 showed P[8] specificity indicating the G1P[8] rotavirus circulating in Goa. In Meghalaya state, 3 strains showed P[8] and 2 showed P[4] genotype specificity. The majority of the G and P genotypes were closely related to each other and G1 genotypes appeared in two separate clusters, while, P[8] and P[4] appeared in the respective clusters. CONCLUSION: The circulation of G1P[8], G1P[6] genotypes in Goa and the presence of G1P[8] and G1P[4] genotypes in Meghalaya was observed.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of target ADME gene expression.

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

Detection of human rotavirus in hospitalized diarrheic children in central India.

During the present study, group A human rotaviruses were detected among diarrheic children using polyacrylamide gel electrophoresis (PAGE) technique, with a typical RNA migration pattern of 4:2:3:2, suggestive of group A rotavirus. During the study, a total of 46 fecal samples collected from hospitalized children with acute diarrhea as well as children inhabiting nearby animal farms with history of presence of animal rotaviruses on the farms were processed for detection of human rotavirus. Out of 33 diarrheic children, 12 showed presence of rotavirus infection (36.36%), however, none of the children from animal farm areas showed presence of rotavirus. Female children were more susceptible to rotavirus infection (46.15%) than males (30%). Majority of the cases of rotavirus gastroenteritis belonged up to one year of the age, with an incidence of 40.91%. RNA profile of rotaviruses suggested circulation of 5 different electropherotypes in this geographical locale of the country, indicating existence of genomic diversity among human rotaviruses. Majority of the isolates were of long pattern (66.67%), whereas short pattern was detected only in one third of the viruses. This preliminary study emphasizes for further detailed studies on the molecular characterization of rotaviruses circulating in this part of country and their relationship with other human rotavirus strains and animal strains in the country.

Detection of Rotavirus Infection in Bovine Calves by RNA-PAGE and RT-PCR.

A total of 128 diarrhoeic faecal samples were collected from cattle and buffalo calves from Pantnagar and Dehradun during winter months. Of the 110 cattle calves screened by RNA-PAGE, rotavirus was detected in 13 samples (11.81%) while no sample from buffalo calves was found positive. All samples were found to have long electropherotype and two distinct electropherotypes having segment variation were observed. The overall prevalence of rotavirus was 10.15% (13/128). RT-PCR targeting group specific VP6 gene confirmed Group A rotavirus in 10 out of 13 samples, while three samples remained un-groupable.