Evaluation of different genomic regions of rotavirus B and rotavirus C for development of real-time RT‒PCR assays.

BACKGROUND: The causative agents of diarrhea, rotavirus B (RVB) and rotavirus C (RVC) are common in adults and patients of all age groups, respectively. Due to the Rotavirus A (RVA) vaccination program, a significant decrease in the number of gastroenteritis cases has been observed globally. The replacement of RVA infections with RVB, RVC, or other related serogroups is suspected due to the possibility of reducing natural selective constraints due to RVA infections. The data available on RVB and RVC incidence are scant due to the lack of cheap and rapid commercial diagnostic assays and the focus on RVA infections. The present study aimed to develop real-time RT‒PCR assays using the data from all genomic RNA segments of human RVB and RVC strains available in the Gene Bank. RESULTS: Among the 11 gene segments, NSP3 and NSP5 of RVB and the VP6 gene of RVC were found to be suitable for real-time RT‒PCR (qRT‒PCR) assays. Fecal specimens collected from diarrheal patients were tested simultaneously for the presence of RVB (n = 192) and RVC (n = 188) using the respective conventional RT‒PCR and newly developed qRT‒PCR assays. All RVB- and RVC-positive specimens were reactive in their respective qRT‒PCR assays and had Ct values ranging between 23.69 and 41.97 and 11.49 and 36.05, respectively. All known positive and negative specimens for other viral agents were nonreactive, and comparative analysis showed 100% concordance with conventional RT‒PCR assays. CONCLUSIONS: The suitability of the NSP5 gene of RVB and the VP6 gene of RVC was verified via qRT‒PCR assays, which showed 100% sensitivity and specificity. The rapid qRT‒PCR assays developed will be useful diagnostic tools, especially during diarrheal outbreaks for testing non-RVA rotaviral agents and reducing the unnecessary use of antibiotics.

Evolutionary analysis of all eleven genes of species C rotaviruses circulating in humans and domestic animals.

Species C rotaviruses (RVC) are the second most common rotavirus species known to cause gastroenteritis in humans and pigs and with occurrence documented in cattle, dogs, ferrets, and sloth bears. Despite the host-specific nature of RVC genotypes, cross-species transmission, reassortment, and recombination events are also documented. In the present study, we inferred the evolutionary history of globally circulating RVC strains, including time scale stasis, the most probable ancestral country, and the most probable source host using Bayesian methods implemented in BEAST v.1.8.4. The human-derived RVC strains were majorly monophyletic and further grouped into two lineages. The RVC strains derived from pigs were monophyletic for the VP1 and the remaining genes were classified into 2 to 4 groups based on the high posterior support. The root mean age for all the genes indicated the circulation of RVC for over 800 years. Overall, the time to Most Recent Common Ancestor of human RVC strains dated back to the beginning of the 20th century. The VP7 and NSP2 genes had the lowest rates of evolution compared to other genes. The majority of the genes of RVC showed their origin in Japan except for VP7 and VP4 genes in South Korea. The phylogeographic analysis with the country as a trait showed the role of Japan, China, and India in the dispersion of the virus. In the current study, significant transmission links between different hosts were analyzed for the first time using the host as a trait. Significant transmission links between pigs and other animal species as well as humans indicate possible transmission from the pig as a source host and suggest monitoring of proximity with animals.

Enteric and non-enteric adenoviruses in children with acute gastroenteritis in Western India.

Human adenoviruses (HAdVs) are the viral agents responsible for a wide spectrum of acute and chronic diseases. HAdVs are the most important etiological agents of acute gastroenteritis (AGE) and are identified as the major contributor to the deaths of diarrheal children globally. The significant rise in HAdV infections in rotavirus-vaccinated children documented in multiple studies demands continuous monitoring of HAdV strains. After the inclusion of rotavirus vaccines in the immunization schedule of India, public health research regarding prevalence, etiology, and risk factors is highly necessary for evidence-based policies and their implementation to sustain diarrhea prevention programs. In the present study, children admitted for AGE between 2013 and 2016 in seven different hospitals in Maharashtra and Gujrat states of Western India were subjected for investigation. HAdVs were found in 5.2% of the fecal specimens with the dominance of species-F (52.4%) strains, followed by the occurrence of non-enteric adenoviruses of species A (17.4%), C (11.4%), B (8.2%), and D (3.2%). The species-F strains were predominant in Ahmadabad (78.5%), Mumbai (61.5%), and Surat (57.1%) cities, followed by species-A strains. In Pune city, species B strains were detected in all HAdV patients, with none of the species A strains. Clinically, patients infected with enteric and non-enteric HAdV strains were indistinguishable. However, a high viral load was observed in species-F specimens as compared to non-species-F. The present study on fecal specimens collected in the pre-rotavirus vaccination era from hospitalized AGE patients will be important for future comparative analysis to know the exact impact of vaccination in children of Western India.

Rotavirus C infections in asymptomatic piglets in India, 2009-2013: genotyping and phylogenetic analysis of all genomic segments.

Asymptomatic infection with rotavirus C (RVC) was observed in pigs in India, with a detection rate of 20%. Sequencing of the VP6, VP7, and NSP4 genes of RVC strains identified the genotypes I7/I10, G1, and E5, respectively. Full genome sequencing of one of these strains revealed that the genotypes of the VP4, VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5 genes were P1, R1, C1, M3, A1, N5, T5, and H1, respectively. The detection of porcine RVC strains at two different locations in India at different time points strongly suggests that they are circulating continuously in the pig population through asymptomatic infections.

Prolonged Shedding of SARS-CoV-2 in Feces of COVID-19 Positive Patients: Trends in Genomic Variation in First and Second Wave.

The main route of the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are through respiratory pathways and close contact of human-to-human. While information about other modes of transmission is comparatively less, some published literature supporting the likelihood of a fecal-oral mode of transmission has been accumulating. The diagnosis of SARS-COV-2 infected cases is based on the real-time reverse transcription-PCR (RT-PCR). The fecal excretion of SARS-COV-2 has been reported frequently, however, the role of fecal viral load with the severity of disease is not yet clear. Our study focused on the investigation of SARS-CoV-2 shedding in the fecal samples of patients with coronavirus disease 2019 (COVID-19). A total of 280 RT-PCR-positive patients were enrolled, among them 15.4% had gastrointestinal (GI) symptoms. It was shown that 62% of the patients were positive for SARS-CoV-2 RNA in fecal specimens. This positivity was not related to the presence of GI symptoms and the severity of disease. The next generation sequencing [NGS] of SARS-CoV-2 from fecal samples of patients was performed to analyze mutational variations. Findings from this study not only emphasized the potential presence of SARS-CoV-2 in feces, but also its continuing mutational changes and its possible role in fecal-oral transmission.

Outbreak of cholera in a remote village in western India.

Background & objectives: Atypical El Tor strains of Vibrio cholerae are frequently implicated in outbreaks of cholera. It is important to understand genetic variations of such strains which impact clinical and epidemiological outcomes. The present study was carried out to characterize an outbreak of cholera which occurred between July 8 and 13, 2018, in a remote settlement in Nashik district, Maharashtra. Methods: A large number of acute diarrhoea cases were reported in Rahude village, Nashik, Maharashtra since July 8, 2018. Molecular characterization of the isolated strains of V. cholerae was done. Results: 195 cases of cholera were detected from a population of 850 (attack rate 22.9%) with two deaths (Case Fatality Ratio of 1.03). A non-haemolytic polymyxin B sensitive strain of V. cholerae O1 Ogawa was isolated from 5/14 fecal samples. Molecular characterization of the isolates indicated that this strain was an altered El Tor (AET) strain. Deletion of the trinucleotide 'GTA' in the rstB gene, a unique feature of classical strains, was observed. Interpretation & conclusions: A cholera outbreak caused by a non-haemolytic polymixin B sensitive AET strain, occurred from July 8 to 13, 2018, in a remote settlement in western India. The molecular characterization of the outbreak strains highlighted an assortment of genetic determinants, stressing the need to monitor the genetic attributes of V. cholerae O1 in outbreaks for better understanding and mapping of clinical and epidemiological changes.

Prevalence and genetic diversity of gastroenteritis viruses in hospitalized children < 5 years of age in Maharashtra state, Western India, 2017-2019.

Four gastroenteritis viruses were responsible for 54% of the acute gastroenteritis (AGE) cases in children hospitalized between May 2017 and December 2019 in Pune city of Maharashtra state, Western India. The majority (79%) of the children were <2 years of age. The prevalence of Rotavirus A (RVA) was 30.5% followed by 14.3% for norovirus, 8.4% for adenovirus, and 5.5% for astrovirus. The severity of the disease was highest in patients with coinfections compared with the patients with a single infection or negative for all (p = 0.024). Genotyping analysis showed that the majority of the RVA-positive samples (66%) could be typed as G3P[8], 63.6% of the norovirus as GII.4 Sydney [P16], 44% of the adenovirus as type 41%, and 56.2% of the astrovirus as astrovirus type 1. The almost equivalent prevalence of rotavirus and nonrotaviruses and acute gastroenteritis (AGE) cases without known etiology in around 46% of the cases was noted in the present study. Our data highlight that after the recent inclusion of rotavirus vaccines as a part of the National Immunization schedule in India, conducting extensive AGE surveillance in children should include nonrotaviruses such as norovirus.

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.

Whole-genome-based characterization of three human Rotavirus C strains isolated from gastroenteritis outbreaks in Western India and a provisional intra-genotypic lineage classification system.

The number of whole-genome sequences of human rotavirus C (RVC) strains available in public databases is recently increasing. Thus far from India only a single whole genome of human RVC of a sporadic case was available. In this study, nearly full-length genome sequencing and phylogenetic analyses of three RVC strains isolated from three different gastroenteritis outbreaks during 2010-2014 in Western India was carried out. Further, an intra-genotypic lineage classification system for human RVCs based on the nucleotide divergence cut-off values was proposed by using the algorithm of the Rotavirus Classification Working Group. Two lineages could be defined for all the genes except the VP7 gene and the M3 VP3 genotype. Provisional classification of the lineages indicated the absence of reassortment events in the genomic constellation of Indian strains, contrary to earlier reports. The comparatively higher variability of the NSP1, NSP3, NSP5 and M2 VP3 genotype, emphasizes their utility in lineage classification.

Evaluation of different genomic regions of Rotavirus A for development of real time PCR.

The nucleotide alignment of all 11 genes of human Rotavirus A (RVA) strains revealed suitability of NSP2, NSP3 and VP6 genes for the development of real time PCR (qRT-PCR). Evaluation of qRT-PCR assays using known rotavirus ELISA positive and negative fecal specimens showed non-overlapping ranges of Mean ±3SD cycle threshold (Ct) values for NSP3 and VP6 based assays. Using serial dilutions of purified RVA, high sensitivity of VP6 qRT-PCR assay (1.95 × 10-5 pg/µL of RNA) was recorded as compared to NSP2 and NSP3 qRT-PCR assays (1.95 × 10-4 pg/µL of RNA). Further, evaluation of the VP6 qRT-PCR assay involving 266 fecal specimens and frequency polygon analysis of the data indicated cut-off value of 35 for Ct with high sensitivity (126/131, 96%) and specificity (12/12, 100%). This VP6 qRT-PCR assay will be a useful diagnostic tool to evaluate clinical presentations in rotaviral gastroenteritis under different conditions such as breast feeding and administration of rotavirus vaccines.

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.

Exploring the concurrent presence of hepatitis A virus genome in serum, stool, saliva, and urine samples of hepatitis A patients.

The use of saliva and urine as an alternative to serum samples for detection of anti-hepatitis A virus (HAV) IgM antibodies has been documented. However, these samples remain underreported or unexplored for shedding of HAV. To address this issue, paired serum, stool, saliva, and urine samples collected from hepatitis A patients were screened by reverse transcription polymerase chain reaction for detection of HAV RNA. HAV RNA was detected in 67.6% (44/65), 52.3% (34/65), 8.7% (5/57), and 12.3% (8/65) of the serum, stool, saliva, and urine samples, respectively. Phylogenetic analysis of nucleotide sequences obtained for partial RNA polymerase region grouped HAV strains from all of the clinical samples of the study in subgenotype IIIA. Low frequency of HAV nucleic acid in saliva and urine samples indicates limited utility of these samples in genomic studies on HAV but suggests its potential for transmission and infection of hepatitis A.

Longer duration of viremia and unique amino acid substitutions in a hepatitis A virus strain [corrected] associated with Guillain-Barré syndrome (GBS).

The molecular characteristics of hepatitis A virus (HAV) have been studied widely though there is a paucity of data on the correlation with virological and serological findings. In the present study, the whole genome of an Indian HAV strain associated with Guillain-Barré syndrome (GBS) was characterized vis-à-vis two other Indian HAV genotype IIIA strains, associated with a self-limiting disease. The percentage nucleotide divergence displayed by the Indian strains (CP-IND, PN-IND, and GBS-IND) varied from 3 to 6, whereas the percentage amino acid divergence varied from 0.1 to 0.7 as compared to the other HAV IIIA strains (n = 5) available in the GenBank. The GBS-IND strain showed an increased rate of nonsynonymous substitutions as well as a larger number of unique and heterologous amino acid substitutions compared to the HAV IIIA GenBank strains. These amino acid substitutions in the GBS-IND strain were detected in a nonstructural protein (2C-251F) and the B-cell epitope regions of structural proteins (VP1-29E, VP1-91S, VP3-50Y, and VP4-5S). In a comparative analysis of HAV strains, homology-based models of the capsid proteins indicated a localized alteration in the surface charge distribution on the VP1 protein of GBS-IND strain and involvement of its unique amino acid substitutions in the predicted antigenic determinants. Overall, the study suggests that the unique amino acid substitutions in the GBS-IND strain may have contributed to neutralization escape of the virus leading to a longer duration of viremia.

Evaluation of genomic regions of hepatitis A virus for phylogenetic analysis: Suitability of the 2C region for genotyping.

The use of different genomic fragments of hepatitis A virus (HAV) has been described for classification of strains available globally. However, the limitations of these fragments have been reported in several studies. The present study was conducted to evaluate the genomic fragments of HAV, spanning from the 5'NCR to 3'NCR to employ them in molecular diagnosis and genotyping. The different phylogenetic methods confirmed the use of the 5'NCR and the VP4 region in diagnosis due to their conserved nature. The entire genome, 2A, 2C and 3D were identified as the suitable genomic regions comparable to the VP1 region recommended earlier for genotyping. Likelihood mapping analysis indicated the full-length genome sequence as the region of choice for genotyping of HAV. This was followed by a short 2C region (1005nt), which needs to be explored.

Case report: Hepatitis A preceding Guillain-Barré syndrome.

A case of acute hepatitis A with Guillain-Barré Syndrome subtype AMAN (acute motor axonal neuropathy) in a 17-year-old male is reported. Serum and cerebrospinal fluid were positive for anti-hepatitis A virus (HAV) IgM, IgG, and IgA. The onset of the syndrome was evident in week 3 of illness. The remarkably high titers of serum anti-HAV IgG appeared unique to a hepatitis A patient with the syndrome. Phylogenetic analysis of the HAV genome detected in the serum and feces revealed genotype IIIA, circulating commonly in Pune, western India.

Evaluation of urine as a clinical specimen for diagnosis of hepatitis a.

The present study pertains to the evaluation of urine as a specimen for detection of anti-hepatitis A virus (anti-HAV) antibodies. Immunoglobulin M (IgM), IgG, and IgA capture enzyme-linked immunosorbent assays for hepatitis A were performed on paired serum and urine specimens collected from hepatitis A patients (n = 92), healthy individuals (n = 100), non-A hepatitis patients (n = 70), and patients with nonhepatic diseases (n = 64, including 37 renal disease patients). Hepatitis A patients seropositive for anti-HAV IgM showed 95.65% uropositivity. No false-positive reactions were observed in control groups. The uropositivity of anti-HAV IgM persisted during the convalescent phase of the disease. Anti-HAV IgG uropositivity correlated well with corresponding seropositivity in all groups (P > 0.05 for each). No significant difference between the proportions of serum and urine positivity for anti-HAV IgA was noted (P > 0.05 for each). Using seroreactivity as a "gold standard," the sensitivity and specificity for anti-HAV IgM, anti-HAV IgG, and anti-HAV IgA tests with urine as a specimen were found to be 95.65 and 100%, 97.76 and 76.47%, and 92.23 and 88.18%, respectively. Urine appears to be comparable to serum for diagnosis of recent and past infection with hepatitis A.