key: cord-257004-zpyms1b7 authors: Joshi, Madhuri S.; Ganorkar, Nital N.; Ranshing, Sujata S.; Basu, Atanu; Chavan, Nutan A.; Gopalkrishna, Varanasi title: Identification of group B rotavirus as an etiological agent in the gastroenteritis outbreak in Maharashtra, India date: 2017-08-11 journal: J Med Virol DOI: 10.1002/jmv.24901 sha: doc_id: 257004 cord_uid: zpyms1b7 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. GAR is the leading cause of gastroenteritis in children and has been linked to diarrheal outbreaks among hospitalized infants, young children attending day care centers, and old age individuals. 1 GBR and GCR infections have been reported more frequently in adult cases of gastroenteritis outbreak. 2, 3 Globally, outbreaks due to norovirus, adenovirus, and astrovirus have also been reported. [4] [5] [6] An outbreak of acute gastroenteritis started on 29th November, 2015 with epicenter around the Bhimashankar sugar factory, A total of 32 stool samples were collected from patients (n = 32) within 24 h of hospitalization. In the present study, a case of acute gastroenteritis was defined as the passage of ≥3 watery stools in a day with or without associated symptoms such as vomiting, fever, and abdominal pain. All patients were examined for fever, number of episodes, and duration of vomiting and diarrhea, extent of dehydration, and treatment for the assessment of disease severity score (DSS). 7 According to the scores obtained, the disease condition of each of the patients was categorized as mild (scores 0-5), moderate (scores 6-10), severe (scores [11] [12] [13] [14] [15] , and very severe (scores [16] [17] [18] [19] [20] . The mean vesikary score between the two groups was compared using t-test. All specimens were examined for virus like particles by Electron microscopy (EM) using clarified 10% stool supernatant by negative staining as described earlier. 8 The electropherotyping of viral RNA was carried out in 10% PAGE at 100 V using Tris-Glycine buffer. The gel was stained with silver nitrate as described earlier. 9 2.5 | RNA extraction, PCR/RT-PCR, and nucleotide sequencing 15 GBR positive stool specimens were genotyped using primers published earlier for VP7 gene. 16 RNA was denatured at 97°C for 5 min and was rapidly chilled on ice for 5 min. Briefly, the RT-PCR reaction was carried out with an initial reverse transcription step at 50°C for 30 min followed by PCR activation at 95°C for 5 min followed by 35 cycles of amplification (60 s at 94°C, 30 s at 55°C, and 60 s at 68°C) with final extension at 68°C for 7 min in a thermal cycler. The SuperScript ® III One-Step RT-PCR System with Platinum ® Taq DNA Polymerase kit (Invitrogen) was used for both cDNA synthesis and PCR amplification in a single tube for detection of RNA viruses. All the PCR products were electrophoresed in 2% agarose gel containing ethidium bromide (0.5%) and visualized under UV transiluminator. PCR amplicons were excised from the gel for purification (QIAquick, Qiagen, Hilden Germany) and cycle sequencing was carried out using Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and ABI 3130XL genetic analyzer (Applied Biosystems). Nucleotide sequence identity was determined through BLAST (www.ncbi.nlm.nih.gov/blast). The phylogenetic tree was generated with Maxiumum Likelihood method using MEGA 6 software. 17 The nucleotide sequences of the strains of GBR infections observed in adults (median age 30 years) was in concordance with earlier reports. 18, 19 The low prevalence in children compared to adults has been suggested to be either due to low exposure of children to GBR or faecal shedding below the detection limit of the assay employed in the study. 20 In China, a series of widespread gastroenteritis epidemics affecting millions of people were reported to be caused by GBR. 18 Over the period, epidemics settled to sporadic focal outbreaks and after 1987, no such reports were available from China. In India, circulation of GBR in sporadic cases of gastroenteritis was revealed from the retrospective analysis of stool samples collected in 1993. 21 RNA PAGE analysis has been reported to be 100 000 times less sensitive as compared to RT-PCR assay 22 The correlation between viral load and severity of the disease has been shown earlier among gastroenteritis patients infected with GAR using quantitative real time PCR assay. 23 In view of this, GBR RNA PAGE positive and negative faecal specimens need to be tested by quantitative Real time PCR assay. The study strains were closer to Indian Bangladeshi strains of GBR and highly conserved in nature as has been demonstrated previously. 24 However, further studies are necessary to ascertain the role of subtle genetic substitutions observed in the study strains coupled with nucleotide analysis of remaining genes to understand the genetic evolution of the virus over the period, its role in pathogenicity and epidemiology of the disease. 25 The state/sub divisional Health Laboratory declared that the piped well water with a high coli form count was the common source of infection and that the leakage in the pipeline and irregularity in chlorination of water led to gastroenteritis outbreak. Even though, the analysis of piped and well water samples for viral agents was not conducted in the present study, the predominance of GBR infections among outbreak cases and the common source of water with high coli form count indicate spread of GBR through fecal contaminated water. The use of well water was completely stopped and the alternative arrangement was made to supply the drinking water. To prevent the GBR spread, regular chlorination of well water was performed. Training was also provided to the family member of every house for hygienic practices and decontamination of water. 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