Serologic and molecular characterization of rotavirus from children with acute gastroenteritis in Chennai, Tamil Nadu, India

Background: Rotavirus gastroenteritis is the leading cause of diarrhea in infants and young children worldwide. Although, Rotavirus vaccine has been introduced in 2017 in states like Tamil Nadu, there are reports of the role of Rotavirus as one of high disease burden agents with genetic variants arising, especially from low-income countries like India.Methods: Authors evaluated stool samples from 507 children with acute gastroenteritis Rotavirus A among the hospitalized children (>5 years) to provide baseline information on changing profile in this state. The stool samples were collected and screened for Rotaviral Antigen by Enzyme Immuno Assay and use of semi-multiplex RT PCR technique was conceded out in order to conclude the P and G genotypes of human rotavirus in rotavirus-positive samples from January 2014 to December 2016 in and around Chennai, India.Results: Of 507 samples collected 213 (42.01%) were positive for rotavirus antigen by Enzyme Immuno Assay (EIA). The maximum positivity (75%) was in the age group of one to two years. Rotavirus positives were subjected to further VP7 and VP4 molecular characterization and the predominant genotypes identified were G9P[4] followed by G9P[8], G1P[8], G3P[8], G2P[4] and mixed types of G2G9 with P[4] and G4P[6][11] with few untypable strains.Conclusions: This study had demonstrated the Rota Virus Gastro Enteritis (RVGE) is a common disease affecting the pediatric population and G9P[4], G9P[8] circulating types among the gastroenteritis cases reported in the city and its suburban area. This study in comparison to previous ones shows that the dominant serotypes and circulating genotypes changes from time to time within country. The results have reemphasized the need of rotavirus vaccines with broad serotype coverage which may help in decreasing the disease burden in this region of the country.

Molecular characterization of rotaviruses isolated from calves with bovine neonatal diarrhea (BND) in Colombia / Caracterizacion molecular de rotavirus aislados a partir de terneros con diarrea neonatal bovina (dnb) en colombia

Bovine rotaviruses are one of the main agents involved in the presentation of Bovine Neonatal Diarrhea (BND), a disease that affects calves in the first month of life. Objective: The present study aimed to determine the types of bovine rotaviruses that affect dairy herds in the Sabana region of Bogotá, Colombia. Materials and methods: 132 fecal samples were obtained from calves of less than five weeks of age and subjected to an ELISA test. MA104 cell cultures were infected with positive samples in order to isolate rotaviruses. The presence of the viral genome was confirmed by amplification and sequencing of a region of the viral VP7 protein-encoding gene. Results: Of the 132 samples, 26 (19, 7%) were ELISA-positive and nine samples were used for viral isolation. PCR amplification was achieved in all infected cultures. Sequencing showed homology of five samples to the G6 genotype. In addition, the presence of the G10 genotype was first determined for the country. Discussion: A greater presence of the G6 genotype from lineage V was found in the Sabana region of Bogota, showing a high prevalence in cattle and association with the presence of BND. The presence of the G10 genotype is a new report for the country and constitutes a new element of investigation in these viruses.

Los rotavirus bovinos son unos de los principales agentes involucrados en la presentación del síndrome de Diarrea Neonatal Bovina (DNB), una enfermedad que afecta terneros en el primer mes de vida. Objetivo: El presente estudio buscó determinar los tipos de rotavirus que afectan los hatos ganaderos de leche en la región de la Sabana de Bogotá, Colombia. Materiales y métodos: Se evaluaron a través de una prueba de ELISA, 132 muestras de materia fecal provenientes de terneros de menos de cinco semanas de edad. Con las muestras positivas, se infectaron células MA 104 con el fin hacer aislamiento. La presencia del genoma viral se verificó por amplificación de una región del gen que codifica para la proteína viral VP-7 y luego se secuenció. Resultados: De las 132 muestras evaluadas, 26 (19,7%) fueron positivas por ELISA. De estas, 9 muestras se emplearon para aislamiento viral. La amplificación de genoma viral por PCR se obtuvo en todos los cultivos infectados. La secuenciación evidenció una homología de 5 muestras con el genotipo G6 y la presencia del genotipo G10, que se encontró por primera vez en el país. Discusión: En la Sabana de Bogotá se encontró una mayor presencia del genotipo G6, linaje V, que tiene alta prevalencia en bovinos y está asociado mayoritariamente con la presencia de DNB. La presencia del genotipo G10 constituye un elemento nuevo de investigación en estos virus.

Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.

Severe diarrhea outbreak in beef calves (Bos indicus) caused by G6P[11], an emergent genotype of bovine rotavirus group A / Surto de diarreia neonatal em bezerros de corte (Bos indicus) ocasionado por um genotipo emergente G6P[11] de rotavírus bovino grupo A

The episodes of diarrhea caused by neonatal bovine rotavirus group A (BoRVA) constitute one of the major health problems in the calf rearing worldwide. The main G (VP7) and P (VP4) genotypes of BoRVA strains involved in the etiology of diarrhea in calves are G6P[1], G10P[11], G6P[5], and G8P[1]. However, less frequently, other G and P genotypes have been described in BoRVA strains identified in diarrheic fecal samples of calves. This study describes the identification and molecular characterization of an emerging genotype (G6P[11]) in BoRVA strains involved in the etiology of a diarrhea outbreak in beef calves in a cattle herd of high production in extensive management system. The diarrhea outbreak, which showed high morbidity (60%) and lethality (7%) rates, occurred in calves (n= 384) Nelore (Bos indicus) up to 30-day-old from the State of Mato Grosso do Sul, Brazil. BoRVA was identified in 80% (16/20) of the fecal samples analyzed by polyacrylamide gel electrophoresis (PAGE) technique. In all PAGE-positive fecal samples were amplified products with 1,062-bp and 876-bp in the RT-PCR assays for VP7 (G type) and VP4 (VP8*) (P type) of BoRVA, respectively. The nucleotide sequence analysis of VP7 and VP4 genes of four wild-type BoRVA strains showed G6-III P[11]-III genotype/lineage. The G6P[11] genotype has been described in RVA strains of human and animal hosts, however, in calves this genotype was only identified in some cross-sectional studies and not as a single cause of diarrhea outbreaks in calves with high morbidity and lethality rates as described in this study...

Os episódios de diarreia neonatal ocasionados pelo rotavírus bovino grupo A (BoRVA) constituem-se em um dos principais problemas sanitários na criação de bezerros em todo o mundo. Os principais genotipos G (VP7) e P (VP4) de cepas de BoRVA envolvidos na etiologia da diarreia em bezerros são G6P[1], G10P[11], G6P[5] e G8P[1]. No entanto, com menor frequência, outros genotipos G e P têm sido descritos em cepas de BoRVA identificadas em amostras de fezes diarreicas de bezerros. Este estudo descreve a identificação e caracterização molecular de um genotipo emergente (G6P[11]) em cepas de BoRVA envolvidas na etiologia de um surto de diarreia em bezerros de um rebanho bovino de corte de alta produção em sistema de manejo extensivo. O surto, que apresentou altas taxas de morbidade (60%) e de letalidade (7%), ocorreu em bezerros (n=384) da raça Nelore (Bos indicus) com até 30 dias de idade, provenientes do estado do Mato Grosso do Sul, Brasil. O BoRVA foi identificado em 80% (16/20) das amostras fecais analisadas pela técnica de eletroforese em gel de poliacrilamida (PAGE). Em todas as amostras fecais PAGE-positivas foi possível a amplificação por RT-PCR de produtos com 1.062 pb e 876 pb referentes aos genes VP7 (G tipo) e VP4 (VP8*) (P tipo), respectivamente, de BoRVA. A análise da sequência de nucleotídeos dos genes VP7 e VP4 de quatro cepas de BoRVA demonstrou a presença do genotipo/linhagem G6-III P[11]-III. O genotipo G6P[11] tem sido descrito em cepas de RVA de hospedeiros humanos e animais. Contudo, em bezerros, este genotipo foi apenas identificado em alguns estudos transversais e não como a única causa de surtos de diarreia em bezerros com altas taxas de morbidade e...

The Molecular Epidemiology of Circulating Group A Rotavirus in Gwangju Metropolitan City, Korea: 2008~2012

Group A rotaviruses are a major cause of acute gastroenteritis in young children worldwide. For the proper management of rotavirus infections, knowledge of the distribution of G and P genotypes including detection of emerging genotype is crucial. Therefore, the aim of this study is to describe epidemiological changes in rotavirus gastroenteritis in Gwangju metropolitan city, South Korea. Stool samples were collected from 14,314 patients with diarrhea, who visited hospitals in Gwangju from 2008 to 2012. Samples were screened for rotavirus with Enzyme-Linked Immunosorbent Assay (ELISA) method and rotavirus P (VP4), G (VP7) genotypes were determined by reverse-transcription polymerase chain reaction. And we performed nucleotide sequencing analysis. Among a total of 14,314 samples investigated 1,982 samples (13.8%) were ELISA positive. Genotyping of Rotavirus was performed using 526 rotavirus samples. The most prevalent circulating G genotype was G1 (40.5%), followed by G2 (27.6%), G3 (19.4%), G9 (9.7%), G4 (2.5%) and G12 (0.4%). The predominant type of P genotypes was P[8] (69.6%), followed by P[4] (27.8%) and P[6] (2.3%). In this study, 13 G-P combinations were detected. From 2008 to 2010, G1P[8] was the most prevalent, followed by G3P[8]. Whereas, 2011 and 2012, G2P[4] was the most common, followed by G1P[8]. Rotavirus gastroenteritis is a common disease associated with significant morbidity, mortality and economic burden. Ongoing rotavirus surveillance to understand the distribution of G and P genotypes will be critical for the development of effective prevention measurements.

Detection and characterization of group a rotaviruses (RVA) detected in diarrhoeic children in Madhya Pradesh, Central India.

Background and objectives: Rotavirus is the leading cause of viral gastroenteritis throughout the world and is associated with up to 600000 deaths worldwide every year, of which more than 150,000 occur in India. This study was undertaken to detect and analyze the human rotavirus A (RVA) isolates from Madhya Pradesh, central India, between 2007 and 2008.Methods: Forty diarrhoeic samples from children up to the age of 5 years, admitted or visited the hospital, were screened using RNA-viral electrophoresis (PAGE), reverse transcription (RT)-PCR and selected isolates were further analyzed by sequencing.Results and interpretation: Incidence of RV was 32.5% in children (13/40) and all the isolates showed a typical migration pattern of 4:2:3:2, suggestive of group A RVs. All the PAGE positive samples yielded positive amplification in RT-PCR, confirming them to be human RVA. The VP7 gene sequence analysis of the selected isolates (H-14 and H-16) identified as G1 type revealed that these isolates form a cluster with Indian G1 isolates (mani63-06 and mani 365-07) and strain from Bangladesh (DH378) with sequence identity of more than 97% at amino acid levels. Simplot and boot scan analysis showed no recombination with other G1 strains.Conclusions: The G1 was detected to be the predominant genotype in this area of the country, which is helpful in selecting the vaccine strain.

Investigation of VP_7 From Rotaviruse Strains in Ningbo City,China / 中国疫苗和免疫

Objective To know the currently prevalent genotype of rotavirus strains in Ningbo City,which will help to the study of vaccine of group A rotavirus.Methods The VP7 fragments were amplified by RT-PCR and sequence analysis was conducted from 2 of the 11 virus strains.Results VP7 amino acid sequences homogeneity of the two strains Ningbo 06-1 and Ningbo 06-3 was 90.2%.They all shared higher amino acid sequences homogeneity with G1 strain Wa(95.1% and 87.7%,respectively)than with other serotypes(G types)rotavirus(

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells / 中国病毒学·英文版

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Isolation and Characterization of G9 Human Rotaviruses

Group A rotaviruses are the most common causes of gastroenteritis among infants and young children. The outer capsid layer of the virus is composed of two structural proteins, VP4 and VP7, and they play important roles in protection by eliciting neutralization antibodies. Group A rotaviruses are subdivided into distinct G and P serotypes according to the antigenic differences of the VP7 and VP4, respectively. Rotavirus G9 serotype was thought to be the fifth most common serotype circulating among the population worldwide. In this study, G9 human rotaviruses (HRV) were isolated from fecal samples using MA104 cells and characterized. Characteristic cytopathic effects of rotavirus were observed and rotaviral antigens were confirmed by indirect immunofluorescence antibody test in MA104 cells inoculated with isolated HRV strains. The nucleotide sequences of the VP7 gene of Korean G9 HRV isolated in this study were determined and compared with those of other recent and prototype G9 rotavirus strains from other parts of the world. Also, the nucleotide sequences of VP4 and NSP4 gene of Korean G9 HRV were determined and compared with those of other rotavirus strains from other countries. The results showed that the Korean HRV isolates belong to a G9, P[8] and NSP4 B genotype. The Korean G9 HRV isolates and their nucleotide sequence data would be usefully applied for the vaccine development of HRV in the near future.

VP4 and VP7 Genotyping of Group A Rotavirus Isolated from Diarrhea Patients in Seoul by Multiplex PCR

The incidence and distribution of the human rotavirus G types (VP7 associated: G1~G4) and P types (VP4 associated: P[4], P[6], P[8], P[10]) were determined from 89 rotavirus strains isolated from diarrhea patients between 2001 and 2002 using reverse transcription and multiplex polymerase chain reaction. G types were identified from 83 (95.5%) and P types were from 82 (92.1%) strains. The predominant genotypes were P[4]G2 (28.1%) and P[6]G4 (27%) with much lower incidence of genotypes P[10]G1 (1.1%) and P[10]G3 (1.1%). P[9] type was not detected. A significant genotypic shift was observed that P[4] was the most prevalent genotype that accounted for 75% during the spring season of 2001, coinfection with P[4] and P[6] for 17.5%. P[6] increased gradually to account for 53% of the strains analysed in the following 2002 spring season. Mixed G types revealing coinfections G2/G3 and G3/G4 were found at low frequency (2.2%).

Distribution of Rotavirus G Serotypes in ChungJu Area / 대한소아소화기영양학회지

PURPOSE: It is important to have the epidemiologic data of rotavirus serotypes for the application of polyvalent rotavirus vaccines. Epidemiological studies of rotavirus serotypes in Korea have been reported only in limited areas with small number of cases. Authors tried to investigate the distribution of rotavirus G serotypes in ChungJu area with RT-PCR. METHOD: Stool specimens were collected from 202 children with acute diarrheal symptoms, who admitted to or visited Kon-Kuk University Hospital in ChungJu from June 1998 to May 1999. Samples were screened for rotavirus with EIA method (TestPack Rotavirus, Abbott Laboratories) and rotavirus G Serotypes were determined by RT-PCR. RESULTS: Rotavirus was positive in 46.6%. The incidence of G serotypes was as follows; G1 10%, G2 10%, G3 28%, G4 26%, and G9 20%. There were three cases of multiple serotypes; G1 with G9, G2 with G9, and G4 with G9. Serotype of G8 was not found. CONCLUSION: The proportion of G serotypes in ChungJu is much different from previous reports. Serotype of G9 was found which had not been reported in Korean children till now. Long term plans for the investigation of rotavirus serotypes must be needed in wide area.

Molecular Cloning and Nucleotide Sequence of the Gene Encoding Fusion(F) Protein of the Thermostable Newcastle Disease Virus Isolated from a Diseased Pheasant

The gene encoding F protein of CBP-1 strain, a heat-stable Newcastle disease virus (NDV) isolated from the diseased pheasants in Korea, was characterized by reverse transcription-polymerase chain reaction (RT-PCR), nucleotide and amino acid sequences. Virus RNA was prepared from the chorioallatoic fluid infected with NDV CBP-1 virus and cDNA was amplified by RT-PCR, cloned and sequenced to analyze. The PCR was sensitive as to detect the virus titer above 25 hemagglutination unit. 1.7kb (1,707bp) size of the cDNA was amplified and cloned into BamHI site of pVL1393 Baculo transfer vector. The nucleotide sequences for F protein were determined by dye terminator cyclic sequencing using four pairs of primers, and 553 amino acid sequences were predicted. In comparison of the nucleotide sequence of F gene of CBP-1 with those of other NDV strains, the homology revealed 88.8%, 98.5% and 98.7% with Kyojungwon (KJW), Texas GB and Beaudette C strains, respectively. As the deduced 553 amino acid sequences of F protein of CBP-1 were compared with those of other NDV strains, the homology appeared 89.9%, 98.7% and 98.9% with KJW, Texas GB and Beaudette C strains, respectively. The putative protease cleavage site (112-116) was R-R-Q-K-R, indicating that CBP-1 strain is velogenic type. The amino acid sequences include 6 sites of N-asparagine-linked glycosylation and 13 cysteine residues. These data indicate that the genotype of CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than KJW strain.