Rotavirus Isolates Research Articles

Detection of rare reassortant G5P[6] rotavirus, Bulgaria

During the ongoing rotavirus strain surveillance program conducted in Bulgaria, an unusual human rotavirus A (RVA) strain, RVA/Human/BG/BG620/2008/G5P[6], was identified among 2200 genotyped Bulgarian RVAs. This strain showed the following genomic configuration: G5–P[6]–I1–R1–C1–M1–A8–N1–T1–E1–H1. Phylogenetic analysis of the genes encoding the neutralization proteins and backbone genes identified a probable mixture of RVA genes of human and porcine origin. The VP1, VP6 and NSP2 genes were more closely related to typical human rotavirus strains. The remaining eight genes were either closely related to typical porcine and unusual human–porcine reassortant rotavirus strains or were equally distant from reference human and porcine strains. This study is the first to report an unusual rotavirus isolate with G5P[6] genotype in Europe which has most likely emerged from zoonotic transmission. The absence of porcine rotavirus sequence data from this area did not permit to assess if the suspected ancestral zoonotic porcine strain already had human rotavirus genes in its genome when transmitted from pig to human, or, the transmission was coupled or followed by gene reassortment event(s). Because our strain shared no neutralization antigens with rotavirus vaccines used for routine immunization in children, attention is needed to monitor if this G–P combination will be able to emerge in human populations. A better understanding of the ecology of rotavirus zoonoses requires simultaneous monitoring of rotavirus strains in humans and animals.

Changing trend of persistent diarrhoea in young children over two decades: observations from a large diarrhoeal disease hospital in Bangladesh

We studied the changing trend and factors associated with persistent diarrhoea (PD) in under-five children presenting to a large diarrhoeal disease hospital in urban Dhaka, Bangladesh, over the last two decades. We used an unmatched case-control design, with a case (persistent diarrhoea; n=944) to control (acute diarrhoea; n=2832) ratio of 1:3 attending the Dhaka Hospital of icddr,b between 1991 and 2010. The proportion of children with PD decreased significantly from 8% in 1991 to 1% in 2010. The proportion of breastfeeding practices, measles vaccination coverage and vitamin A supplementation among 12-59 months old improved from 79% to 85%, 69% to 85% and 26% to 74%, respectively, which were significant. Although the isolation of rotavirus from stool in children with PD and acute diarrhoea increased, the isolation of Shigella spp., and Vibrio cholerae O1 decreased significantly. In a logistic regression analysis, wasting (OR=1.62), use of antibiotic before attending hospital (OR=5.94), absent clinical dehydration (OR=1.53) and bloody/mucoid stool (OR=3.33) were significantly associated with persistent diarrhoea. There, thus, is a need to integrate an appropriate and sustainable deterrent strategy to take the benefit of the significant reduction in prevalence as well as risks of PD in such population.

Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses

ABSTRACTDirected differentiation of stem cell lines into intestine-like tissue called induced human intestinal organoids (iHIOs) is now possible (J. R. Spence, C. N. Mayhew, S. A. Rankin, M. F. Kuhar, J. E. Vallance, K. Tolle, E. E. Hoskins, V. V. Kalinichenko, S. I. Wells, A. M. Zorn, N. F. Shroyer, and J. M. Wells, Nature 470:105-109, 2011). We tested iHIOs as a new model to cultivate and study fecal viruses. Protocols for infection of iHIOs with a laboratory strain of rotavirus, simian SA11, were developed. Proof-of-principle analyses showed that iHIOs support replication of a gastrointestinal virus, rotavirus, on the basis of detection of nonstructural viral proteins (nonstructural protein 4 [NSP4] and NSP2) by immunofluorescence, increased levels of viral RNA by quantitative reverse transcription-PCR (qRT-PCR), and production of infectious progeny virus. iHIOs were also shown to support replication of 12/13 clinical rotavirus isolates directly from stool samples. An unexpected finding was the detection of rotavirus infection not only in the epithelial cells but also in the mesenchymal cell population of the iHIOs. This work demonstrates that iHIOs offer a promising new model to study rotaviruses and other gastrointestinal viruses.

Phylogenetic Analysis of the Rotavirus Genotypes Originated from Children < 5 Years of Age in 16 Cities in South Korea, between 2000 and 2004

ObjectivesThe purpose of this study was to examine the diversity of the G and P types of human rotavirus strains isolated in South Korea during 2000 to 2004. MethodsWe selected 38 Group A rotavirus isolates among 652 fecal samples, which were collected from infants and children < 5 years of age with acute gastroenteritis or diarrhea admitted in 8 hospitals representative of five provinces of South Korea between 2000 and 2004. Rotavirus P- and G-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed. ResultsOne G1P[4] consisted G1-Id-P[4]-V; one G1P[6] consisted G1-Id-P[6]-Ia; nine G1P[8] consisted G1-Ib-P[8]-Ia (n=3), G1-Ic-P[8]-Ia (n=1), and G1-Id-P[8]-Ia (n=5); 13 G2P[4] consisted G2-V-P[4]-V; two G3P[4] consisted G3-IIId-P[4]-V; five G3P[8] consisted G3-IIId-P[8]-Ia; four G4P[6] consisted G4-Ie-P[6]-Ia; two G4P[8] consisted G4-Ie-P[8]-II; one G9P[6] consisted G9-III-P[6]-Ia. ConclusionsA considerable amount of rotavirus genotypic diversity was detected in South Korea from 2000 to 2004. These findings are important to develop the effective vaccines and to undertake epidemiologic studies.

Identification of a G8P[14] Rotavirus Isolate Obtained from a Taiwanese Child: Evidence for a Relationship with Bovine Rotaviruses

Identification of a G8P[14] Rotavirus Isolate Obtained from a Taiwanese Child: Evidence for a Relationship with Bovine Rotaviruses

Cloning and phylogenetic analysis of the NSP1~5 genes of giant panda rotavirus strain CH-1 isolated in China

Rotavirus (RV), a member of the genus Rotavirus of the family Reoviridae, is an important causative agent of diarrhoea diseases of human and animals worldwide. The Asian Rotavirus Surveillance Network reported that overall 45% of diarrhoea admissions in Asian region were positive for rotavirus in nine countries and regions of Asia (2008). But very limited knowledge about animal diarrhoea diseases caused by rotavirus was published until now. Valuation of genetic relations between human and animal rotavirus isolates is very limited. To better understand the rotavirus CH-1 strain isolated from diarrheic faeces of giant panda in 2008, we cloned the non-structural protein (NSP) 1~5 complete coding sequence of the giant panda rotavirus (GPRV), and sequenced NSP1~5 genes (GenBank accession number: NSP1, GU205762; NSP2, GU188281; NSP3, GU329525; NSP4, GU188282; NSP5, GU329526). Based on these information and data from GenBank of other genus of RV NSP1~5 genes, phylogenetic analysis were realized. The phylogenetic tree revealed that GPRV NSP1~5 genes were close to that of porcine rotavirus, bovine rotavirus and human rotavirus. This research may provide some useful information helping us understanding giant panda rotavirus. Key words: Giant panda rotavirus isolate strain CH-1, NSP1~5 genes, cloning, phylogenetic analysis.

Molecular detection, isolation and characterization of avian rotavirus from broiler chicken

Molecular detection, isolation and characterization of avian rotavirus from broiler chicken

DETECÇÃO E ISOLAMENTO DE ROTAVÍRUS CAPRINO DO GRUPO A

RESUMO Rotavírus é a principal causa de diarreia em animais jovens de várias espécies. Os rotavirus estão classificados na família Reoviridae e contêm um genoma formado por 11 segmentos de RNA de cadeia dupla, envolvidos em três camadas proteicas. Neste estudo, foi detectado rotavírus pela eletroforese em gel de poliacrilamida (PAGE) em amostras de fezes de caprinos, com 80 dias de idade, durante surto de diarreia em um rebanho leiteiro. O perfil eletroforético das amostras, denominadas cap01/2007 e cap02/2007, foi caracterizado no PAGE como sendo de rotavírus do grupo A. Para o isolamento viral, suspensão fecal a 20% em tampão tris 0,1M pH 7,3 foi filtrada e tratada com tripsina cristalina na concentração de 5mg/mL. A amostra caprina cap01/2007 foi inoculada em cultura de células MA104 (Rim de Macaco Rhesus). Foram testados dois tratamentos (T1 e T2): após adsorção (estufa 37º C por uma hora) adicionou-se meio de manutenção (Dulbecco-MEM) sem tripsina, mantendo o inóculo (T1). No tratamento 2 (T2), foi realizado o mesmo pro cedimento anterior, porém o inóculo foi dispensado. O efeito citopático foi observado na primeira passagem para ambos os tratamentos (T1 e T2) com 48 horas de inoculação e o isolamento viral foi confirmado no PAGE. O perfil de migração manteve-se inalterado após três passagens sucessivas. Este é o primeiro relato de isolamento de rotavírus em caprinos no Brasil.

Molecular characterization of G and P rotavirus genotypes, G9 strain, from children with acute gastroenteritis in the Metropolitan Region of Belém, Pará State, from 1999 to 2007

ABSTRACT Rotavirus (RV) is the major viral agent associated with gastroenteritis and is responsible for 39% of all diarrheal cases requiring hospitalization, as well as approximately 520 thousand deaths among children under five years of age. Rotavirus belongs to the Reoviridae family, genus Rotavirus and its genome consists of 11 segments of double-stranded RNA which encode six structural viral proteins (VPs) and six non-structural viral proteins (NSPs). VP4 and VP7 proteins make up the outer layer of the RV particle and define the two genotypes, P and G, respectively. To date, at least 23 G genotypes and 31 P genotypes have been identified. G9-type RV strains, which are associated with a more severe disease in humans, have emerged globally. Dual type-specificities have been reported, and G9 strains most often belong to the P[8] genotype. While G9 genotype includes six distinct lineages, four distinct lineages are currently recognized as belonging to the P[8] genotype. The aim of this study was to characterize the VP4 and VP7 genes of the RV G9 genotypes that circulated in the Metropolitan Region of Belem, Para, in northern Brazil from 1999 to 2007. The viral dsRNA of 38 samples was extracted from fecal suspensions and analyzed using polyacrylamide gel electrophoresis to determine electropherotypes, followed by sequencing reactions. Overall, 32 selected G9P[8] samples were analyzed, all of which possessed long electropherotype. Phylogenetic analysis of the VP7 gene showed that all G9 strains belonged to lineage 3 with a high degree of similarity between them; only eight nucleotide changes have been detected in this lineage. However, only three of these amino acid substitutions were observed in our study, occurring at positions 43 (l→V), 66 (A→V) and 73 (Q→R). Of interest, the substitutions at positions 43 and 73 were only found in samples from 2007. Phylogenetic analysis of the VP4 gene revealed that all of the P[8] strains belonged to lineage 3, with 15 nucleotide changes corresponding to four amino acid substitutions at positions 108 (V→l), 172 (R→K), 173 (l→V) and 275 (K→R). The amino acid substitutions at positions 172 and 275 were observed only in RV isolates from 1999 to 2002. Overall, the RV G9 samples showed high homology throughout the entire study period. Interestingly, strains isolated in 2007 were the most divergent with respect to both VP4 and VP7 genes. Our data underscore the need for continuous surveillance of circulating RV strains in our region to detect the emergence of new genetic variants that would pose a challenge to current RV immunization strategies. This should include close monitoring of the genetic diversity of emerging G9 strains.

Genetic characterization of bulgarian rotavirus isolates and detection of rotavirus variants: challenges for the rotavirus vaccine program?

Annually 20-70% of all hospital admissions and 20% of fatal diarrhea cases among children less than 5 years of age occur due to severe rotavirus diarrhea. Universal immunization is the major strategy aimed at controlling rotavirus infection. The main objective of the present study was to elucidate the evolutionary relationships of the most common rotavirus strains co-circulating in Bulgaria. The sequence and phylogenetic analysis revealed strain diversity and circulation of different rotavirus variants belonging to a single genotype. A mutated G4P[8] strain with the insertion of an asparagine residue in position 76; G2, G9, and G1 variants with amino acid substitutions in the antigenic regions A, B, and/or C were all identified in this study in the absence of an immunization program. Rotavirus strain surveillance in both the pre- and post-vaccine eras is of increasing importance in order to assess the effectiveness of the rotavirus vaccines for protection against disease associated with a diverse population of rotavirus strains.

Molecular characterization of rare G12P[6] rotavirus isolates closely related to G12 strains from the United States, CAU 195 and CAU 214

Two human G12 rotaviruses, CAU 195 and CAU 214, were isolated from South Korea using cell culture and characterized on the basis of sequence divergence in the VP7, VP4, and NSP4 genes. Phylogenetic analysis of the VP7 gene sequences indicated that these strains clustered into lineage III and were most closely related to G12 rotaviruses isolated in the United States. The VP4 and NSP4 gene sequences showed that two strains belonged to the P[6]-Ia lineage and genotype [B]. This finding provides information that can be used to evaluate G12 strains and aid in the development of effective vaccines in the future.

Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus

Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ≥10(4) pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this "two-hit" reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.

Multiple‐gene characterization of rotavirus strains: Evidence of genetic linkage among the VP7‐, VP4‐, VP6‐, and NSP4‐encoding genes

A total of 162 rotavirus strains detected between 1996 and 2006 among individuals with diarrhea in Rio de Janeiro, Brazil, were analyzed by multiple-gene genotyping. Characterization of strains was done by RT-PCR assay for amplification and typing of the VP7-, VP4-, VP6-, and NSP4-encoding genes. Overall, 139 (85.8%) strains belonged to the common group A rotavirus combinations: 67 (41.4%) belonged to genotype G1-P[8]-I1-E1; 18 (11.1%) were G2-P[4]-I2-E2; 11 (6.8%) were G3-P[8]-I1-E1; 12 (7.4%) were G4-P[8]-I1-E1; and 31 (19.1%) were G9-P[8]-I1-E1. Two samples presented mixed genotypes (G1 + G3-P[8]-I1-E1 and G1 + G9-P[9]-I1-E1) and rare combinations, such as G2-P[6]-I2-E2 and G9-P[6]-I2-E2, were detected in six (3.7%) strains. The results suggest a linkage among all four genes. Genotypes G1/G3/G4/G5/G9-P[8] were correlated strongly to I1-E1 genotypes and G2-P[4]/P[6] were correlated to I2-E2 genotypes. Unusual combinations of genes, such as G3-P[9]-I2-E2, G9-P[9]-I1-E2, and G3-P[9]-I3-E3, were observed in 15 (9.3%) strains. The characterization of multiple genes allows a more complete analysis of the rotavirus isolates and provides evidence of natural reassortment of strains.

Isolation of Foal Rotavirus in MA-104 Cells

Isolation of Foal Rotavirus in MA-104 Cells

A novel multiplex RT-PCR for identification of VP6 subgroups of human and porcine rotaviruses

VP6 protein antigens allow classification of rotaviruses into at least four subgroups, depending on the presence or absence of SG-specific epitopes: SG I, SG II, SG (I + II), and SG non-(I + II). However, MAbs against epitopes on the VP6 protein of human and porcine rotaviruses, sometimes, do not recognize SG-specific epitopes or recognize irrelevant-SG epitopes and therefore result in the incorrect assignment of subgroups. In order to solve this problem, a novel multiplex RT-PCR was developed as an alternative tool to identify VP6 genogroups using newly designed primers which are specific for genogroup I or II. The sensitivity and specificity of the newly developed multiplex RT-PCR method was evaluated by testing with human and porcine rotaviruses of known SG I, SG II, SG (I + II), and SG non-(I + II) strains in comparison with monoplex RT-PCR and VP6 sequence analysis. The results show that the genogroups of both human and porcine rotaviruses as determined by the new multiplex RT-PCR method were in 100% agreement with those determined by monoplex RT-PCR and VP6 sequence analysis. The method was shown to be specific, sensitive, less-time consuming, and successful in genogrouping clinical isolates of rotaviruses circulating in children and piglets with acute diarrhea.

Rotavirus genotypes in children in the Basque Country (northern Spain) over a 13-year period (July 1996–June 2009)

To describe the circulation dynamics of human rotavirus genotypes in a region of southern Europe over a 13-year period. The G- and P-types of rotavirus isolates of patients aged less than 5 years were analyzed using multiplex, reverse transcription polymerase-chain reaction. Of 1,538 isolates investigated, a combination of individual G- and P-types was obtained in 1,368. The most prevalent combination was G1[P8] (57.5% of the genotyped strains), which circulated in all seasons and predominated in nine out of 13 seasons. The strains G2[P4] (14.4%), G3[P8] (8.3%), G4[P8] (5.5%) and G9[P8] (13.4%) circulated intermittently. G4[P8] strains were frequently detected in the 1990s but only sporadically after 2000. G9[P8] strains emerged from 1997-1998 and became dominant in the winters of 2005-2007. G2[P4] strains were predominant in 2003-2004, before the rotavirus vaccines were commercialized. Unusual combinations of common G- and P-types and the presence of unusual G- and/or P-types (G6[P14], G8[P8], G8[P14] and G12[P8]) were rarely observed (<1%). We found no differences in hospitalization due to distinct genotypes. G-types G1-G4 and G9 represented >99% of circulating rotaviruses over a 13-year period. Therefore, vaccine efficacy in this region can be expected to be high.

Ovine rotavirus strain LLR-85-based bovine rotavirus candidate vaccines: Construction, characterization and immunogenicity evaluation

Group A bovine rotaviruses (BRVs) are the most important cause of diarrheal diseases in neonatal calves and cause significant morbidity and mortality in the young animals, and epidemiologic surveillance of bovine rotavirus G genotypes conducted in various cattle populations throughout the world has shown that approximately 90% of the bovine rotavirus isolates belong to G6 and G10. Based on the modified Jennerian approach to immunization, we constructed and characterized a reassortant rotavirus stain, which bears a single bovine rotavirus VP7 gene encoding G genotype 6 specificity while the remaining 10 genes are derived from the ovine attenuated rotavirus LLR-85. The reassortant rotavirus strain, named as R191, and its parental virus strain LLR-85 were combined as bivalent vaccine candidates to inoculate the colostrums-deprived neonatal calves for evaluation of the immunogenicity. The calves were orally inoculated with the reassortant R191 (group 1), the parental rotavirus LLR-85 (group 2), or combined the R191 and LLR-85 (group 3), and serum specimens were detected to determine the immune response of IgG and IgA antibodies. Results showed that seroconversion to positivity for IgG and IgA antibodies occurred at postinoculation day (PID) 10 in all of the inoculated calves, and the highest titers of the serum IgG (range 1:800 to 1:6400) and IgA (range 1:800 to 1:3200) antibodies were obtained at PID 21 for all calves. Meanwhile, virus shedding was detected after inoculation, showing that the inoculated virus was positive in 2 of 77 fecal specimens (2.6%) collected from the inoculated calves during the first 7 days of oral inoculation with the rotavirus vaccine candidates. The results suggested that the rotavirus strains R191 and LLR-85 are promising bivalent vaccine candidates for the prevention of bovine G6 and G10 rotavirus infection.

Rotavirus-associated diarrhoea in foals in Greece

Severe outbreaks of diarrhoeic syndrome occurred in young foals at the same stud farm during two consecutive breeding periods namely spring 2006 and 2007. Rotavirus-like particles were detected by electron microscopy in the faeces of the affected foals and group A rotavirus infection was confirmed by Reverse-Transcription (RT)-PCR with selected sets of rotavirus-specific primers. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 enabled classification of the viruses as G3AP[12] and revealed that the viruses were highly similar to recently reported equine rotavirus strains circulating in Europe. All Greek equine rotavirus isolates were genetically identical, suggesting persistence of the same viral strain in the stud farm, over the two consecutive foaling periods.

Évolution des souches de Rotavirus du groupe A en circulation en Tunisie sur une période de trois ans (2005–2007)

Background Rotaviruses are the most frequent agents associated with diarrhoea in children worldwide. Analysis of mobility of the 11 segments of genomic RNA by polyacrylamide gel electrophoresis (PAGE) yields a pattern which is characteristic for a particular rotavirus isolate. The group A rotaviruses can be further characterized by analysis of VP7 and VP4 genes specificities, responsible for rotavirus classification into G and P genotypes, respectively. The aim of the present study was to determine the evolution of group A Rotavirus strains circulating in Tunisia over a 3-year period (2005–2007). Material and methods A total of 1503 stool samples collected from children less than five years old, consulting or hospitalised in Tunisia for diarrhoea between 2005 and 2007, were screened for the presence of group A Rotaviruses. Rotavirus-positive specimens were further analyzed by PAGE and G/P-genotyped by multiplex semi-nested RT-PCR. Results Rotaviruses were detected in 323 stool samples over 1503 (21 %). Long electropherotypes predominated in Tunisia during the whole period of study ( N = 158 vs N = 82 short electropherotypes). VP7 genotyping showed the cocirculation of five different genotypes: G1, G2, G3, G4 and G9. VP4 typing detected four different P-genotypes: P[8], P[4], P[6] and P[11]. Rotavirus strains with G3P[8] specificity were predominating in Tunisia in 2005 and 2006, replaced by G2P[4] strains in 2007.

Hospital‐Based Surveillance of Rotavirus Diarrhea in the People’s Republic of China, August 2003–July 2007

Rotaviruses cause acute diarrhea worldwide. Previous studies of rotavirus diarrhea in China found that rotavirus infection is the most common cause of severe diarrhea in young children. In the present study, surveillance of rotavirus diarrhea was conducted involving 9549 children aged <5 years who were admitted for treatment of diarrhea at 11 sentinel hospitals in China from August 2003 through July 2007. Group A rotavirus was detected in 3749 (47.8%) of the 7846 fecal specimens by using enzyme-linked immunosorbent assay. Rotavirus isolates were characterized by reverse-transcriptase polymerase chain reaction to determine G and P genotypes. All the strains that are common worldwide were detected; G3P[8] was the most common. An unusual G5 strain was detected in 2 specimens. Of all episodes of rotavirus diarrhea, 94% occurred during the first 2 years of life, peaking at 6-23 months of age. Our findings indicate that globally common rotavirus strains continue to be a major cause of severe childhood diarrhea in China. Introduction of routine immunization with effective rotavirus vaccines would substantially reduce this burden.