One unusual human G3P[3] group A rotavirus (RVA) strain M2-102 was identified in stool sample collected from a child with diarrhea in Guangxi Province, China in 2014. It is well known that G3P[3] is a genotype commonly identified in feline and canine RVAs. However, the preliminary phylogenetic analyses of the VP7 and VP4 genes of strain M2-102 indicated that these two genes were closely related to bat RVA strain MYAS33 and simian strain RRV, respectively, whereas both clustered distantly to feline/canine-like RVA strains. In this study, full genome sequencing and molecular analyses were conducted to obtain the true origin of strain M2-102. It was revealed that strain RVA/Human-wt/CHN/M2-102/2014/G3P[3] exhibited a G3-P[3]-I3-R3-C3-M3-A9-N3-T3-E3-H6 genotype constellation for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes. Phylogenetic analyses revealed that 5 genes (VP7, VP1, VP2, NSP2 and NSP3) from strain M2-102 were closely related to those of bat strain MYAS33 from Yunnan Province which was thought a true bat RVA strain rather than a virus transmitted between species, while another 5 genes (VP4, VP3, NSP1, NSP4 and NSP5) clustered closely with those of simian strain RRV, yet the VP6 gene was closely related to that of human G3P[9] strain AU-1 and AU-1-like RVAs. The epidemiological data indicated that the child infected with M2-102 came from a countryside village, located in Dong Autonomous County of Sanjiang (subtropical hilly wooded area), Liuzhou city in Guangxi Province which might provide natural environment for reassortment events occurring among animal and human RVAs. Therefore, the data suggest that human strain M2-102 might originate from multiple reassortment events among bat, simian and human AU-1-like RVAs, yet it is not clear whether the genomic backbone based on bat MYAS33 (5 genes) and simian RRV (5 genes) like rotaviruses had been obtained through reassortment before being transmitted to the human. This is the first report on whole genome analysis of human G3P[3] RVA from China.
Child, Preschool ; China ; Genome, Viral ; Genomics ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Reassortant Viruses ; classification ; genetics ; isolation & purification ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Infections ; virology ; Viral Proteins ; genetics

Rotavirus is the leading causal agent of severe acute gastroenteritis in children aged <5 years. A specific pharmacologic agent for the treatment of rotavirus-infected children is lacking. In China, only the Luo Tewei oral vaccine (Lanzhou Institute of Biological Products, Shanghai, China), which is produced from Lanzhou lamb rotavirus vaccine (LLR), is available. Studies have hypothesized that the genotype of LLR is G10P[12], To identify the genotype of LLR by reverse transcription-polymerase chain reaction, we showed that the VP7 and VP4 genotypes of LLR were G10 and P[15], respectively, based on sequencing, alignment and phylogenetic analyses. In conclusion, we identified the genotype of rotavirus strain LLR to be G10P[15].
China ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Rotavirus ; chemistry ; classification ; genetics ; isolation & purification ; Rotavirus Infections ; virology ; Rotavirus Vaccines ; chemistry ; classification ; genetics ; isolation & purification ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics

We wished to select a cold-adapted genotype G1P[8] ZTR-68 rotavirus (China southwest strain) in MA104 cells for possible use as a live vaccine. ZTR-68 was recovered originally from children with diarrhea. The virus was cultivated at 37 degrees C at the first passage. Then, the cultivation temperature was decreased stepwise by 3 degrees C per eight passages. In total, the virus was passaged 32 times, and cultivation was terminated at 28 degrees C. Biological characteristics of the virus were analyzed during serial passages. There was no difference between the migration patterns of genomic dsRNA segments according to polyacrylamide gel electrophoresis of original and cold-adapted viruses. Infectious and red cell-agglutination titers of cold-adapted virus were lower than those of the parent virus. Also, the virus formed small-size plaques with irregular shapes at 31 degrees C and 28 degrees C. These results suggested that a genetically stable attenuated virus can be obtained through serial cold-adapted passages. Thus, an alternative strategy is provided by cold-adaption for development of attenuated live rotavirus vaccines.
Adaptation, Physiological ; China ; Cold Temperature ; Diarrhea ; virology ; Female ; Genotype ; Humans ; Infant ; Male ; Rotavirus ; genetics ; growth & development ; isolation & purification ; physiology ; Serial Passage ; Virus Cultivation ; Virus Replication

Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations.
Child, Preschool ; Feces/virology ; Gastroenteritis/immunology/prevention & control/virology ; Genotype ; Humans ; Infant ; *Mass Vaccination ; RNA, Viral/genetics ; Republic of Korea ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus/*classification/*genetics/isolation & purification ; Rotavirus Infections/immunology/*prevention & control/virology ; Rotavirus Vaccines/*immunology ; Vaccines, Attenuated/immunology

This study aimed to amplify major genome segments (VP7, VP4, VP6, VP2 and NSP2-5) of porcine G3 group A rotavirus (GARV) LLZ212 isolated in our laboratory, determine their genotypes, and explore the evolutionary relationships between G3 GARV strains isolated from humans and pigs in Lulong County, Hebei Province, China. Major genome segments of seven GARV strains were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the segments were sequenced. The genome segments of seven GARV strains were determined by the online RotaC genotyping tool (RotaC v2.0). The reference sequences of each GARV genome segment were downloaded from GenBank. Homology and phylogenetic evolutionary analyses were conducted using the MEGA 5.0 and DNAStar software packages. LLZ212 isolated from pigs in Lulong had the following genotype: G3-P[8]-I5-C1-N1-T1-E1-H1. All human GARV strains had the following genotype: G3-P[8]-I1-C1-N1-T1-E1-H1. The VP7, VP4, NSP4 and NSP5 genes of the LLZ212 strain had the highest nucleotide identities with the human GARV E885, CMH014/07, Wa and RMC321 strains, respectively, and these clustered together in a sublineage. The VP6, NSP4 and NSP5 genes of the LLZ212 strain shared the highest nucleotide identities with the porcine GARV PRG921 strain, while VP2 associated most closely with porcine GARV OSU strain, and these also clustered in a sublineage. A rare porcine G3-P[8]-I5-C1-N1-T1-E1-H1 GARV strain was identified, which may represent a reassortment between porcine and human viruses. In conclusion, the VP7, VP4, NSP4 and NSP5 genes of LLZ212 share high levels of sequence identity with human GARV, while VP2, VP6, NSP2 and NSP3 cluster with porcine GARV.
Animals ; Capsid Proteins ; genetics ; Child, Preschool ; China ; epidemiology ; Evolution, Molecular ; Genotype ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Infections ; epidemiology ; veterinary ; virology ; Swine ; Swine Diseases ; epidemiology ; virology ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo investigate the etiological characteristics of human rotavirus (HRV), human calicivirus (HuCV), human astrovirus (HAstV) and human enteral adenovirus (HAdV) in Ningxia province during 2011.

METHODSStool specimen was collected from acute diarrhea case of Ningxia during 2011. HRV was detected by ELISA and serotype/genotype identified on those RT-PCR positive specimens. HuCV, HAstV and HAdV were detected by RT-PCR.

RESULTSIn this study, a total of 690 specimens were detected, with the infection rates of HRV, HuCV, HAstV and HAdV as 2.17%, 21.74%, 3.19% and 6.52%, respectively. Co-infections were found in 4.20% of all the samples being tested. Among 15 HRV positive cases, serotypes G1, G3 and P[4] were the most predominant strains.

CONCLUSIONChildren who were under 2 years of age were the majority among patients infected by diarrhea viruses while HuCV was recognized as the main pathogen responsible for the viral diarrhea cases in Ningxia, 2011.

Adenoviruses, Human ; isolation & purification ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Caliciviridae ; isolation & purification ; Child ; Child, Preschool ; China ; epidemiology ; Diarrhea ; virology ; Humans ; Infant ; Infant, Newborn ; Mamastrovirus ; isolation & purification ; Middle Aged ; Rotavirus ; isolation & purification ; Young Adult

BACKGROUND: We evaluated the analytical and clinical performances of the SD BIOLINE Rota/Adeno Rapid kit (SD Rota/Adeno Rapid; Standard Diagnostics, Inc., Korea), an immunochromatographic assay (ICA), for the simultaneous detection of rotaviruses and adenoviruses in human stool samples. METHODS: We tested 400 clinical stool samples from patients with acute gastroenteritis and compared the ICA results with the results obtained by using ELISA, enzyme-linked fluorescent assays (ELFA), PCR, and multiplex reverse transcription-PCR (mRT-PCR). To assess the analytical performance of the SD BIOLINE Rota/Adeno Rapid kit, we determined its detection limit, reproducibility, cross-reactivity, and analytical reactivity for adenovirus subtypes, and performed interference studies. RESULTS: The overall agreement rates among the tested methods were 91.5% for rotavirus and 85.5% for adenovirus. On the basis of mRT-PCR, the overall agreement, positive agreement, and negative agreement rates of the ICA were 95.6%, 100%, and 94.9% for rotavirus, and 94.0%, 71.4%, and 94.8% for adenovirus, respectively. Using the ICA, we detected all the subtypes of adenovirus tested, but the analytical reactivities for adenovirus subtypes were different between the 4 adenovirus detection methods. The high reproducibility was confirmed, and no cross-reactivity or interference was detected. CONCLUSIONS: The SD BIOLINE Rota/Adeno Rapid kit showed acceptable analytical and clinical performances. However, interpretation of adenovirus positive/negative result should be cautious because of different detectability for adenovirus subtypes among adenovirus detection methods.
Acute Disease ; Adenoviridae/genetics/*immunology/isolation & purification ; Cross Reactions ; DNA, Viral/analysis ; Enzyme-Linked Immunosorbent Assay ; Feces/*virology ; Gastroenteritis/diagnosis/virology ; Humans ; *Immunochromatography ; Multiplex Polymerase Chain Reaction ; RNA, Viral/analysis ; Reagent Kits, Diagnostic ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus/genetics/*immunology/isolation & purification

Abstract:This study aims to investigate the genetic characteristics of group A rotavirus (GARV) G9P[8] strains from infantile diarrhea samples in Hebei Lulong region from 2009 to 2011. We randomly selected five GARV G9P[8] strains in Hebei Lulong region from 2009 to 2011, amplified the 11 gene fragments of GARVs by RT-PCR, and analyz their full-genome sequences by homology and phylogenetic analysis with DNAStar and MEGA. The nucleotide homology between strains LL11131077 and LL11131083 in 2011 was significantly higher than hat etween them and the other three strains in 2009 and 2010. The G9P[8] GARVs circulating in Hebei Lulong region from 2009 to 2011 elenged to the same genotype as the prevalent G9P[8] GARVs in other parts of the world. However,the two strains in 2011, compared with those in 2009 and 2010, were located in a different sub-branch of the phylogenetic tree and had amino acid mutations at many sites.
China ; Feces ; virology ; Genome, Viral ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Infections ; virology ; Viral Proteins ; genetics

OBJECTIVETo establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.

METHODSPrimers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.

RESULTSThe 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.

CONCLUSIONThe established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

Astroviridae ; genetics ; isolation & purification ; Diarrhea ; virology ; Feces ; virology ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Norovirus ; genetics ; isolation & purification ; RNA, Viral ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rotavirus ; genetics ; isolation & purification ; Sapovirus ; genetics ; isolation & purification

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.
Adult ; Aged ; Antibodies, Viral/*blood ; Antigens, Viral/genetics/immunology/metabolism ; Capsid Proteins/genetics/immunology/metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin A/blood ; Immunoglobulin G/blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Recombinant Proteins/biosynthesis/genetics/immunology ; Republic of Korea/epidemiology ; Rotavirus/isolation & purification/*metabolism ; Rotavirus Infections/*epidemiology/virology ; Seroepidemiologic Studies ; Time Factors ; Young Adult