G5型人A组轮状病毒LL36755株的VP4、VP6和NSP4编码基因的分子特征

最近在亚洲首次发现并报道了感染人的G5型人A组轮状病毒LL36755株,为进一步探讨其进化来源,克隆了G5型人A组轮状病毒LL36755株的VP4、VP6、NSP4编码基因,并分析其基因序列的分子特征。结果发现卢龙株LL36755为罕见的G5P[6]型,其VP6的亚群为SGⅡ型,NSP4的基因型为B型。系统进化树分析表明,卢龙株LL36755的VP7、VP4编码基因与猪来源的毒株关系密切,而VP6、NSP4编码基因与人来源的毒株紧密相联系。可以推断新的人腹泻A组轮状病毒LL36755株是猪的VP7,VP4编码基因与人的VP6,NSP4编码基因的自然重组;而且该毒株不是G5的原型,很可能是人类轮状病毒与猪轮状病毒毒株的自然重组后逐步进化而来。     

Reassortant DS‐1‐like G1P[4] Rotavirus A strains generated from co‐circulating strains in Vietnam, 2012/2013

One major mechanism by which Rotavirus A (RVA) evolves is genetic reassortment between strains with different genotype constellations. However, the parental strains of the reassortants generated have seldom been identified. Here, the whole genome of two suspected reassortants, RVA/Human‐wt/VNM/SP127/2013/G1P[4] and RVA/Human‐wt/VNM/SP193/2013/G1P[4], with short RNA electropherotypes were examined by Illumina MiSeq sequencing and their ancestral phylogenies reconstructed. Their genotype constellation, G1‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, indicated that they were G1 VP7 mono‐reassortants possessing DS‐1‐like genetic backbones. The two strains were ≧99.7% identical across the genome. While their VP7 genes were ≧99.7 identical to that of a Wa‐like strain RVA/Human‐wt/VNM/SP110/2012/G1P[8] which co‐circulated during the 2012/2013 season, 10 genes were ≧99.8% identical to that of the DS‐1‐like strains RVA/Human‐wt/VNM/SP015/2012/G2P[4] (and SP108) that co‐circulated during the season. The identities were consistent with the phylogenetic relationships observed between the genes of the reassortants and those of the afore‐mentioned strains. Consequently, the G1P[4] strains appear to have been generated by genetic reassortment between SP110‐like and SP015‐like strains. In conclusion, this study provides robust molecular evidence for the first time that G1P[4] strains detected in Hanoi Vietnam were generated by inter‐genogroup reassortment between co‐circulating G1P[8] and G2P[4] strains within the same place and season.

     

Monitoring and evaluation of infectious rotaviruses in various wastewater effluents and receiving waters revealed correlation and seasonal pattern of occurrences

Aims: Sewage systems are important nodes to monitor enteric pathogens transmitted via water. The aim of this study was to assess the presence of rotaviruses in effluents from wastewater treatment plants (WWTPs) and receiving streams in Beijing, China, to evaluate the reductions of rotaviruses in WWTPs and to provide viral fate and transport data for further epidemiological studies. Methods and Results: Two PCR‐based methods, including an RT‐qPCR and another quantitative RT‐PCR (ICC‐RT‐qPCR), which was integrated with cell culturing, were applied to conduct a 1‐year monitoring of infectious rotaviruses and viral genes in effluents from three WWTPs and the receiving waters in Beijing, China. The ICC‐RT‐qPCR was able to detect more positive samples than RT‐qPCR, showing positive results for 67% of primary effluents, 47% of secondary effluents and 14% of tertiary effluents, in comparison with 44, 22 and 6% by RT‐qPCR, respectively. Seasonal variations of rotaviruses were observed in all effluents with higher occurrences in winter than in summer, which correlated well with the seasonal pattern of rotaviruses in the river receiving wastewater effluents. The reduction efficiencies by different treatment processes were assessed. Secondary treatments can remove most of infectious rotaviruses in primary sewage, with annual average reduction values of 2·08 ± 0·63, 2·83 ± 0·49 and 2·00 ± 1·10 log₁₀ for the three WWTPs, respectively. Tertiary treatments were able to further remove infectious rotaviruses. Conclusions: The results showed a year‐round distribution of rotaviruses in three WWTPs in Beijing and provided important information regarding the transport and susceptibility of rotaviruses to different levels of wastewater treatment processes. Significance and Impact of the Study: This study, for the first time, revealed the whole year prevalence and reductions of rotaviruses in WWTPs and the corresponding receiving waters in China, and demonstrated the impact of wastewater discharge on the potential spreading of infectious rotaviruses and public health.     

我国1998～1999年流行的婴幼儿腹泻轮状病毒的分型研究

轮状病毒是世界范围内引起婴幼儿腹泻的主要病原。根据病毒外壳蛋白VP4和VP7抗原性的不同可区分为不同型：P（VP4,protease sensitive）型和G（VP7,glycoprotein）型。1998－1999年在中国8个城市（长春、秦皇岛、北京、杭州、福州、广州、成都、昆明）采集了急性腹泻患儿的1093份非细菌性腹泻粪便标本,先进行A组轮状病毒（HRV）的筛选,其中阳性标本433份（39.6％）,电泳型长型占优势（96％）。对HRV标本,再利用血清型特异的MAbELISA和／或RT－PCR进行G分型。结果表明,在1998－1999年,在上述8城市非细菌性腹泻行季节,以HRV G1型为主要流行株,占阳性的83.4％,其次为G3（12.0%）、G4（3.5%)和G2(3.2%）。此外,有3份（0.7％）HRV标本未能分型,12（2.8％）份标本为混合感染,还结合1982－1996年全国12个地区1382份HRV标本的分型资料,分析了我国HVR G血清型的流行规律。实验中又抽样选取了124份GHRV标本,用RT－PCR进行P分型,其中P[8]型76份（61.3％）,P[4]型14份（11.3%）,P[6]型12份(9.7%),P[9]型8份(6.4%)。另外15份（12.1%)未能分出P型,有待进一步检定,实验中HRV分离株除了觉见的P[8]G1(51.4%)、P[4]G2(4.6%)毒株外,还检测到P[8]G3(11.0%)、P[8]G4(6.4%) 和其它较少见的病毒型。以上结果为我国轮状病毒疫苗的应用和开发提供了较系统、清晰的流行病学背景资料。     

Genotypes of rotavirus associated with acute gastroenteritis in Seoul, Korea

Acute viral gastroenteritis is one of the most common infectious diseases in infants and young children. Rotavirus is mainly important in childhood. The present study determined the detection rate, seasonality and G and P genotypes of rotaviruses in children hospitalized for acute gastroenteritis in Seoul, Korea in 2009. A total of 1,423 stool specimens were screened by ELISA for the presence of rotavirus antigens and the rotavirus-positive stools genotyped by RT-PCR. The G genotype was determined for 90% of samples (242/269) and the P genotype for 93.3% (251/269). During the study, 25 G-P combinations were detected with G1P[8] in 38.3% (n= 103) and G4P[6] in 5.9% (n= 16) cases. These data provided information on rotavirus in patients with acute gastroenteritis in Seoul, Korea and provided baseline data to motivate for the implementation of control measures for rotavirus disease.     

Detection and characterization of rotavirus G and P types from children participating in a rotavirus vaccine trial in Belém, Brazil

This study sought the characterization of rotaviruses in a trial with a tetravalent rhesus-human rotavirus vaccine in Belém, Brazil in children who received three doses of vaccine or placebo in the 1st, 3rd and 5th months of life. Rotavirus electropherotypes, subgroups, G serotypes, G, [P] and [P], G genotypes were determined in 93.3%, 95.9%, 93.3%, 73.3%, 95.5% and 92.2% of isolates, respectively. Serotypes G1, G2 and G4 were detected in 58.9%, 30% and 4.4% of the cases, respectively. Rotavirus genotype G5 was detected for the first time in Northern region in 4.4% of the infections. Rotavirus genotypes P[8], P[4], P[6] and P[8 + 6] were detected in 54.5%, 26.7%, 12.2%, and 2.2% of the cases, respectively. The predominant genotypes were P[8], G1 and P[4], G2 with 53% and 26.6% of the infections, respectively. Unusual strains accounted for 20.5% including P[4], G1, P[6], G1, P[6], G4, P[6], G5, P[8], G2, P[8], G5. Mixed infections involving P[8 + 6], G2 and P[8 + 6], G1 were also noted. The neonatal P[6] strains associated with diarrhea were detected among children aged 9-24 months. To our knowledge, this study represents the first in Brazil to analyse, on molecular basis, rotavirus genotypes from children participating in a rotavirus vaccine trial. These results are of potential importance regarding future rotavirus vaccination strategies in Brazil.     

Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Isolation of human monoclonal antibodies that neutralize human rotavirus

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 micro g/ml.     

Evidence of the co‐circulation of enteric viruses in sewage and in the population of Greater Cairo

Aims: To characterize major enteric viruses (enterovirus, rotavirus, norovirus, astrovirus and adenovirus) in the sewage of Greater Cairo and to compare the results with clinical data collected during the same period. Methods and Results: Seventy‐two sewage samples from two waste water treatment plants were collected from April 2006 through February 2007. Enteroviruses, noroviruses (NoVs) and rotaviruses (RVs) were detected by RT‐PCR in 22%, 18% and 8·3% of the samples, respectively. No adenovirus and astrovirus was detected. G2P[8], G9P[8], G1P[8], G2P[4] and rare G12 RV isolates were detected in the environment as well as a bovine RV. The environmental NoV strains mostly belonged to genogroup I (84%). Rotaviruses and some of the NoVs were similar to those found in the clinical samples at the same time. Conclusions: The comparison of environmental and clinical data suggests that similar RV and NoV isolates were circulating in the environment and in the population during the same period. Significance and Impact of the Study: Few studies have investigated the prevalence and the epidemiology of RVs and NoVs in Cairo. This work is the first to establish a correlation between viral gastroenteritis and the concomitant presence of enteric viruses in the environment for Greater Cairo where combined environmental and clinical surveys should help to prevent infections caused by these major pathogens.     

The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses.     

<Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> and <Emphasis Type="Italic">Giardia intestinalis</Emphasis> in Calf Diarrhoea in Sweden

The objective of this study conducted in 75 herds was to investigate the presence and significance of Cryptosporidium parvum and Giardia intestinalis in Swedish dairy calves in comparison with rotavirus, coronavirus and Escherichia coli K99+. The farmers were asked to collect faecal samples from each heifer calf that had diarrhoea between birth and 90 days of age, and also from a healthy calf of the same age. In total, 270 samples were collected and analysed. C. parvum, either alone or together with G. intestinalis and/or rotavirus, was detected in 16 (11%) and 6 (5%) of the samples from diarrhoeic and healthy calves, respectively. Even though a higher proportion of diarrhoeic calves shed C. parvum, the difference between the groups was not statistically significant (p = 0.067), possibly due to the low number of positive samples. G. intestinalis was found in 42 (29%) of the diarrhoea samples and in 29 (23%) of the samples from healthy calves. Rotavirus and coronavirus were demonstrated in 24% and 3% of the diarrhoea samples, respectively, whereas E. coli K99+ was only found in samples from 2 healthy calves. C. parvum and G. intestinalis were found in samples from calves 7 to 84 days of age and during all seasons. The results confirm that C. parvum is present in Swedish dairy herds and might have clinical significance. G. intestinalis was the most common agent found but the importance of this parasite remains unclear. Both parasites have suggested zoonotic potential and thus warrant further attention. In addition, rotavirus is a major pathogen in neonatal enteritis in Sweden, whereas coronavirus and E. coli K99+ seem to be of less importance.     

Emergence and Characterization of Unusual DS-1-Like G1P[8] Rotavirus Strains in Children with Diarrhea in Thailand

The emergence and rapid spread of unusual DS-1-like G1P[8] rotaviruses in Japan have been recently reported. During rotavirus surveillance in Thailand, three DS-1-like G1P[8] strains (RVA/Human-wt/THA/PCB-180/2013/G1P[8], RVA/Human-wt/THA/SKT-109/2013/G1P[8], and RVA/Human-wt/THA/SSKT-41/2013/G1P[8]) were identified in stool specimens from hospitalized children with severe diarrhea. In this study, we sequenced and characterized the complete genomes of strains PCB-180, SKT-109, and SSKT-41. On whole genomic analysis, all three strains exhibited a unique genotype constellation including both genogroup 1 and 2 genes: G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This novel genotype constellation is shared with Japanese DS-1-like G1P[8] strains. Phylogenetic analysis revealed that the G/P genes of strains PCB-180, SKT-109, and SSKT-41 appeared to have originated from human Wa-like G1P[8] strains. On the other hand, the non-G/P genes of the three strains were assumed to have originated from human DS-1-like strains. Thus, strains PCB-180, SKT-109, and SSKT-41 appeared to be derived through reassortment event(s) between Wa-like G1P[8] and DS-1-like human rotaviruses. Furthermore, strains PCB-180, SKT-109, and SSKT-41 were found to have the 11-segment genome almost indistinguishable from one another in their nucleotide sequences and phylogenetic lineages, indicating the derivation of the three strains from a common origin. Moreover, all the 11 genes of the three strains were closely related to those of Japanese DS-1-like G1P[8] strains. Therefore, DS-1-like G1P[8] strains that have emerged in Thailand and Japan were assumed to have originated from a recent common ancestor. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like G1P[8] strains that have emerged in an area other than Japan. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] rotaviruses.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Investigation of CTLA‐4 and CD86 gene polymorphisms in Iranian patients with brucellosis infection

The protective immune response against Brucella involves T‐cell‐mediated immunity. T‐lymphocyte receptors, CD28 and cytotoxic T‐lymphocyte‐associated protein‐4 (CTLA‐4), bind the same ligands, CD80 (B7‐1) and CD86 (B7‐2) on antigen‐presenting cells and regulate T cell activation. CD28 delivers stimulatory signals whereas CTLA‐4 provides inhibitory signals for T cell activation. Here, we investigated the association of four polymorphisms in CTLA4 (+49A/G [rs231775] and ?318 C/T [rs5742909]) and its ligand CD86 (+1057 G/A [rs1129055] and +2379G/C [rs17281995]) with brucellosis infection. The study included 153 Iranian patients with active brucellosis and 128 healthy individuals as the control group. Genotyping of the CTLA4 and CD86 variants was performed using tetra amplification refractory mutation system‐polymerase chain reaction (T‐ARMS‐PCR) and PCR–restriction fragment length polymorphism analysis, respectively. It was found that the CTLA4 ?318 CT genotype and T allele were present more frequently in cases than in controls and are therefore associated with an increased risk for brucellosis (?318 TT genotype; OR = 2.544, P = 0.002). Likewise, the CD86 +1057 GA and AA genotypes and A allele were associated with an increased risk of brucellosis (+1057 AA genotype; OR = 3.81, P = 0.001). However, no statistically significant difference between brucellosis patients and controls in the allele and genotype distributions of CTLA4, +49A/G (P = 0.859) and CD86, +2379G/C (P = 0.476) was found. In conclusion, CTLA4 ?318 CT genotype and T allele and the CD86 +057 GA and AA genotypes and A allele play roles as risk factors for developing brucellosis infection in Iran.

     

Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999

The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394-4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.