The Distribution of G (VP7) and P (VP4) Serotypes among Human Rotaviruses Recovered from Japanese Children with Diarrhea

Both the G (VP7) and P (VP4) serotypes of human rotaviruses collected over a 10-year period from Japanese children with diarrhea were determined by recently-developed polymerase chain reaction-based typing assays. The combination of G1 and P8 was found in 65.2% and the combination of G2 and P4 was found in 15.2%. For the rest of the specimens, only a few other combinations occurred and their relative frequencies were less than 10%. The viruses carrying P9 were always associated with G3 as is the prototype strain AU-1.     

Detection of human rotavirus in hospitalized diarrheic children in central India

During the present study, group A human rotaviruses were detected among diarrheic children using polyacrylamide gel electrophoresis (PAGE) technique, with a typical RNA migration pattern of 4:2:3:2, suggestive of group A rotavirus. During the study, a total of 46 fecal samples collected from hospitalized children with acute diarrhea as well as children inhabiting nearby animal farms with history of presence of animal rotaviruses on the farms were processed for detection of human rotavirus. Out of 33 diarrheic children, 12 showed presence of rotavirus infection (36.36%), however, none of the children from animal farm areas showed presence of rotavirus. Female children were more susceptible to rotavirus infection (46.15%) than males (30%). Majority of the cases of rotavirus gastroenteritis belonged up to one year of the age, with an incidence of 40.91%. RNA profile of rotaviruses suggested circulation of 5 different electropherotypes in this geographical locale of the country, indicating existence of genomic diversity among human rotaviruses. Majority of the isolates were of long pattern (66.67%), whereas short pattern was detected only in one third of the viruses. This preliminary study emphasizes for further detailed studies on the molecular characterization of rotaviruses circulating in this part of country and their relationship with other human rotavirus strains and animal strains in the country.     

Sequential rotavirus infections: characterization of serotypes and electrophoretypes

Few studies have characterized the rotaviruses observed in repeated infections in the same patient. Rotavirus serotypes and electrophoretypes were determined from faecal samples of 24 children hospitalized between 1984 and 1989 in a Clermont-Ferrand hospital and who had had repeated rotavirus shedding.The patients were aged between 3 days and 4 years. Fifty infections were recorded. Of the 48 serotypes obtained, 18 were serotype 1 (37.5 %), 10 serotype 2 (20.8 %) and 20 serotype 4 (41.7 %). Serotype 3 was not found. Seven serotype 2 infections were characterized during rehospitalization. Six patients had two subsequent infections with the same serotype.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Gastroenteritis caused by human rotaviruses (serotype three) in a suckling mouse model

The pathogenic potential of human rotaviruses of serotypes 1 through 4 was evaluated in suckling mice. Oral inoculation of three different human rotaviruses of serotype 3 into 5-6 day old CD-1 mice caused disease characterized by diarrhea and dehydration. The mean 50% diarrhea inducing dose (DD50) was 5 X 10(5) pfu. Histopathological examination of small intestines revealed villus epithelial cell vacuolization localized to the distal one-third of the villus. Only Serotype 3 rotaviruses exhibited a rapid phase of viral growth in the intestine between 7 and 12 hours post-inoculation. Larger inocula of rotavirus serotypes 1, 2, and 4 did not cause disease or typical histopathologic changes. However, immunoperoxidase staining for rotavirus antigen was positive in all serotypes tested indicating that infection can occur without apparent disease and is not serotype specific. This convenient in-vivo model can be used to evaluate attenuation of human origin vaccine candidates of serotype 3.     

Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.     

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.     

Use of PCR and nucleotide sequence analysis of the VP7 gene for detection of bovine rotaviruses and identification of their G serotypes

A Vp7 gene fragment PCR protocol was developed to detect the bovine rotaviruses and to identify their G serotypes. The most widespread bovine rotaviruses of G serotypes (G6, G8 and G10) can be distinguished on the basis of the PCR fragment size, while other G serotypes can be differentiated through a comparative analysis of the VP7 gene fragment nuclcotide sequence. Twenty-four bovine rotavirus field isolates were detected, and their G stereotypes were determined by using the method in question. Fourteen isolates were shown to be of G6 serotype; four of them were of G8, five--of G10, and one isolate was of G11 serotype. A possibility of detecting more than one isolate by this method was shown. Finally, a feasibility of using the method for searching for rotaviruses of new bovine rotavirus G serotypes and for rotaviruses, which do not belong to a so far described G serotypes, is discussed.     

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。     

昆明婴幼儿腹泻的轮状病毒分子基因特征研究

目的了解昆明婴幼儿腹泻中轮状病毒感染情况。方法收集昆明医学院第一附属医院儿科2004年10-12月住院的腹泻的患儿粪便标本60份,采用聚丙烯酰胺凝胶电泳（PAGE）法和RT—PCR法进行轮状病毒分型。结果60份粪便标本中,29份检测到A组轮状病毒RNA基因,阳性检出率为48.3％,未发现B组及C组轮状病毒。其中RNA长型有29份（100％）,未发现短型和混合型。对29份有明确编号的轮状病毒阳性标本来源的患儿分析显示,平均发病年龄10.5个月。29份标本中,有20份可用RT—PCR分型,均为G3型,未发现其他型。结论A组轮状病毒是昆明地区5岁以下婴幼儿腹泻病的主要病原,3月龄-2岁婴幼儿是轮状病毒的易感人群,以冬季10月份至12月份为流行高峰,基因组以长型为主,血清型为G3型。     

Diversity of Rotavirus Strains Causing Diarrhea in <5 Years Old Chinese Children: A Systematic Review

Background

We conducted a systematic review of the diversity and fluctuation of group A rotavirus strains circulating in China.

Methods and Findings

Studies of rotavirus-based diarrhea among children less than 5 years, published in English or Chinese between 1994 and 2012, were searched in PubMed, SinoMed, and CNKI and reviewed by applying standardized algorithms. The temporal and spatial trends of genotyping and serotyping were analyzed using a random-effects model. Ninety-three studies met the inclusion/exclusion criteria and were included in the meta-analysis. Overall, 22,112 and 10,660 rotavirus samples had been examined for G and P types, respectively. The most common G types were G1 (39·5%), G3 (35·6%), G2 (1·3%), and G9 (0·1%). Among P types, P[8] (54·6%) was the predominant type, followed by P[4] (11·1%) and P6 (0·1%). The most common G-P combinations were G3P[8] (32·1%) and G1P[8] (24·5%), followed by G2P[6] (13·2%) and G2P[4] (10·1%). Before 2000, serotype G1 was the predominant strain and accounted for 74·3% of all rotavirus infections; however, since 2000, G3 (45·2%) has been the predominant strain. Rotavirus P types showed little variation over the study period.

Conclusion

Despite the variation of serotypes observed in China, the G1, G2, G3, and G4 serotypes accounted for most rotavirus strains in recent decades. These results suggest that Chinese children will be adequately protected with currently available or forthcoming rotavirus vaccines.     

Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus.

Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.     

Data on the study of the epidemiology of rotavirus infection in Leningrad

The results obtained in the study of rotavirus infection in Leningrad in 1984-1987 are presented. Enzyme immunoassay techniques were used for the examination of 4,715 children aged 0-14 years and 1,162 adults with diagnosed acute enteral infection of unknown etiology, as well as the control group of 556 of healthy children aged 0-6 years and 77 healthy adults. The rotavirus antigen was detected in 1.210 sick children (25.7%) and 133 sick adults (11.4%), as well as in 6 healthy children (1.1%), but not detected in healthy adults. The following epidemiological regularities of rotavirus infection were established: the highest sick rate among children aged 0-2 years and a low level of rotavirus carriership among healthy persons; the seasonal character of rotavirus infection, its outbreaks occurring in winter and the epidemic periods varying in their character and duration in different years; the prevailing role of rotaviruses in the development of winter rises of acute enteral infection of unknown etiology in the city among children aged 0-2 years. The problem of the respiratory syndrome in rotavirus diarrhea is discussed. The rotavirus antigen was detected in 39 out of 144 children (27%) with diagnosed acute respiratory virus diseases and in 4 out of 99 nasopharyngeal washings (4%) from diarrhea patients. Adenoviruses were shown to play an insignificant role in the etiology of diarrhea (10% of cases).     

婴幼儿腹泻A群轮状病毒G和P的基因分型研究

目的研究浙江萧山医院婴幼儿童腹泻标本中人轮状病毒（Human Rotavims）毒株的感染情况及G和P基因型流行特点。方法收集该院2009年8月至2010年8月腹泻儿童15233份粪便标本采用酶联免疫吸附试验、逆转录-巢式聚合酶链反应进行轮状病毒病原检测,将128份阳性标本进行VP7和VP4基本分型。结果15233份婴幼儿腹泻标本中有2706份标本为轮状病毒阳性,阳性率17．8％;男孩和女孩检出率差异无统计学意义,以6-12月龄段检出率最高;对128份阳性标本进行G血清分型和P基因分型,G1型53份（41．4％）、G3型38份（29．7％）、G1G3型17份（13．3％）、G未分型20份（15．6％）;P[8]型72份（56．3％）、P[4]型16份（12．5％）、P[8]P[4]型3份（2．3％）、P未分型37份（28．9％）,G血清型和P基因型的组合以G1P[8]为主,占29．7％（38／128）。结论浙江萧山医院A群轮状病毒G血清以G1型为主,其次为G3型,P基因型以P[8]型为主。     

Rotaviruses in younger children in Novosibirsk in 2005-2007: detection and genotyping

Examination of 1898 patients with acute enteric infection from March 2005 to February 2007 showed that group A rotaviruses were the most frequent cause (35%) of acute gastroenteritis among children under 3 years of age. Majority of cases of rotavirus infection was detected in infants under 1 year of age (71.8%). The peak of sporadic incidence was observed between February and May. High rate of mixed infection (45.6%) was observed - associations of rotaviruses with other viruses (noroviruses, astroviruses) and bacteria (Salmonella, Shigella, enteroinvasive Escherichia coli, Campylobacter, and opportunistic species) were detected. P- and G-genotypes of 337(50.8%) isolates of group A rotaviruses were determined by RT-PCR. The most prevalent strain was P[8]G1 (54.6%) followed by P[8]G3 (10.7%), P[8]G9 (8.6%), P[4]G2 (8.3%), and P[8]G4 (4.5%) genotypes.     

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.     

Epidemiology and Evolution of Rotaviruses and Noroviruses from an Archival WHO Global Study in Children (1976–79) with Implications for Vaccine Design

Prompted by the discovery of new gastrointestinal viruses, the NIH, NIAID and WHO investigated the etiology of acute diarrhea that occurred from 1976–1979 in a global cohort of infants and young children. Rotaviruses were found to be major pathogens worldwide, whereas the Norwalk virus could not be detected using a radioimmunoassay. The aim of this study is to re-evaluate the role and diversity of rotaviruses and noroviruses in the original cohort using more sensitive current technologies. Stools collected from Asia, Africa, and South America (n = 485) were evaluated for viral genotypes by RT-PCR and sequencing. Rotaviruses were detected in 28.9% and noroviruses in 9.7% of the specimens, with G1 rotaviruses and GII noroviruses accounting for the majority of each respective virus. Various strains in this study predated the currently assigned dates of discovery for their particular genotype, and in addition, two noroviruses (KL45 and T091) could not be assigned to current genotypes. Phylogenetic analyses demonstrated a relative constancy in circulating rotavirus genotypes over time, with several genotypes from this study becoming established in the current repertoire of viral species. Similarly, GII noroviruses have maintained dominance, with GII.4 noroviruses continuing as a predominant genotype over time. Taken together, the complex molecular epidemiology of rotaviruses and noroviruses circulating in the 1970’s is consistent with current patterns, an important consideration in the design of multivalent vaccines to control these viruses.