Prevalence and genomic analysis of t203-like G9 (G9-VI) rotaviruses circulating in children with gastroenteritis in Beijing, China.

Our previous surveillance revealed that t203-like G9 (tentatively designated subtype G9-VI) rotaviruses re-emerged in 2010 in Beijing and rapidly prevailed over the G9-III subtype (the most common G9 subtype globally) and previously predominant G genotypes over the following two years. G9-VI belongs to the VP7 evolutionary lineage VI, which includes unusual and sporadic human rotaviruses from China (t203) and Japan. To obtain insight into the epidemiology, evolution, and transmission advantages of G9-VI rotavirus, we performed follow-up surveillance (2014-2017) and whole-genome analysis of 12 representative G9 strains. The results showed that the G9 genotype was predominant (77.4%), with a marked increase in prevalence (previously 43.5%). Within the G9 genotype, subtype G9-VI accounted for the majority (98.3%) of cases. The most prevalent P-genotype was P[8] (93.7%), within which subtype P[8]b was rare (0.7%). Phylogenetically, the G9-VI subtype strains in this study clustered closely with contemporary emerging human rotaviruses from many other countries in VP7 lineage VI, indicating that this subtype is capable of spreading globally. These currently emerging G9-VI rotaviruses formed a distinct monophyletic subcluster when compared to early G9-VI rotaviruses. Furthermore, four specific amino acid substitutions and synonymous codon substitutions were observed in the VP7 genes between the current G9-VI and globally common G9-III rotaviruses. The remaining nine genes of all of the analyzed representative G9 strains, whether G9-VI or G9-III, combined with the P[8]a, P[8]b, or P[6] genotype and exhibited the same Wa-like backbone constellation.

G2 rotavirus within an emergent VP7 evolutionary lineage circulating in children with acute diarrhea in Guangxi Province of China, 2014.

Routine surveillance revealed that the prevalence of P[4] rotaviruses circulating in children with acute diarrhea in Guangxi Province, China, increased in 2014. However, VP7 genotyping for these P[4] rotaviruses was unsuccessful. Exhaustive database searching and sequence analysis indicated that the G genotype of these P[4] rotaviruses was G2, and the VP7 genes clustered with recently emerging G2 strains in several countries within an emergent evolutionary lineage that was distinct from the previously designated lineages I-IV as well as lineage V including porcine rotaviruses. Further studies are essential to monitor the potential global spread of this emerging G2 rotavirus.

Human bocavirus 1 and 2 genotype-specific antibodies for rapid antigen testing in pediatric patients with acute respiratory infections.

BACKGROUND: Previous serological studies of human bocavirus (HBoV) 1 could not exclude cross-reactivity with the other three HBoVs, particularly HBoV2. METHODS: To search for genotype-specific antibodies against HBoV1 and HBoV2, the divergent regions (DRs) located on the major capsid protein VP3 were defined through viral amino acid alignment and structure prediction. DR-deduced peptides were used as antigens to harvest corresponding anti-DR rabbit sera. To determine their genotype specificities for HBoV1 and HBoV2, these sera samples were used as antibodies against the antigens VP3 of HBoV1 and HBoV2 (expressed in Escherichia coli) in western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and bio-layer interferometry (BLI) assays. Subsequently, the antibodies were evaluated with clinical specimens from pediatric patients with acute respiratory tract infection by indirect immunofluorescence assay (IFA). RESULTS: There were four DRs (DR1-4) located on VP3 with different secondary and tertiary structures between HBoV1 and HBoV2. Regarding the reactivity with VP3 of HBoV1 or HBoV2 in WB and ELISA, high intra-genotype cross-reactivity of anti-HBoV1 or HBoV2 DR1, DR3, and DR4, but not anti-DR2, was observed. Genotype-specific binding capacity of anti-DR2 sera was confirmed by BLI and IFA, in which only anti-HBoV1 DR2 antibody reacted with HBoV1-positive respiratory specimens. CONCLUSION: Antibodies against DR2, located on VP3 of HBoV1 or HBoV2, were genotype specific for HBoV1 and HBoV2, respectively.

Molecular epidemiology of coxsackievirus A16 circulating in children in Beijing, China from 2010 to 2019.

BACKGROUND: Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD). This study aimed to investigate the molecular epidemiology and evolutionary characteristics of CVA16. METHODS: Throat swabs were collected from children with HFMD and suspected HFMD during 2010-2019. Enteroviruses (EVs) were detected and typed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR. The genotype, evolutionary rate, the most recent common ancestor, population dynamics and selection pressure of CVA16 were analyzed based on viral protein gene (VP1) by bioinformatics software. RESULTS: A total of 4709 throat swabs were screened. EVs were detected in 3180 samples and 814 were CVA16 positive. More than 81% of CVA16-positive children were under 5 years old. The prevalence of CVA16 showed obvious periodic fluctuations with a high level during 2010-2012 followed by an apparent decline during 2013-2017. However, the activities of CVA16 increased gradually during 2018-2019. All the Beijing CVA16 strains belonged to sub-genotype B1, and B1b was the dominant strain. One B1c strain was detected in Beijing for the first time in 2016. The estimated mean evolutionary rate of VP1 gene was 4.49 × 10-3 substitution/site/year. Methionine gradually fixed at site-23 of VP1 since 2012. Two sites were detected under episodic positive selection, one of which (site-223) located in neutralizing linear epitope PEP71. CONCLUSIONS: The dominant strains of CVA16 belonged to clade B1b and evolved in a fast evolutionary rate during 2010-2019 in Beijing. To provide more favorable data for HFMD prevention and control, it is necessary to keep attention on molecular epidemiological and evolutionary characteristics of CVA16.

Prevalence and genetic diversity of noroviruses in outpatient pediatric clinics in Beijing, China 2010-2012.

Norovirus is a major cause of diarrheal disease with epidemic, outbreak or sporadic patterns in humans of all ages worldwide. This study aimed to determine the genotypic characteristics of noroviruses from infants and children in Beijing. Stool samples (n=1128) were collected from patients with symptoms of acute gastroenteritis in the past 3 years from 2010 to 2012. The norovirus positivity rate was 16.1% (182/1128) by using RT-PCR, including 122 with primer set covering polymerase region, 177 with primer set covering capsid region, and 117 with both polymerase and capsid regions. By sequence analysis for capsid genes, all the noroviruses identified were belonging to genogroup II (GII). Among these positive samples, GII.4 (61.0%) was the most common genotype detected, followed by GII.3 (35.0%). The new variant GII.4 Sydney_2012 strains emerged in this study in September and became the predominant genotype later. Those 117 from 182 RT-PCR positive samplers were able to be genotyped based on the sequences of both polymerase and capsid genes. The result was interesting that 59 out of these 117 positive specimens (50.4%) had mismatched genotypes between polymerase and capsid genes, including 7 suspected recombinants patterns. Among them, GII.P12/GII.3 was the most common combination which accounts for 54.2% (32/59), followed by GII.Pe/GII.4 Sydney_2012 which was 23.7% (14/59). Two novel recombinants, GII.P22/GII.5 and GII.21/GII.3 were first detected in this study. In summary, this study provides a detailed description based on laboratory data of the genetic diversity of norovirus in young children with acute gastroenteritis in Beijing. Moreover the data revealed that in the evolution of norovirus, new variant and novel recombination emerged frequently.

Identification of circulating porcine-human reassortant G4P[6] rotavirus from children with acute diarrhea in China by whole genome analyses.

P[6] group A rotavirus (RVA) strains identified in four stool specimens collected from children with acute diarrhea in Guangxi Province, southern China in 2010, with unknown G type were further analyzed by full genomic analysis. It was revealed by whole genome sequencing that 11 genomic cognate gene segments of these P[6] RVA strains shared almost 100% nucleotide identities and all exhibited an identical G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 genotype constellation. Phylogenetic analyses of VP7, VP1-VP4, NSP1, NSP2, NSP4 and NSP5 genes revealed that these Guangxi G4P[6] RVA strains were closely related to porcine and porcine-like human RVAs, while VP6 and NSP3 were closely related to those of common human RVAs. Interestingly, the four infants from whom these specimens were collected had come from different villages and/or towns. They had not contacted with each other and had had acute diarrhea before admitted into the same hospital. The genomic analyses and the clinical data revealed that these four Guangxi G4P[6] RVA strains from China were reassortants possessing VP6 and NSP3 gene segments of human origin yet all other nine gene segments of porcine origin. It is the first report on porcine-human reassortant G4P[6] RVA with identical genome configuration circulating in children.

[Detection and genotyping of human bocavirus 2 in children with acute diarrhea].

To investigate the prevalence of HBoV2 in pediatric patients with acute diarrhea in Beijing and the characteristic of the genome of the virus, 553 stool specimens were collected from pediatric outpatients with acute diarrhea in Affiliated Children's Hospital of Capital Institute of Pediatrics during Nov. 2010 to Oct. 2011. TaqMan-based Real-time polymerase chain reaction was performed to detect HBoV2 DNA from these specimens. Two positive specimens with high viral loads were selected for segmented amplification and then the amplified fragments were cloned into the plasmid vector pGEM-T, transformed into Escherichia coli DH5alpha and sequenced. Then genomic sequences assembled from those DNA fragments were compared with other parvovirus genomic sequences in the GenBank. Among these 553 specimens tested, 15 (2.7%) were HBoV2 DNA positive. The highest positive rate was shown in July (7.0%) through the whole year and in 3-6 month age group (4.1%) among different age groups. All these 15 specimens positive for HBoV2 DNA were collected from patients younger than 2 years old, including 4 simultaneously positive for norovirus, 3 positive for rotavirus and 1 positive for adenovirus. By sequence analysis, 2 almost complete HBoV2 genomic sequences assembled from gene fragments amplified from specimens BJQ19 and BJQ390 were typical HBoV2. And they shared high homology with each other (99.2%), while they shared the highest homology with FJ375129 from Shanghai China (99.1% and 99.2%) among other parvoviruses. These data suggest that some of acute diarrhea in pediatric patients in Beijing were associated with HBoV2, and infants and young children aged from 3 months to 2 years, are more likely to be infected by HBoV2.

[Investigation of adenovirus infection in hospitalized children with diarrhea during 2010 in Beijing, China].

OBJECTIVE: The study was designed to evaluate adenovirus infection in hospitalized children with diarrhea. METHOD: Stool specimens were collected from 519 hospitalized children with diarrhea during 2010, including those defined as community-acquired diarrhea (CAD) who developed diarrhea symptoms within 48 hours after admission, and those defined as hospital-acquired diarrhea (HAD) whose symptoms of diarrhea occurred beyond 48 hours after admission. PCR was employed to identify adenovirus in fecal samples by using universal primers for adenoviruses of all types, and specific primers for adenovirus group F. PCR products with expected size were sequenced for adenovirus typing. Clinical data for children with adenovirus positive specimens were analyzed. RESULT: A total of 519 hospitalized children, including 289 with CAD and 230 with HAD, were enrolled in the study. Out of 519 stool specimens, 76 showed PCR products with expected 301 bp and identified as adenovirus by sequencing, and the overall positive rate was 14.6%. Out of 289 CAD samples, 43 were positive (positive rate was 14.9%). Of them, 20 were identified as enteric adenovirus infection (adenovirus type 41, Ad41). Thirty-three out of 230 HAD samples were positive (positive rate was 14.3%). Of them, 13 were characterized as enteric adenovirus infection (one was Ad40 and others were Ad41). Ad41 in this study could be divided into two genotypes by phylogenetic tree analysis. Non-enteric adenoviruses were identified in 43 specimens (43/76, 56.6%) including 5 of serotype 1, 8 of serotype 2, 15 of serotype 3, 10 of serotype 7, 1 of serotype 12, and 4 of serotype 31. In this study, the positive rate of adenovirus between CAD children and HAD children did not differ (χ(2) = 0.03, P > 0.05), while the positive rate of enteric adenovirus was high in CAD children. CONCLUSION: Adenovirus infection was the main cause of diarrhea in hospitalized children. In this study, the positive rate of adenovirus was similar between children with CAD and with HAD. Enteric adenovirus (adenovirus group F) was the most common adenovirus serotype detected in 2010 in Beijing, and Ad41 was the dominant type.

[Investigation of a novel VP4 subgenotype of rotavirus in children with diarrhea in Beijing during 2009-2010].

P[8]b is a newly discovered sub-genotype for VP4 gene of group A human rotaviruses (HRV) worldwide. This study was to develop an effective method to identify P[8]a, P[8]b, P[4] and P[6] (sub) genotypes of VP4 genes of HRV and to investigate the prevalence of P[8]b sub-genotype and its G/P combinations of HRV in outpatient and inpatient children with diarrhea in Children's Hospital affiliated to Capital Institute of Pediatrics from 2009 to 2010. By analyzing the collected nucleotide sequences of VP4 gene for all known P genotypes of HRV including P[8]b subtype from GenBank and using softwares of DNAS-tar and MegAlign to align and analyze multiple sequences, probes for P[8]a, P[8]b, P[4] and P[6] (sub) genotypes in the corresponding regions which are highly divergent among genes from different genotypes and conserved within genes of VP4s in same genotypes were designed. Then four sets of primers for PCR amplified DIG labeled probes were designed and corresponding DIG-labeled specific P genotype probes were synthesized with PCR by using VP8* genes of Beijing field HRV strains representing P-genotypes P[8]a, P[8]b, P[4] and P[6], respectively, as templates. Dot-blot hybridization was developed based on cDNA of VP4 genes. The dot-blot hybridization assay for P genotyping was reliable which was confirmed by sequencing of RT-PCR products of VP4 genes amplified from corresponding clinical samples. P genotyping for VP4 genes from 88 HRV positive specimens from the Outpatient Department (55%, 88/160) and 79 HRV positive specimens from the hospitalized (70.5%, 79/112) children with diarrhea indicated that P[8] a subtype was still the most prevalent sub-genotype, which was 96.6% (85/88) and 62.0% (49/79) respectively. The positive rate for P[8]b subtypes in hospitalized children with HRV diarrhea was higher (27.9%, 22/79) than that of in outpatient (2.3%, 2/88) HRV infected children. HRV with P[4] genotype was only found in one of the hospitalized children (1.3%, 1/79), and HRV with P[6] genotype was not detected from specimens either from outpatient or inpatient. G9P[8]b was the predominant combination among the P[8]b subtype of HRV positive specimens in this study. The results in this study indicated that G9P[8]b HRV circulated in children with diarrhea in Beijing.

Human bocavirus infections are common in Beijing population indicated by sero-antibody prevalence analysis.

BACKGROUND: Human bocavirus (HBoV) is a newly identified human parvovirus that was originally detected in the respiratory secretions of children with respiratory infections. This study aimed to learn about the importance of HBoV infections by revealing the prevalence of serum antibodies against HBoV in Beijing population. METHODS: Two batches of serum specimens collected in different periods were tested by Western blotting for specific IgG against HBoV using recombinant VP2 as antigen. RESULTS: Out of 677 serum specimens collected during April 1996 to March 1997, 400 (59.1%) were positive and antibody positive rate for another batch of 141 serum specimens collected in August, 2005 from adults aged from 20 years to over 60 years was 78.7% (111/141). Comparison of the sero-prevalence profiles for serum specimens collected during 1996 - 1997 to those collected in 2005 indicated that the antibody positive rate for specimens collected in 2005 was higher than that of the corresponding age groups collected during 1996 - 1997. CONCLUSIONS: The data suggest that HBoV has been circulating in Beijing population for at least over 10 years, and most of children had been exposed to HBoV by age of 7 years. Higher HBoV antibody positive rate shown in the serum specimens collected in 2005 suggested that infections by HBoV have been increased in Beijing population in recent years.

[Analysis on VP7 and VP4 genes of human rotavirus G9 identified from children with diarrhea in Beijing, from 2007 to 2008].

OBJECTIVE: To characterize the outer capsid protein VP7 and VP4 encoding genes of human rotavirus G9 strains detected in Beijing, from 2007 to 2008. METHODS: Full length of VP7 genes of G9 rotaviruses from 12 fecal specimens previously detected by dot-blot hybridization assay were amplified by RT-PCR and sequenced after being cloned into T vector. The sequences of these VP7s were compared to VP7 genes of rotavirus G9 prototype strains and recently circulating strains around the world. VP4 genes of these 12 G9 strains were amplified by nested-PCR for P genotyping. RESULTS: Sequence analysis for the full length of VP7 genes from these 12 specimens confirmed that they were G9 rotaviruses. P genotyping for VP4 genes revealed that both P[8]G9 and P[6]G9 were circulating in Beijing in the last 2 years. Sequence and phylogenetic analysis demonstrated that VP7 genes of G9 strains from Beijing in this study were clustered in the lineage III which resembled the G9 strains circulating in other places around the world, indicated by high identities of nucleotide and deduced amino acid sequences and were distant with the first reported G9 strain T203 identified in China in 1994. It was found that there were some consistent amino acid substitutes at the corresponding positions among VP7s from these 12 specimens and from Xinjiang and Wuhan, both in G9P[8] and G9P[6] strains. CONCLUSION: The rotavirus G9 strains both in combination of G9P[8] and G9P[6] were circulating in Beijing in the past years. It seemed that rotavirus G9 should be included in the list of surveillance for rotavirus in China.

[Seroprevalence of antibody against human bocavirus in Beijing, China].

OBJECTIVE: To find out the importance of human bocavirus (HBoV) as an infectious agent for population in Beijing, China, seroprevalence study was conducted by using expressed recombinant major capsid VP2 protein as an antigen. METHODS: Serum specimens collected from infants and children who visited the Children's Hospital Affiliated to the Capital Institute of Pediatrics for health check up and adults visited the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for investigation. The major capsid protein VP2 from HBoV was expressed in E. coli strain BL21 (DE3) with the transformed PET30b vector inserted with full-length VP2 gene of HBoV and the specific antigenicity of this expressed protein was validated by previous study. Western blot was used to detect specific IgG antibody against HBoV in collected serum specimens diluted to 1:200. Mock expressed protein was E. coli cells strain BL21 (DE3) with the transformed PET30b vector without insert. Anti-His monoclonal antibody and rabbit anti-HBoV VP2 polypeptides hyper-immune serum were used as positive control for antibody detection. RESULTS: Out of 677 serum specimens tested, 400 (59.1%) were positive by Western blot. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were lower in the age groups of 1 and 2 months (41.4% and 31.3%, respectively) and were higher in the following ages from 6 months to 7 years (from 45.6% to 69.7%). The antibody positive rates were at a relatively constant level (about 70%) in the age groups from 7 years to 40 years and became lower (61.8% - 62.8%) in groups of age over 50 years. CONCLUSION: The high seroprevalence against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in Beijing, China as early as in 1996 and most of children had been exposed to HBoV by the age of 7 years. Infants under the age of 6 months were susceptible to infection with this virus.