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.

Genetic Evolution and Variation of Human Adenovirus Serotype 31 Epidemic Strains in Beijing, China, during 2010-2022.

Human adenovirus serotype 31 (HAdV-31) is closely associated with gastroenteritis in children and can cause fatal systemic disseminated diseases in immunocompromised patients. The lack of genomic data for HAdV-31, especially in China, will greatly limit research on its prevention and control. Sequencing and bioinformatics analyses were performed for HAdV-31 strains from diarrheal children in Beijing, China, during 2010-2022. Three capsid protein genes (hexon, penton, and fiber) were obtained in 37 cases, including one in which the whole genome was sequenced. HAdV-31 strains clustered into three distinct clades (I-III) in a phylogenetic tree constructed based on concatenated genes and the whole genome; the endemic strains only gathered into clade II, and most of the reference strains clustered into clade I. Compared with penton and hexon, fiber had a faster evolutionary rate (1.32 × 10-4 substitutions/site/year), an earlier divergence time (1697), lower homology (98.32-100% at the amino acid level), and greater genetic variation (0.0032). Four out of the six predicted positive selection pressure codons were also in the knob of fiber. These results reveal the molecular evolution characteristics and variations of HAdV-31 in Beijing, and fiber may be one of the main evolution driving forces.

Comparison of Pre-existing Anti-AAV8 Total Antibody Screening and Confirmatory Assays with a Cell-Based Neutralizing Assay in Normal Human Serum.

Pre-existing adeno-associated viruses (AAV) neutralizing antibodies (NAb) can prevent AAV vectors from transducing target tissues. The immune responses can include binding/total antibodies (TAb) and neutralizing antibodies (NAb). This study is aimed at comparing total antibody assay (TAb) and cell-based NAb assay against AAV8 to help inform the best assay format for patient exclusion criteria. We developed a chemiluminescence-based enzyme-linked immunosorbent assay to analyze AAV8 TAb in human serum. The specificity of AAV8 TAb was determined using a confirmatory assay. A COS-7-based assay was used to analyze anti-AAV8 NAbs. The TAb screening cut point factor was determined to be 2.65, and the confirmatory cut point (CCP) was 57.1%. The prevalence of AAV8 TAb in 84 normal subjects was 40%, of which 24% were NAb positive and 16% were NAb negative. All NAb-positive subjects were confirmed to be TAb-positive and also passed the CCP-positive criteria. All 16 NAb-negative subjects did not pass the CCP criterion for the positive specificity test. There was a high concordance between AAV8 TAb confirmatory assay and NAb assay. The confirmatory assay improved the specificity of the TAb screening test and confirmed neutralizing activity. We proposed a tiered assay approach, in which an anti-AAV8 screening assay should be followed by a confirmatory assay during pre-enrollment for patient exclusions for AAV8 gene therapy. This approach can be used in lieu of developing a NAb assay and can be also implemented as a companion diagnostic assay for post-marketing seroreactivity assessments due to ease of development and use.

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.

Genetic diversity and molecular evolution of human adenovirus serotype 41 strains circulating in Beijing, China, during 2010-2019.

Human adenovirus serotype 41 (HAdV-F41) is an important pathogen that causes diarrhea in children. However, the data on its molecular genetic characteristics and evolutionary history are still neither comprehensive nor sufficient. Four capsid protein genes from 58 HAdV-F41-positive specimens taken from diarrheal children in Beijing during 2010-2019 were amplified and analyzed. Variant amino acids in the hexon gene (18 sites) and short fiber gene (4 sites) clustered these strains into two clades and four subclades. The deletion of 15 amino acids found in the gene seemed to have little effect on the genomic strain cluster same as to penton gene. The HAdV-F41 strains had high diversity, as assessed from the intraspecific recombination of hexon, short fiber and long fiber. The molecular evolutionary rate of HAdV-F41's concatenated genes was 4.07 × 10-5 substitutions/site/year, and it diverged from the most recent common ancestor in 1720. Apart from in the penton gene, positive selection codons were predicted in the other three genes, which may play a synergistic role in the evolution of HAdV-F41. These results provide new insights for understanding the characteristics of infectivity and developing vectors and vaccine vehicles for HAdV-F41.

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.

Keeping all secondary structures of the non-coding region in the circular genome of human bocavirus 2 is important for DNA replication and virus assembly, as revealed by three hetero-recombinant genomic clones.

The episomal structures of all human bocavirus (HBoV) genotypes have been deciphered, including the circular genome of HBoV2 (HBoV2-C1). To discern the role of the circular HBoV2 genome, three distinct linearized HBoV2-C1 genomes were cloned into pBlueScript SKII(+) to obtain pBlueScript HBoV2 5043-5042 (retaining all secondary structures), pBlueScript-HBoV2 5075-5074 (retaining hairpin number 2 and the 5' terminal structure), and pBlueScript-HBoV2 5220-5219 (retaining only the 5' terminal structure at the 5' -genome end). The recombinant plasmids were separately transfected HEK293 cells, revealing that more HBoV2 DNA had accumulated in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells at 72 h post-transfection, as determined by real-time PCR. However, more mRNA was transcribed by pBlueScript-HBoV2 5075-5074 than by the other constructs, as determined by dot-blot hybridization and RNAscope. No significant differences in NS1-70 protein expression were observed among the three HBoV2 genomic clones. However, electron microscopy showed that HBoV2 virus particles were only present in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells. By using three hetero-recombinant HBoV2 genomic clones in HEK293 transfected cells, only the genome with intact secondary structures produced virus particles, suggesting that retaining these structures in a circular genome is important for HBoV2 DNA replication and virus assembly.

Risk of acute gastroenteritis associated with human bocavirus infection in children: A systematic review and meta-analysis.

Human bocaviruses (HBoVs), which were first identified in 2005 and are composed of genotypes 1-4, have been increasingly detected worldwide in pediatric patients with acute gastroenteritis. To investigate if HBoV infection is a risk factor of acute gastroenteritis in children younger than 5 years old, we searched PubMed, Embase (via Ovid), the Chinese Biomedical Literature Database (CBM), and the Cochrane Library for studies assessing the prevalence of HBoVs in individuals from Oct 25, 2005 to Oct 31, 2016. We included studies using PCR-based diagnostics for HBoVs from stool specimens of patients with or without acute gastroenteritis that carried out research for over 1 year on pediatric patients aged younger than 5 years old. The primary outcome was the HBoV prevalence among all cases with acute gastroenteritis. Pooled estimates of the HBoV prevalence were then generated by fitting linear mixed effect meta-regression models. Of the 36 studies included, the pooled HBoV prevalence in 20,591 patients with acute gastroenteritis was 6.90% (95% confidence interval (95% CI): 5.80-8.10%). In the ten studies with a control group, HBoVs were detected in 12.40% of the 3,620 cases with acute gastroenteritis and in 12.22% of the 2,030 control children (odds ratio (OR): 1.44; 95% CI: 0.95-2.19, p = 0.09 between case and control groups). HBoV1 and HBoV2 were detected in 3.49% and 8.59% of acute gastroenteritis cases, respectively, and in 2.22% and 5.09% of control children, respectively (OR: 1.40; 95% CI: 0.61-3.25; p = 0.43 and OR: 1.68; 95% CI: 1.21-2.32; p = 0.002, respectively). Current evidence suggests that the overall HBoV prevalence in children younger than 5 years old is not significantly different between groups with or without acute gastroenteritis. However, when HBoV1 was excluded, the HBoV2 prevalence was significantly different between these two groups, which may imply that HBoV2 is a risk factor of acute gastroenteritis in children younger than 5 years old.

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples.

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

High prevalence of GII norovirus in hospitalized children with acute diarrhea, in Beijing.

This study was addressed to the relationship between norovirus and acute diarrhea in hospitalized children, including hospital-acquired infection (HAI) and community-acquired infection (CAI) in a children's hospital in Beijing. RT-PCR was used to detect norovirus in stool specimen, followed by sequence analysis for PCR products. From 2010 to 2013, a total of 1248 specimens, including 661 from the HAI group and 587 from the CAI group were tested for norovirus. Norovirus were detected in 380 of 1248 (30.4%) diarrheal specimens. The positive rate for norovirus detection was higher in children within HAI group than CAI group (35.3%, 232/661 vs. 25.6%, 148/587), and the difference was significant (X2 = 14.35, P<0.05). For age distribution, the highest positivity rates of norovirus were in age of 0-5 months for HAI group and 12-23 months for CAI group. In the study, 262 amplicons of the VP1 region from norovirus-positive specimens were sequenced, which showed GII.3 and GII.4 norovirus were the most common genotypes detected in 50.0% (n = 131) and 48.9% (n = 128) of the positive specimens, respectively. Regarding the wards distribution, GII.3 norovirus was mainly detected in ward for neonatal diseases (36/85 in HAI group; 19/46 in CAI group), GII.4 norovirus was mainly detected in ward for respiratory and digestive diseases (21/85 in HAI group; 15/33 in CAI group). CONCLUSION: The data elaborated the importance of norovirus in hospital associated infectious diarrhea. The prevalence of norovirus is higher from HAI group than CAI group, and the norovirus from the patients in CAI group could be the source of infection in HAI group.

Prevalence and Phylogenetic Analysis of Human Bocaviruses 1-4 in Pediatric Patients with Various Infectious Diseases.

OBJECTIVES: Viral infections caused by human bocaviruses 1-4 (HBoV1-4) are more complicated than previously believed. A retrospective, large-scale study was undertaken to explore the prevalence of HBoV1-4 in pediatric patients with various infectious diseases and delineate their phylogenetic characteristics. METHODS: Clinical samples from four specimen types, including 4,941 respiratory, 2,239 cerebrospinal fluid (CSF), 2,619 serum, and 1,121 fecal specimens, collected from pediatric patients with various infectious diseases were screened for HBoV1-4. A 690-nt fragment in each specimen was then amplified and sequenced for phylogenetic analysis. Clinical characteristics of HBoV-positive patients with different specimen types available were evaluated. RESULTS: Approximately 1.2% of patients were confirmed as HBoV-positive, with the highest positive rate in patients with gastrointestinal infection (2.2%), followed by respiratory (1.65%), central nervous system (0.8%), and hematological infections (0.2%). A single genetic lineage of HBoV1 circulated among children over the 8-year period, while a new cluster of HBoV2, via intra-genotype recombination between HBoV2A and HBoV2B, was prevalent. Some patients had HBoV1-positive respiratory and serum specimens or fecal specimens. Several cases became HBoV1-positive following the appearance of respiratory infection, while several cases were positive for HBoV2 only in CSF and serum specimens, rather than respiratory specimens. CONCLUSIONS: A single genetic lineage of HBoV1 is speculated as a viral pathogen of respiratory infection and causes both comorbid infection and acute gastroenteritis. Additionally, a new cluster of HBoV2 is prevalent in China, which may infect the host through sites other than the respiratory tract.

[Genomic Characterization of an Unusual Human G3P[3] Rotavirus with Multiple Cross-species Reassortment].

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.

[Rotavirus G9-â ¥ Re-emerging and Circulating Predominantly in Children with Diarrhea in Beijing].

The first reported group A rotavirus(RVA)G9 strain T203 in mainland China detected from an infant in Beijing in 1994 clustered within VP7 lineage Ⅵ(G9-Ⅵ),whereas the common RVA G9 belong to VP7 lineage Ⅲ(G9-Ⅲ) worldwide. Interestingly, since it was first reported in 1994 there was no G9-Ⅵ circulating in Beijing, until an unexpected G9-Ⅵ strain was identified by sequencing in 2010 RVA surveillance. This present study was to develop a convenient and effective dot-blot hybridization method for differentiating G9-Ⅲ and G9-Ⅵ rotaviruses, to investigate the re-emerging circulating distribution of G9-Ⅵ RVAs in outpatient children with diarrhea from 2011 to 2012in Beijing. By multiple-sequence aligning and analyzing the collected VP7 gene nucleotide sequences of G9 RVAs worldwide from GenBank database using Clustal W software, a region within VP7 gene which is highly divergent between G9-Ⅲ and G9-Ⅵ rotaviruses and conserved within themselves was selected as probe. One pair of common primers at both side of this probe region was designed, and used for synthesizing DIG labeled G9-Ⅲ and G9-Ⅵ probes with PCR, respectively. Subsequently, RVA G and P genotypes were identified as previously described. Then G9-Ⅲ and G9-Ⅵ were further differentiated from G9 RVAs using dot-blot hybridization method established in this study. It was showed that G9 was the most prevalent genotype(43.5%),followed by G3(30.5%)、G1(12.2%)and G2(11.5%),and no G4 genotype was detected.Interestingly,G9-Ⅵ was the most predominant(96.5%) type, while only 3.5% was G9-Ⅲ among these G9 rotaviruses.Phylogenetically,G9-Ⅵ rotaviruses in this study clustered closely with human G9-Ⅵ rotaviruses which were more recently re-emerging in several countries including China around the world as well as porcine G9-Ⅵ rotavirus strain F7P4 in Canada, whereas they clustered distantly to worldwide common G9-Ⅲ strains.G9-Ⅵ rotaviruses were re-emerging in the world, whether it gained stronger spreading ability or virulence than ever common G9-Ⅲ during evolution with porcine G9-Ⅵ rotaviruses need further analysis.

Development of a Generic Anti-PEG Antibody Assay Using BioScale's Acoustic Membrane MicroParticle Technology.

Immunogenicity testing for PEGylated biotherapeutics should include methods to detect both anti-protein and anti-PEG antibodies (anti-PEG). Although some methods have been published for the detection of anti-PEG antibodies, the information is incomplete and, in some cases, reagents used (such as Tween-20) are known to interfere with detection. This rapid communication describes the use of BioScale's Acoustic Membrane MicroParticle (AMMP®) technology using the ViBE® Workstation to measure anti-PEG antibodies in human serum samples. Briefly, a sample spiked with monoclonal human IgG anti-PEG antibody is diluted in buffer and incubated with paramagnetic beads coated with linear chain mPEG to capture anti-PEG antibodies. The complex is then captured on an acoustic membrane coated with Protein A. The change in mass on the membrane caused by the binding of the complex to the membrane results in a signal proportional to the mass of anti-PEG antibodies. The data indicate that an assay with a sensitivity of less than 1000 ng/mL for IgG is achievable. This level of sensitivity is better than current published reports on IgG anti-PEG antibody detection.

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.

Prevalence analysis of different human bocavirus genotypes in pediatric patients revealed intra-genotype recombination.

BACKGROUND: Human bocavirus (HBoV) genotypes 1-4 have been detected worldwide in respiratory samples and stool samples, and are increasingly associated with respiratory and intestinal infections of previously unknown etiology in young children. Several studies revealed evidence of extensive recombination among HBoV genotypes at the NP1 and VP1 gene boundary region. This study explored the prevalence of HBoV genotypes in pediatric patients in Beijing, and studied their phylogeny. METHODS: A total of 4941 respiratory specimens and 1121 fecal specimens were collected from pediatric patients with respiratory infections from January 2006 to December 2013, or with acute diarrhea from October 2010 to December 2012. Conventional PCR was used to detect HBoV1-4 within these samples. Gene fragments at the NP1 and VP1 gene boundary were amplified from HBoV-positive specimens, sequenced, and their phylogenetic inferences constructed using MEGA 6.0 software. Recombination events were identified with SimPlot software. RESULTS: Human bocavirus 1, 2, and 3 were detected in 9 (0.80%), 15 (1.33%), and 1 (0.08%) of 1121 stool samples, respectively. However, only HBoV1 (82, 1.65%) was detected in respiratory specimens. Phylogenetic analysis of gene fragments at the HBoV NP1 and VP1 gene boundary indicated that HBoV1 sequences obtained from fecal or respiratory specimens across 8years were highly conserved (99-100%), while 15 HBoV2 sequences collected across 2years in Beijing were more diverse with up to 4.40% variation. Of the 15 HBoV2 sequences, 14 clustered into a new lineage divergent from other HBoV2 sequences in GenBank. Five HBoV2 genomic sequences were analyzed for recombination, revealing intra-genotype recombination between HBoV2A and HBoV2B. CONCLUSIONS: More HBoV1 were detected in children with respiratory tract diseases, and HBoV2 in patients with acute diarrhea. Phylogenetic analysis revealed a new cluster of HBoV2 was prevalent in China, which may be the result of intra-genotype recombination between HBoV2A and HBoV2B.

Adenoviruses associated with acute diarrhea in children in Beijing, China.

Adenoviruses have been recognized as important causal pathogens of community-acquired diarrhea (CAD) among children, but their role in hospital-acquired diarrhea (HAD) is not well-understood. Hospitalized children with acute diarrhea and children who visited the outpatient department due to diarrhea were investigated from 2011 to 2012. Adenovirus was detected in stool specimens by PCR and further characterized by sequencing and phylogenetic analysis. SPSS software (version 19.0) was used for statistical analyses. A total of 2233 diarrheal children were enrolled in this study; this sample was comprised of 1371 hospitalized children, including 885 with CAD (IP-CAD) and 486 with HAD, and 862 outpatients with CAD (OP-CAD). Among these 2,233 patients, adenovirus was detected in 219 cases (9.8%). The positive rates for adenovirus were significantly different between the IP-CAD (9.3%), HAD (13.8%) and OP-CAD (8.1%) cases (X²â€Š= 11.76, p = 0.003). The positive rate of adenovirus was lower in infants under six months of age compared to the positive rates in the other age groups. Of the 219 of adenovirus positive patients, 91 (41.6%) were identified as having serotype 41. Although enteric adenovirus (group F) was the most frequently detected adenovirus among children with either CAD or HAD, the role of non-enteric adenoviruses, especially the adenovirus 31 type (19.7%), cannot be ignored in diarrheal children.

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.

A generic template for automated bioanalytical ligand-binding assays using modular robotic scripts in support of discovery biotherapeutic programs.

BACKGROUND: Sample dilution and reagent pipetting are time-consuming steps in ligand-binding assays (LBAs). Traditional automation-assisted LBAs use assay-specific scripts that require labor-intensive script writing and user training. RESULTS: Five major script modules were developed on Tecan Freedom EVO liquid handling software to facilitate the automated sample preparation and LBA procedure: sample dilution, sample minimum required dilution, standard/QC minimum required dilution, standard/QC/sample addition, and reagent addition. The modular design of automation scripts allowed the users to assemble an automated assay with minimal script modification. The application of the template was demonstrated in three LBAs to support discovery biotherapeutic programs. CONCLUSION: The results demonstrated that the modular scripts provided the flexibility in adapting to various LBA formats and the significant time saving in script writing and scientist training. Data generated by the automated process were comparable to those by manual process while the bioanalytical productivity was significantly improved using the modular robotic scripts.

Detection of a bocavirus circular genome in fecal specimens from children with acute diarrhea in Beijing, China.

To determine if human bocavirus 2 (HBoV2) has a circular genome similar to the head-to-tail sequence of HBoV1 and the episomal form of HBoV3, 15 HBoV2 positive samples identified from 553 stool specimens from children with acute diarrhea were tested for a head-to-tail sequence using TaqMan-based real-time PCR. A circular genome with a head-to-tail sequence was identified in one (BJQ435) out of 15 samples tested by nested PCR. The complete circular genome of HBoV2-C1 (BJQ435) was 5307 nt in length and was flanked with a 520 nt-long terminal non-coding region (NCR). The secondary structure of HBoV2 -C1 had some differences compared to HBoV3-E1 (JN086998). Our study indicates that the HBoV genome exists in the form of a head-to-tail monomer and provides more information for understanding the HBoV replication mechanism.