Identification and genetic characterization of a recently identified enterovirus C116 in China.

Enterovirus C116 (EV-C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV-C116 in clinical samples worldwide, there is currently limited information available. In this study, two EV-C-positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta-transcriptome sequencing. Based on the prototypes of several EV-Cs, two lineages were observed. Lineage 1 included many types that could not cause EV-like cytopathic effect in cell culture. Three genogroups of EV-C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV-C116 still diffuses worldwide. Obvious inter-type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV-Cs biodiversity. The EV-C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV-like cytopathic effects were not observed. HEp-2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV-C116 growth. These results challenge the traditional recognition of the uncultured nature of EV-C116 strains and explain the difficulty of clinical detection.

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.

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.

Changes in endemic patterns of respiratory syncytial virus infection in pediatric patients under the pressure of nonpharmaceutical interventions for COVID-19 in Beijing, China.

A series of nonpharmaceutical interventions (NPIs) was launched in Beijing, China, on January 24, 2020, to control coronavirus disease 2019. To reveal the roles of NPIs on the respiratory syncytial virus (RSV), respiratory specimens collected from children with acute respiratory tract infection between July 2017 and Dec 2021 in Beijing were screened by capillary electrophoresis-based multiplex PCR (CEMP) assay. Specimens positive for RSV were subjected to a polymerase chain reaction (PCR) and genotyped by G gene sequencing and phylogenetic analysis using iqtree v1.6.12. The parallel and fixed (paraFix) mutations were analyzed with the R package sitePath. Clinical data were compared using SPSS 22.0 software. Before NPIs launched, each RSV endemic season started from October/November to February/March of the next year in Beijing. After that, the RSV positive rate abruptly dropped from 31.93% in January to 4.39% in February 2020; then, a dormant state with RSV positive rates ≤1% from March to September, a nearly dormant state in October (2.85%) and November (2.98%) and a delayed endemic season in 2020, and abnormal RSV positive rates remaining at approximately 10% in summer until September 2021 were detected. Finally, an endemic RSV season returned in October 2021. There was a game between Subtypes A and B, and RSV-A replaced RSV-B in July 2021 to become the dominant subtype. Six RSV-A and eight RSV-B paraFix mutations were identified on G. The percentage of severe pneumonia patients decreased to 40.51% after NPIs launched. NPIs launched in Beijing seriously interfered with the endemic season of RSV.

Human bocavirus 1 is a genuine pathogen for acute respiratory tract infection in pediatric patients determined by nucleic acid, antigen, and serology tests.

Background: Human bocavirus 1 (HBoV1), first discovered in 2005, was positive in symptomatic and healthy children and co-detected with other respiratory viruses. It is a long journey to decisively demonstrate the unique viral pathogenic function of acute respiratory tract infection (ARTI) in pediatric patients. Methods: Respiratory specimens collected from pediatric patients with ARTI from January 2017 to December 2021 were screened by a capillary electrophoresis-based multiplex PCR (CEMP) assay, then genotyped by PCR and sequencing for HBoV1. For the antigen test, a part of HBoV1 DNA positive nasopharyngeal aspirates (NPAs) was used as an antigen, while a rabbit anti-HBoV1 DR2 specific to HBoV1 was used as an antibody in the indirect-immunofluorescence assay (IFA). Finally, the levels of IgG specific to HBoV1 in acute and convalescent sera selected retrospectively from only HBoV1 DNA-positive patients were evaluated by IFA. Results: Among 9,899 specimens, 681 were positive for HBoV1 DNA (6.88%, 681/9899), which included 336 positives only for HBoV1 (49.34%, 336/681) and 345 (50.66%, 345/681) positives also for other pathogens. In the antigen test, there were 37 among 47 NPAs determined as HBoV1 antigen-positive (78.72%, 37/47), including 18 (48.65%, 18/37) positives solely for HBoV1 DNA. Among 4 pediatric patients with both acute and convalescent sera, there was one positive for HBoV1 antigen (D8873) and 2 lack the antigen results (D1474 and D10792), which showed seroconversion with a ≥ 4-fold increase in IgG levels. Conclusions: The combination results of nucleic acid, antigen, and serology tests answered that HBoV1 is a genuine pathogen for ARTI in pediatric patients.

Surges of hospital-based rhinovirus infection during the 2020 coronavirus disease-19 (COVID-19) pandemic in Beijing, China.

BACKGROUND: A series of public health preventive measures has been widely implemented in Beijing to control the coronavirus disease-19 (COVID-19) pandemic since January 2020. An evaluation of the effects of these preventive measures on the spread of other respiratory viruses is necessary. METHODS: Respiratory specimens collected from children with acute respiratory infections were tested by NxTAG™ respiratory pathogen panel assays during January 2017 and December 2020. Specimens characterized as rhinoviruses (RVs) were sequenced to identify the RV species and types. Then, the epidemiology results of respiratory pathogens in 2020 were compared with those from 2017 to 2019 using SPSS statistics 22.0. RESULTS: The positive rates of adenovirus (ADV), influenza virus (flu), RVs, and respiratory syncytial virus (RSV) dropped abruptly by 86.31%, 94.67%, 94.59%, and 92.17%, respectively, from February to May 2020, compared with the average level in the same period during 2017-2019. Positive rates of RVs then steeply increased from June 2020 (13.77%), to an apex (37.25%) in August 2020, significantly higher than the average rates (22.51%) in August 2017-2019 (P = 0.005). The increase, especially in group ≥ 3 years, was accompanied by the reopening of schools and kindergartens after the 23rd and 24th week of 2020 in Beijing. CONCLUSIONS: Whereas the abrupt drop in viral pathogen positive rates from February to May 2020 revealed the remarkable effects of the COVID-19 preventive measures, the sharp increase in positive rates of RVs from the 23rd week of 2020 might be explained by the reopening of schools and kindergartens in Beijing.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection.

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 ( P < 0.05), respectively. In comparison with those of qPCR, the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22% and 80.00%, respectively; the specificity was 100.00%; and the Kappa values were 0.764 and 0.878 ( P < 0. 05), respectively. Thus, rapid and specific detection of EBV and CMV is possible using ICR-RAA assays.

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.

Protective effect of an alpha 7 nicotinic acetylcholine receptor agonist against enterovirus 71 infection in neuronal cells.

Enterovirus 71, as one of the dominant pathogens associated with severe hand, foot, and mouth disease, has been well reported to trigger severe neurological symptoms among young children over the last decade, particularly among children in the Asia-Pacific region. To date, no effective antiviral agent has been developed for the treatment of severe enterovirus 71 infection. PNU-282987, a selective alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist, has been reported to have a neuroprotective effect by participating in inflammatory regulation in previous studies. Therefore, in the present study, we aimed to assess the cell-protective effect of PNU-282987 against enterovirus 71 infection in neuronal cells, and to discuss potential mechanisms underlying this cell-protective effect in order to elucidate the potential impact of such agonists in the treatment of neurotropic viral infection. We observed that treatment with PNU-282987 improved cell viability and inhibited viral replication in enterovirus 71-infected SH-SY5Y cells. Further investigation revealed that inhibition of enterovirus 71 production by PNU-282987 is likely associated with events of RNA replication, and that increased levels of INF mRNA and its downstream antiviral proteins stimulated by the JAK-STAT2 pathway may contribute to the antiviral effect of PNU-282987. Moreover, our findings suggest that both the antiviral and anti-inflammatory effects of PNU-282987 may contribute to the neural protective effect of the drug in enterovirus 71-infected cells. Taken together, the results suggest that selective α7nAChR agonists may represent viable candidates for future therapeutic treatment of severe enterovirus 71 infection, and for other cases of neurotropic viral infection.

Association of Different Human Rhinovirus Species with Asthma in Children: A Preliminary Study.

BACKGROUND: Human rhinoviruses (HRVs) are divided into three genetic species: HRV-A, HRV-B, and HRV-C. The association of different HRV species with asthma in children in China has not yet been evaluated. This preliminary study aimed to assess the associations between different HRV species, particularly HRV-C, and asthma in young children in China. METHODS: A total of 702 nasopharyngeal aspirates were obtained from 155 children with asthma (asthma group), 461 children with acute respiratory infection (ARI) without asthma (nonasthma ARI group), and 86 children from the control group. Semi-nested polymerase chain reaction (PCR) was used to detect HRVs, and PCR products were sequenced for species identification. Epidemiological characteristics of HRV-positive cases were analyzed. RESULTS: HRVs were the most common pathogen (15.4%; 108/702) in the patients in this study. The prevalence of HRV was significantly different (F = 20.633, P = 0.000) between the asthma (25.8%) and nonasthma ARI groups (11.1%). Phylogenetic analysis indicated that in the 108 cases positive for HRVs, 41 were identified as HRV-A, 8 as HRV-B, and 56 as HRV-C. Comparing the asthma with the nonasthma ARI group, Spearman's rank correlation analysis revealed an association between HRV-A (P < 0.05) and C (P < 0.01) and asthma, confirmed by regression analysis, with odds ratios of 2.2 (HRV-A) and 4.2 (HRV-C). CONCLUSIONS: Our data revealed a high prevalence of HRVs in children in China, regardless of clinical status. HRV-C was the dominant species and may be one of the key factors in the association of HRVs with asthma.

Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

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.

Parainfluenza Virus Types 1, 2, and 3 in Pediatric Patients with Acute Respiratory Infections in Beijing During 2004 to 2012.

BACKGROUND: Although human parainfluenza virus (HPIV) has been determined as an important viral cause of acute respiratory infections (ARIs) in infants and young children, data on long-term investigation are still lacking to disclose the infection pattern of HPIV in China. METHODS: Nasopharyngeal aspirates were collected from 25,773 hospitalized pediatric patients with ARIs from January 2004 through December 2012 for respiratory virus screen by direct immuno-fluorescence assay. RESULTS: Out of these specimens, 1675 (6.50%, 1675/25,773) showed HPIV positive, including 261 (1.01%, 261/25,773) for HPIV1, 28 (0.11%, 28/25,773) for HPIV2, and 1388 (5.39%, 1388/25,773) for HPIV3, 2 of the samples were positive for both HPIV1 and HPIV3, and 36 were co-detected with other viruses. The positive rates of HPIVs were higher in those younger than 3 years old. HPIV3 was detected from all age groups, predominantly from patients under 3 years of age, and the highest frequency was found in those 6 months to 1-year old (352/4077, 8.63%). HPIV3 was the dominant type in each of the years detected between May and July. HPIV1 showed a peak in every odd year, mainly in August or September. HPIV was detected most frequently from patients with upper respiratory infection (12.49%, 157/1257), followed by bronchitis (11.13%, 176/2479), asthma (9.31%, 43/462), bronchiolitis (5.91%, 150/2536), pneumonia (6.06%, 1034/17,068), and those with underlying diseases (1.0%, 15/1506). HPIV3 is the dominant type in these six disease groups referred above, especially in the asthma group. CONCLUSIONS: HPIV is one of the important viral causes of ARIs in infants and young children in Beijing based on the data from the hospitalized children covering a 9-year term. HPIV3 is the predominant type in all these years and in most of the disease groups. HPIVs with different types show different seasonality.

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.

[Characterization of the whole genome from a human parechovirus type 3 detected from the serum of a child with sepsis in Beijing, China].

Human parechovirus type 3 (HPeV3) is an important pathogen of severe sepsis. HPeV3 is a non- enveloped, single-stranded, positive-sense RNA virus with a linear and continuous genomic RNA. The complete genome of a HPeV3 (BJ-C3174) strain was analyzed from the serum specimen from a child with sepsis hospitalized in Beijing, China, in 2012. The whole genome of BJ-C3174 was 7329 nucleotides (nt) in length excluding a poly (A) tail. One large open reading frame (ORF) of 6531 nt encoding a putative polyprotein precursor of 2177 amino acids (aa) was flanked by a 5' untranslated region (UTR) of 709 nt and 3' UTR of 91 nt. Phylogenetic analysis showed that BJ-C3174 belonged to HPeV3 and was closest to the HPeV3 strain BONN-2 from Germany. Compared with HPeV1-8 reference strains, BJ-C3174 shared the highest similarities with BONN-2 in full length and in each of the gene segments of the genome. The nucleotide and predicted amino acid identities of the whole genome between BJ-C3174 and BONN-2 were 99.3% and 99.8%, respectively, which were higher than those compared with HPeV3 prototype. Recom- bination of the gene segment with other HPeVs types was not identified.

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.

[Detecting human respiratory syncytial virus in respiratory samples collected from children with acute respiratory infections by reverse transcription-loop mediated isothermal amplification].

OBJECTIVE: To establish a rapid, sensitive and specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detecting human respiratory syncytial virus (RSV) in respiratory samples collected from children with acute respiratory infections. METHOD: According to the conserved matrix gene sequences of respiratory syncytial virus subtypes A and B downloaded from GenBank, primers were designed and RT-LAMP assay was developed to detect RNA of RSV sensitivity of the RT-LAMP method was evaluated by using ten-fold serially diluted in vitro-transcribed matrix RNA fragments from RSV A and RSV B, respectively. Specificity of the RT-LAMP method was tested through cross-reaction with other RNA and DNA viruses. Then 5 RSV strains isolated from clinical specimens using tissue cultures were tested by RT-LAMP assay. A total of 101 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 40 positive for RSV and 61 negative for RSV, were tested by RT-LAMP assay and by RT-nested PCR. RESULT: Sensitivity analysis indicated that this RT-LAMP method was able to detect 1 copy/µl of RSV A and RSV B RNA, no amplification was shown in RT-LAMP with DNA or cDNA from other viruses in 60 min, revealed that the RT-LAMP assay is highly specific. Five RSV isolates confirmed as 4 RSV A and 1 RSV B previously were detected by RT-LAMP method as positive in 30 min. For those 101 specimens tested, 37 were RSV positive determined by RT-LAMP assay, as well as 35 RSV positive by RT-nested PCR. The total coincidence rate of RT-LAMP assay with DFA and RT-nested PCR in detecting RSV is 95.0%, 94.1% with Kappa value 0.895 and 0.871, respectively. CONCLUSION: A new, sensitive, accurate and rapid method, RT-LAMP assay for detecting human respiratory syncytial viruses from nasopharyngeal aspirates was developed, which should be helpful in rapid detection of RSV from respiratory tract samples of children.

[Human rhinovirus with different genotypes in children with acute respiratory tract infections in Beijing].

To understand the infections and molecular biological characteristics of different human rhinovirus (HRV) genotypes -A, B, C, especially C in children with acute respiratory tract infections (ARI) in Beijing. Seven hundreds and three respiratory tract specimens were collected from children with ARI during Jan. 2011 to Dec. 2011. Semi-nested PCR was developed for detecting HRVs. Gene fragment of VP4/VP2 capsid protein amplified from HRV positive specimens was sequenced and analyzed by software DNAStar, the phylogenetic tree was then constructed by MEGA 5. 05. Among these 703 specimens tested, 54 (7.7%, 54/703) were HRV positive, including 25 (46.3%, 25/54) positive for HRV-A, 8 (14. 8%, 8/54) for HRV-B, 21 (38. 9%, 21/54) for HRV-C determined by sequence analysis. Most of these children (94. 4%00, 51/54) infected with HRVs were younger than 5 years old, and the highest positive rate was shown in group younger than 1 year (11. 4%). These patients positive for HRVs were diagnosed as bronchiolitis (23.1%), asthma (20.0%), pneumonia (1.0%), bronchitis (4.4%) and upper respiratory tract infections (4. 1%). Sequence analysis of VP4/VP2 gene fragment revealed that 70. 0% to 100. 0% nucleotide identity was shown among the sequences within the same HRV genotype, and 55. 5% to 65. 8% nucleotide identity among the sequences from different HRV genotypes. In conclusion, HRVs, especially HRV-C, are important pathogens for children with ARI in Beijing. The prevalence of HRV-C is similar to that of HRV-A, higher than that of HRV-B. High sequence variation among different HRV genotypes was indicated in this study.

[Detecting human adenoviruses in respiratory samples collected from children with acute respiratory infections by loop-mediated isothermal amplification].

OBJECTIVE: To establish a rapid and reliable loop-mediated isothermal amplification (LAMP) method for detecting adenoviruses (ADV)in respiratory samples collected from children with acute respiratory infections. METHOD: According to the sequences of hexon genes of common adenovirus serotypes (Ad3, Ad7, and Ad14) downloaded from GenBank, primers were designed and LAMP method for detecting adenovirus DNA was developed. Sensitivity of the LAMP method was evaluated by using constructed recombinant plasmid DNA with gene fragment from hexon of ADV3, and specificity was tested through cross-reaction with other viruses. Then 11 ADV strains isolated from clinical specimens using tissue cultures were tested by LAMP method. A total of 108 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 36 for ADV positive and 72 for ADV negative, were tested by both LAMP method and multiplex nested PCR. RESULT: Analysis for sensitivity indicated that this LAMP method can detect 1.9×10(2)copies/ml of DNA, and no amplification was shown in DNA or cDNA of other viruses, which revealed that the specificity of the LAMP method is high. For 108 specimens which had been tested by DFA, 34 out of the 36 ADV positive specimens showed positive signal within 90 minutes using LAMP. Five out of 72 negative specimens by DFA were positive using LAMP; 39 out of the 41 ADV positive specimens by multiplex nested PCR showed positive signal using LAMP, including 19 for Ad3 and 20 for Ad7; 67 negative specimens confirmed by multiplex nested PCR showed negative signal using LAMP. The total consistency rate of DFA and LAMP method for detecting ADV was 93.5%, and the total coincidence rate of multiplex nested PCR and LAMP method for detecting ADV was 98.1%. CONCLUSION: A new, sensitive, accurate and rapid method for detecting human adenovirus from nasopharyngeal aspirates by LAMP was developed, which should be a potential method for rapid detection of ADV from respiratory tract of children in clinical diagnosis of ADV infection.

Common WU polyomavirus infection in a Beijing population indicated by surveillance for serum IgG antibody against capsid protein VP2.

BACKGROUND: WU polyomavirus (WU virus) was identified as a novel polyomavirus in 2007 from specimens of pediatric patients with acute respiratory infection (ARI). A lack of permissive cell lines has limited investigations into WU virus pathogenesis and prevalence. METHODS: The encoding region of the capsid protein VP2 gene was amplified from a WU virus DNA-positive clinical specimen and expressed as a recombinant Histagged protein in Escherichia coli BL21 (DE3). The expressed VP2 was identified by expected molecular weight and immunoreactivity with anti-His monoclonal antibody in Western blotting assay. Serum samples collected from 455 individuals of all ages in Beijing without symptoms of ARI were tested for IgG antibodies against the affinity-purified recombinant VP2 protein by Western blotting to investigate the prevalence of natural WU virus infection. In addition, serum samples from four ARI pediatric patients, whose nasopharyngeal aspirates were positive for WU virus DNA and negative for all other respiratory-related viruses, were tested for IgM antibody against the recombinant VP2. RESULTS: Of the 455 serum samples, 238 reacted with the recombinant VP2, yielding an overall positive rate of 52.3% for IgG against VP2 of WU virus. The positive rate was the highest in serum samples from infants and children between 1 to 4 years of age. One of four ARI pediatric patients was positive for IgM against WU virus VP2, implicating WU virus as the causative disease agent. CONCLUSIONS: The high prevalence of IgG against WU polyomavirus in Beijing-based study population indicates that WU virus infection is common in Beijing. WU virus may be responsible for some pediatric ARI cases, and primary infection of this virus may occur mostly in childhood.