Species A rotaviruses (RVs), which belong to the family Sedoreoviridae and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.7% (8/12) to 91.7 % (11/12). Furthermore, the recombinant LLR stably expressing NLuc was rescued based on the five-plasmid reverse genetics system. Improvements to the rotavirus reverse genetics system will enhance its applicability for studies of rotavirus biology and clinical use.

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The LLR RVA vaccine, which licensed in 2000 and widely used in China, significantly reduced rotavirus disease burden in China. With the changing of RV circulating strains and the emergence of new genotypes, the LLR vaccine against RVGE needed to be upgraded. In this study, we aimed to establish an RG system for the RVA vaccine strain LLR (G10P[15]). Transfection with plasmids expressing 11 genomic RNA segments of LLR along with the pCMV/868CP helper plasmid, resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the LLR strain was successfully established. Furthermore, the plasmid-based reverse genetics system was used to generate lamb RVA reassortants with VP4 or VP7 genes derived from human RVA strains in China, which were not previously adapted to cell culture. We were able to rescue the six VP7 (G1, G2, G3, G4, G8, and G9) mono-reassortants, but no VP4 (P[4] or P[8]) mono-reassortant was rescued. The six VP7 reassortants covered all G-genotypes currently circulating in China and stably replicated in MA104 cells, which should be exploited as the next-generation rotavirus vaccines candidates in China. Furthermore, the LLR RG system in this study will be a useful vaccine vector for intestinal pathogens such as norovirus and Vibrio cholerae.

Human metapneumovirus (HMPV) is a major pathogen of acute respiratory tract infections (ARTIs) in children. Whole genome sequence analyses could help understand the evolution and transmission events of this virus. In this study, we sequenced HMPV whole genomes to improve the identification of molecular epidemiology in Beijing, China. Nasopharyngeal aspirates of hospitalized children aged < 14 years old with ARTIs were screened for HMPV infection using qPCR. Fourteen pairs of overlapping primers were used to amplify whole genome sequences of HMPV from positive samples with high viral loads. The epidemiology of HMPV was analysed and 27 HMPV whole genome sequences were obtained. Sequence identity and the positional entropy analyses showed that most regions of HMPV genome are conserved, whereas the G gene contained many variations. Phylogenetic analysis identified 25 HMPV sequences that belonged to a newly defined subtype A2b1; G gene sequences from 24 of these contained a 111-nucleotide duplication. HMPV is an important respiratory pathogen in paediatric patients. The new subtype A2b1 with a 111-nucleotide duplication has become predominate in Beijing, China.

BackgroundThis study aims to described the epidemiology and genotypic diversity of Human metapneumovirus (HMPV) and the impact of SARS-CoV-2 on the prevalence of HMPV in hospitalized children with Acute respiratory tract infections (ARTIs) in Beijing, China.MethodsFrom April 2018 to March 2019 and from September 2020 to August 2021, nasopharyngeal aspirates (NPAs) from hospitalized children with ARTIs in Beijing were collected and subjected to real-time polymerase chain reaction tests for HMPV. Then genotyping, detection of 15 common respiratory viruses and clinical characteristics were analyzed on HMPV positive samples.Results7.9% (124/1572) enrolled pediatric patients were identified as having HMPV infection, and the majority of children under the age of 5 (78.2%, 92/124), From April 2018 to March 2019. The detection rate of HMPV in spring and winter is significantly higher than that in summer and autumn. The co-infection rate were 37.1% (46/124), the most common co-infected virus were parainfluenza virus type 3 (HPIV-3). The main diagnosis of HMPV infection was pneumonia (92.7%,115/124), most patient have cough and fever. Of 78 HMPV-positive specimens, A2b (82.1%,64/78) were the main epidemic subtypes. Hospitalized children with HMPV genotype A infection had a higher viral load compared to genotype B. During the COVID-19 outbreak, Among 232 samples, only 4 cases were HMPV-positive. After statistical test, the detection rate of HMPV during the COVID-19 pandemic has decreased significantly compared with that before the epidemic (p = 0.001).ConclusionsHMPV is an important cause of ARTIs in children under 5 years old. The epidemic peak is generally in winter and spring, and the A2b subtype is the most common. However, under the prevention and control of the COVID-19 pandemic, the HMPV infection of hospitalized children with ARTIs has decreased significantly.

Objective:To construct 2019 novel coronavirus (2019-nCoV) 614D and 614G pseudovirus by HIV lentivirus packaging system and explore their biological specificity.Methods:The recombinant expression plasmids pCDNA3.1-614D and pCDNA3.1-614G were transiently cotransfected with psPAX2 and pLenti CMV Puro LUC into 293T cells respectively. After 72 hours, the supernatant was collected and ultracentrifuged with 20% sucrose cushion. The titer, morphology, protein expression and neutralizing activity of pseudovirus were determined.Results:S protein specific fluorescence was detected by indirect immunofluorescence test, Western blot analysis showed S protein was expressed, and the spike of pseudovirus was observed under transmission electron microscope. The titers of pseudovirus 614D and 614G were 1.12×10 4 and 2.52×10 4 TCID 50/ml, respectively. The pseudovirus 614D and 614G could be neutralized by S rabbit polyclonal antibody, indicating that the pseudovirus has high specificity. Conclusions:In this study, 2019-nCoV 614D and 614G pseudovirus was successfully constructed, which laid the foundation for the establishment of in vitro neutralizing antibody detection platform based on pseudovirus.

人偏肺病毒(Human metapneumovirus,HMPV)是2001年鉴定出的新发呼吸道病毒,婴幼儿、老人和免疫抑制人群易感,引起上呼吸道和下呼吸道感染,目前尚无疫苗和特异性治疗方案.为获得北京地区HMPV临床流行毒株,本研究将经荧光定量PCR检测为HMPV阳性的鼻咽抽吸物样本分别接种LLC-MK2、Vero-E6和分化良好的人呼吸道上皮细胞(Human Airway Epithelium,HAE),观察细胞病变、检测免疫荧光、电镜观察病毒形态、测定病毒滴度及分析复制特点,对分离获得的HMPV分离株进行鉴定.结果表明,HMPV感染LLC-MK2细胞可形成合胞体,但在Vero-E6中多呈单个细胞感染;经免疫荧光检测,HAE、LLC-MK2和Vero-E6细胞均可见绿色荧光;电镜结果可见病毒为近似球型的颗粒,有包膜和刺突,直径约在150nm～200nm之间;HMPV在HAE和LLC-MK2两种细胞上的复制特点基本相同.本研究成功建立了临床样本在LLC-MK2、Vero-E6和HAE分离培养HMPV的方法,分离并鉴定了 HMPV临床分离株,为HMPV感染机制的研究奠定基础.

Background Acute respiratory tract infections (ARTIs) causes high amounts of morbidity and mortality worldwide every year. Human metapneumovirus (HMPV) is a major pathogen of ARTIs in children. In this study, we aimed to investigate the epidemiology and genotypic diversity of HMPV in children hospitalized with ARTIs in Beijing, China. Methods Hospitalized children aged < 14 years with ARTIs were enrolled from April 2017 to March 2018; nasopharyngeal aspirates were collected and subjected to real-time polymerase chain reaction tests for HMPV. HMPV-positive samples were genotyped based on a partial N gene. Whole genome sequences were determined for samples with high viral loads. Results 4.08% (52/1276) enrolled paediatric patients were identified as having HMPV infection. The epidemic season is winter and early spring, children aged ≤ 4 years were more susceptible to HMPV infection (47/52, 90.38%). The co-infection rate were 36.54% (19/52), the most common co-infected virus were influenza and respiratory syncytial virus. The main diagnoses of HMPV infection were pneumonia (29/52, 55.77%) and bronchitis (23/52, 44.23%), while the main clinical manifestations were cough, fever, rhinorrhoea, and sneeze. Among 48 HMPV-positive specimens, A2b (19/48, 39.58%) and B1 (26/48, 54.17%) were the main epidemic subtypes. Patients with HMPV genotype A infection had a higher viral load compared to genotype B patients (6.07 vs. 5.37 log 10 RNA copies/ml). Five complete sequences of HMPV were obtained. This is the first report of a whole genome sequence of HMPV-B1 isolated in China. Conclusions HMPV is an important respiratory pathogen in paediatric patients. Cases of HMPV infection could burden hospitals in the epidemic season. HMPV viral loads and genotypes have no correlation with co-infection or clinical characteristics.

Objective:To explore the pathogenic spectrum and epidemiological characteristics of respiratory viruses in children with acute lower respiratory tract infection in Changsha, and provide scientific basis for disease prevention and treatment.Methods:A total of 1 092 respiratory tract specimens of children were collected and 12 respiratory viruses were detected by real-time quantitative transcription polymerase chain reaction.Results:Among the samples from 1 092 cases, those from 437 cases (40%) were positive for respiratory syncytial virus (RSV), 337 cases (30.9%) were positive for parainfluenza virus-3 (PIV-3), 263 cases (24.1%) were positive for human bocavirus (HBOV) and 228 cases (20.8%) were positive for adenovirus (ADV). The detection rates of boys and girls were 82.26% and 83.42%. The infection rate of RSV was higher in the group ≤ 6 months of age, the infection rates of PIV-3 and HBOV ≤2 years old were higher, and the infection rate of ADV was higher in the group between 6 months to 5 years old. The detection rates of virus infection of spring, summer, autumn and winter were 90.48%, 83.50%, 62.26% and 82.80%, respectively, there were significant differences among them.Conclusions:The main viruses in children with acute lower respiratory tract infection in Changsha were RSV and PIV-3. Mixed infections were common. Children under 2 years of age were more likely to get acute lower respiratory infections. Viruses had seasonal trends and peaked in winter and spring.

INTRODUCTION:Etiology studies of severe acute respiratory infections (SARI) in adults are limited. We studied potential etiologies of SARI among adults in six countries using multi-pathogen diagnostics. METHODS:We enrolled both adults with SARI (acute respiratory illness onset with fever and cough requiring hospitalization) and asymptomatic adults (adults hospitalized with non-infectious illnesses, non-household members accompanying SARI patients, adults enrolled from outpatient departments, and community members) in each country. Demographics, clinical data, and nasopharyngeal and oropharyngeal specimens were collected from both SARI patients and asymptomatic adults. Specimens were tested for presence of 29 pathogens utilizing the Taqman® Array Card platform. We applied a non-parametric Bayesian regression extension of a partially latent class model approach to estimate proportions of SARI caused by specific pathogens. RESULTS:We enrolled 2,388 SARI patients and 1,135 asymptomatic adults from October 2013 through October 2015. We detected ≥1 pathogen in 76% of SARI patients and 67% of asymptomatic adults. Haemophilus influenzae and Streptococcus pneumoniae were most commonly detected (≥23% of SARI patients and asymptomatic adults). Through modeling, etiology was attributed to a pathogen in most SARI patients (range among countries: 57.3-93.2%); pathogens commonly attributed to SARI etiology included influenza A (14.4-54.4%), influenza B (1.9-19.1%), rhino/enterovirus (1.8-42.6%), and RSV (3.6-14.6%). CONCLUSIONS:Use of multi-pathogen diagnostics and modeling enabled attribution of etiology in most adult SARI patients, despite frequent detection of multiple pathogens in the upper respiratory tract. Seasonal flu vaccination and development of RSV vaccine would likely reduce the burden of SARI in these populations.

BACKGROUND:Washington University polyomavirus (WUPyV) is a novel human polyomavirus detected in childwith acute respiratory infection in 2007. However, the relationship between WUPyV and respiratory diseases has yet to be established for lacking of a suitable in vitro culture system.METHODS:To isolate WUPyV with human airway epithelial (HAE) cells, the positive samples were incubated in HAE, and then the nucleic acid, VP1 protein and virions were detected using real-time PCR, immunofluorescence and electron microscopy respectively.RESULTS:The result showed that WUPyV could replicate effectively in HAE cells and virions with typical polyomavirus characteristics could be observed. Additionally, the entire genome sequence of the isolated strain (BJ0771) was obtained and phylogenetic analysis indicated that BJ0771 belongs to gene cluster I.CONCLUSIONS:Our findings demonstrated clinical WUPyV strain was successfully isolated for the first time in the world and this will help unravel the etiology and pathogenic mechanisms of WUPyV in respiratory infection diseases.

ON1 is a novel genotype of human respiratory syncytial virus (HRSV) subtype A, in children with acute respiratory tract infections (ARTIs). However, there is not much data on the prevalence and clinical and molecular characterization in China. Our study is based on the children who had respiratory infections positive for RSV-A admitted by Gansu Provincial Maternity and Child-care Hospital in Lanzhou (northwestern China) during the last 7 epidemic seasons from 2010 to 2017. In our study, different strains of the novel RSV-A genotype ON1, first identified in Canada in December 2010, were first detected in Gansu Provincial Maternity and Child-care Hospital in August 2012 and then followed by an abrupt expansion in the number of ON1 variants in the beginning of 2014 and eventually replaced all other RSV-A strains from 2015 to 2017. ON1 is characterized by a 72-nt duplication in the C-terminal region of the highly variable attachment glycoprotein (G), predicted to lengthen the polypeptide with 24 amino acids, including a 23-aa duplication, which likely changes antigenicity. New N-glycosylation sites occurred within the 23-aa duplication and 24-aa insertion of the ON1 viruses in our study. Notably, RSV infections occurred later, but peaked sooner from the 2014/2015 to 2016/2017 epidemic seasons, compared with the previous 4 seasons. Our study concluded that genotype ON1 has caused larger outbreaks and became the predominate genotype for HRSV subgroup A in Lanzhou from 2013 to 2017, and became the sole genotype of RSV-A in 2015/2016 and 2016/2017. Our data indicate that northwest of China and the world will eventually be dominated by the ON1 RSV-A genotype, including the possibility for vaccine development. Based on trends seen in RSV-B BA genotype, which predominated for decades, there is a possibility to develop a vaccine for children in the next 10 years.

The discovery and analysis of pathogens carried by non-human primates are important for understanding zoonotic infections in humans. We identified a highly divergent astrovirus (AstV) from fecal matter from a rhesus monkey in China, which has been tentatively named "monkey-feces-associated AstV" (MkAstV). The full-length genome of MkAstV was determined to be 7377 nt in length. It exhibits the standard genomic AstV organization of three open reading frames (ORFs) and is most closely related to duck AstV (28%, 49%, and 35% amino acid sequence identity in ORF1a, ORF1b, and ORF2, respectively). Coincidentally, while this report was being prepared, an astrovirus sequence from Hainan black-spectacled toad became available in the GenBank database, showing 95%, 94% and 92% aa sequence identity in ORF1a, ORF1b and ORF2, respectively, to the corresponding ORFs of MkAstV. Phylogenetic analysis of ORF1a, ORF1b, and ORF2 indicated that MkAstV and the amphibian-related astroviruses formed an independent cluster in the genus Avastrovirus. The host of MkAstV remains unknown. Epidemiological and serological studies of this novel virus should be undertaken in primates, including humans.

Background Lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of LRTI s caused by HMPV and HB oV in China is limited. Objectives Human bocavirus ( HB oV) and human metapneumovirus ( HMPV ) are two important viruses for children with lower respiratory tract infections ( LRTI ). We aimed to assay the correlation between viral load and clinical characteristics of HB oV and HMPV with LRTI in Changsha, China. Methods Nasopharyngeal aspirates ( NPA s) from children with LRTI were collected. Real‐time PCR was used to screen HB oV and HMPV . Analyses were performed using SPSS 16.0 software. Results Pneumonia was the most frequent diagnosis. There was no significant difference between HB oV‐ and HMPV ‐positive patients in age ( P = .506) or hospitalization duration ( P = .280); 24.1% and 18.2% were positive for HB oV and HMPV . HB oV infections peaked in summer (32.2%), and HMPV infections peaked in winter (28.9%). The HB oV‐positive patients had a shorter hospitalization duration than the HB oV‐negative patients ( P = .021), and the HMPV ‐positive patients had a higher prevalence of fever than the HMPV ‐negative patients ( P = .002). The HB oV viral load was significantly higher among patients aged <1 year ( P = .006). The mean HB oV and HMPV viral loads were not significantly different between patients with single infections and coinfections. Patients infected with HB oV only were older than those coinfected with HB oV and other respiratory viruses ( P = .005). No significant difference was found in the clinical characteristics of patients infected with HMPV only and those coinfected with HMPV and other respiratory viruses. Conclusion Pneumonia was the most frequent diagnosis caused by HB oV and HMPV . Neither HB oV nor HMPV viral load was correlated with disease severity.

Pneumonia is a serious public health issue and is concerned around the world. This study is to investigate the association between viral load in children with human adenovirus (HAdV) pneumonia and disease severity.

Objective To investigate the clinical and epidemiological characteristics of Coronavirus HKU1 ( Human CoV-HKU1) and NL63 ( Human CoV-NL63 ) in children with acute respiratory tract infection in Nanjing. Methods From August 2009 to July 2011, 1286 respiratory samples were collected from the outpatient and hospitalized children in the Children′ s Hospital of Nanjing Medical University. Reverse transcription polymerase chain reaction ( RT-PCR) was used to detect HCoV-HKU1 and NL63 genes, besides,positive samples were used for common respiratory virus screening. The positive amplification products were cloned, sequenced, homologous and phylogenetic analysis was conducted by molecular biological method . Results The detection rate of HCoV-HKU1 was 1. 1％ (14 / 1286), the positive sequences shared a 98. 2％ -100％ nucleotide identity with the HCoV-HKU1 strains and mixed infection rate was 92. 9％ . The main clinical diagnoses were bronchitis, bronchopneumonia and bronchiolitis. The clinical manifestations were cough, fever, wheezing. The detection rate of HCoV-NL63 was 1. 5％ (19 / 1286), the positive sequences shared a 95. 6％ -100％ nucleotide identity with the HCoV-NL63 strains and mixed infection rate were 63. 2％ . The main clinical diagnosis were acute upper respiratory tract infection, bronchitis, bronchopneumonia. The clinical manifestations were fever, cough, expectoration. No deaths were found in both HCoV-HKU1 and NL63 infections. Conclusions From August 2009 to July 2011, HCoV-HKU1 and NL63 were detected in children with respiratory tract infection in Nanjing area. HCoV-HKU1 infected cases were lower respiratory tract infection, epidemic in winter and spring, infected cases were mainly under 1 years of age, HCoV-NL63 infected cases including upper respiratory and lower respiratory tract infection, epidemic in the season of summer and autumn. The infected cases were mainly at the age rank from 1 year to 3 years. The clinical manifestations of children infected with coronavirus HKU1 and NL63 are not specificity.

To investigate the current genotypes of circulating human adenovirus (HAdV) strains, we molecularly genotyped HAdV in the nasopharyngeal aspirates (NPAs) of patients with acute lower respiratory tract infections (ALRTIs) and attempted to determine their associations with clinical symptoms. A total of 4751 NPA samples were collected from 4751 patients admitted to Hunan Provincial People's Hospital from September 2007 to March 2014, of which 447 (9.4%) samples were HAdV positive. Fourteen different HAdV types were identified; HAdV types 1 to 7 (HAdV 1-7) were identified in 95.7% of the 447 NPA samples with HAdV-7 and HAdV-3 being the most prevalent. In addition, 93.3% (417 of 447) of patients were younger than 5 years. The incidence of HAdV infection peaked in summer. Different HAdV types showed a predilection for different age groups and different seasonal distribution patterns. Coinfection of HAdVs and other respiratory viruses was detected in 63.3% (283 of 447) of the HAdV-positive samples. The most common clinical diagnosis was pneumonia and the most common symptoms were fever and cough. In comparison with children infected with HAdV-3 alone, those infected with HAdV-7 alone had an increased frequency of severe pneumonia involvement (11.6% vs 32.4%; P = 0.031), higher intensive care unit admission rates (7.0% vs 26.5%; P = 0.019), and a longer length of hospital stay (P = 0.03). Mixed infections in younger children were associated with a longer hospital stay (P = 0.023). Our results demonstrate the recent changes in the trends of circulating HAdV genotypes associated with ALRTIs in Hunan China.

Objective To investigate the human bocavirus (HBoV) infection in the children diagnosed with brochiolitis,incomparison with the clinical characteristics of bronchiolitis of HBoV vs.respiratory syncytial virus (RSV) infection alone.Methods A total of 396 throat swabs were obtained from children diagnosed with bronchiolitis from January 2015 to December 2016 in Pediatric Asthma Center of Gansu Maternal and Child Health Care Hospital,Lanzhou.Specimens were tested for the nucleic acids of HBoV,RSV and other 6 common respiratory viruses by nested-polymerase chain reaction (nested PCR ()and reverse transcription-polymerase chain reaction (RT-PCR),and the epidemic status,clinical characteristics of HBoV and RSV were analyzed and compared with each other.Results The positive rate of virus infection was 53.54％,and HBoV and RSV infections were 9.84％ and 24.49％ in 396 cases.The numbers of cases of HBoV single infection,RSV single infection,HBoV and RSV mixed infection were 29,86 and 5.The number of boys infected with HBoV was 27.There was statistical significance in the difference between the genders.The statistical significance also existed in difference of age.The babies at ages ranging from 6 to 12 months had the hifgest positive rate.The first incidence peak of the infection of HBoV was in October 2015.The children with HBoV infection,compared with RSV patients,presented more often with vomiting,diarrhea (P ＜ 0.05),while symptoms such as the occurrence of dyspnea,hypoxemia and the hospitalization time were all lower.Conclusions HBoV is one of the possible causes of brochiolitis in infants,its infection distributed over the whole year.The clinical symptoms,laboratory and radio-graphic changes are similar between the HBoV and RSV infections,the only difference was that the HBoV infection was comparatively mild.

Rhinovirus C (RV-C), a newly identified group of human rhinoviruses (RVs), is associated with exacerbation of severe asthma. The type I interferon (IFN) response induced by this virus and the mechanisms of evasion of IFN-mediated innate immunity for RV-C remain unclear. In this study, we constructed a full-length cDNA clone of RV-C (LZ651) from a clinical sample. IFN-β mRNA and protein levels were not elevated in differentiated Human bronchial epithelial (HBE) cells at the air-liquid interface infected with RV-C, except in the early stage of infection. The ability to attenuate IFN-β activation was ascribed to 3Cpro of RV-C, and the 40-His site of 3Cpro played an important role. Furthermore, RIG-I was degraded by 3Cpro in a caspase-dependent manner and 3Cpro cleaved MAVS at 148 Q/A, which inhibited IFN signaling. Taken together, our results demonstrate the mechanism by which RV-C circumvents the production of type I IFN in infected cells.

Objective To explore the viral pathogens of acute respiratory tract infection in Shanghai,and to provide reference data for diagnosis and treatment.Methods Between January 2012 and December 2013,643 nasopharyngeal aspiration (NPA) samples were collected from children who had been hospitalized for acute respiratory tract infection at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine,the Ninth People's Hospital,Affiliated to Medical College of Shanghai Jiaotong University School and Putuo District Center Hospital Shanghai,China.Human rhinoviruses,Respiratory syncytial virus,Adenovirus,Influenza viruses A and B,Parainfluenza viruses 1-4,Human metapneumovirus,Human bocavirus,Human coronavirus HKU1 and Human coronavirus NL-63 were detected by reverse transcirption-polymerase chain reaction (RT-PCR) and standard polymerase chain reaction (PCR) methods.Epidemiology and clinical manifestations of each virus were analyzed.Results Six hundred and forty-three NPAs samples were collected from children (402 boys,241 girls) with ARTI,The mean age was 12 months(range from 11 days to 12 years).Viral pathogens were identified in 369 (57.4％) samples.RSV was the highest detection rate in 2012 and ADV was the highest detection rate in 2013,HCoV-NL63 was not detected.6 months to 1 year old age group virus detection rate was highest,along with the age growth,virus detection rate was falling,virus detection throughout the year,diagnosis includes upper and lower respiratory tract infection.Conclusions Viral pathogens are the main etiology of ARTI in children in Shanghai area.RSV and ADV are the most frequent viral pathogens.

Human parainfluenza viruses (HPIVs) are an important cause of acute lower respiratory tract infections (ALRTIs). HPIV‐4, a newly identified virus, has been associated with severe ALRTIs recently. A total of 771 nasopharyngeal aspirate samples were collected from hospitalized children between March 2010 and February 2011. HPIVs were detected by Nest‐PCR, and other known respiratory viruses were detected by RT‐PCR and PCR. All amplification products were sequenced. HPIVs were detected in 151 (19.58%) patients, of whom 28 (3.63%) were positive for HPIV‐4, 12(1.55%) for HPIV‐1, 4 (0.51%) for HPIV‐2, and 107 (13.87%) for HPIV‐3. Only three were found to be co‐infected with different types of HPIVs. All HPIV‐positive children were under 5 years of age, with the majority being less than 1 year. Only the detection rate of HPIV‐3 had a significant statistical difference (χ 2 = 29.648, P = 0.000) between ages. HPIV‐3 and HPIV‐4 were detected during the summer. Sixty (39.74%) were co‐infected with other respiratory viruses, and human rhinovirus (HRV) was the most common co‐infecting virus. The most frequent clinical diagnosis was bronchopneumonia, and all patients had cough; some patients who were infected with HPIV‐3 and HPIV‐4 had polypnea and cyanosis. No significant difference was found in clinical manifestations between those who were infected with HPIV‐4 and HPIV‐3. Two genotypes for HPIV‐4 were prevalent, although HPIV‐4a dominated. HPIV‐4 is an important virus for children hospitalized with ALRTIs in China. HRV was the most common co‐infecting virus. Two genotypes for HPIV‐4 are prevalent, HPIV‐4a dominated. J. Med. Virol. 88:2085–2091, 2016 . © 2016 Wiley Periodicals, Inc.