Immunogenicity and protective efficacy of human metapneumovirus virus-like particles produced by a recombinant baculovirus in mice.

BACKGROUND: Human metapneumovirus (HMPV) causes respiratory tract infections among infant, elderly, and immunocompromised patients, with significant mortality. Currently no licensed vaccines or therapeutic agents of HMPV exist. METHODS: HMPV virus-like particle (VLP) was constructed by co-expressing fusion protein of HMPV and matrix 1 protein of influenza virus using the baculovirus expression. Mice were immunized with VLP with or without aluminum hydroxide (alum) adjuvant by intramuscular route respectively. Sera were determined for titers of IgG and neutralizing antibody. Splenic lymphocytes were determined by IFN-γ and IL-4 ELISPOT. Mice were challenged with HMPV, and protective efficacy was evaluated. RESULTS: We generated HMPV VLP in baculovirus expression system. After three times immunization, IgG antibody titers induced by VLP formulated with or without alum adjuvant group were 273,066 ± 100,331 and 136,533 ± 47,269 respectively, there was no difference (p Ëƒ 0.05); the neutralizing antibody titers vaccinated with VLP plus with alum adjuvant (266 ± 92) were higher than those of the VLP alone group (106 ± 37). For IFN-γ, mice vaccinated with VLP with or without alum adjuvant are 151 ± 36.4 and 77.0 ± 17.1SFC/106 respectively, there was difference (p = 0.03); For IL-4, they are 261.3 ± 38.7 versus 125.67 ± 29.78SFC/106 respectively, the difference was significant (p = 0.009). After challenge, in pathological analysis, the overall lesion scores in the VLP plus with and without alum adjuvant were 3.25 and 5.6 respectively, those of control group is 8. For immunohistochemical analyses, the average optical density of the lungs in the VLP immunized group containing adjuvant (9.07 ± 1.74) was lower than that in the VLP group without adjuvant (12.83 ± 2.31, p = 0.14). CONCLUSIONS: This is the first study to demonstrate that HMPV VLP was successfully prepared in the baculovirus expression system. HMPV VLP could induce specific humoral and cellular immune responses as well as protective efficacy, and aluminum hydroxide may be an effective adjuvant in mice.

Systems pharmacology dissection of pharmacological mechanisms of Xiaochaihu decoction against human coronavirus.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) pandemic is still rage worldwide, there are still very limited treatments for human coronaviruses (HCoVs) infections. Xiaochahu decoction (XCHD), which is one of the traditional Chinese medicine (TCM) prescriptions in Qingfeipaidu decoction (QFPDD), is widely used for COVID-19 treatment in China and able to relieve the symptoms of fever, fatigue, anorexia, and sore throat. To explore the role and mechanisms of XCHD against HCoVs, we presented an integrated systems pharmacology framework in this study. METHODS: We constructed a global herb-compound-target (H-C-T) network of XCHD against HCoVs. Multi-level systems pharmacology analyses were conducted to highlight the key XCHD-regulated proteins, and reveal multiple HCoVs relevant biological functions affected by XCHD. We further utilized network-based prediction, drug-likeness analysis, combining with literature investigations to uncover the key ani-HCoV constituents in XCHD, whose effects on anit-HCoV-229E virus were validated using cytopathic effect (CPE) assay. Finally, we proposed potential molecular mechanisms of these compounds against HCoVs via subnetwork analysis. RESULTS: Based on the systems pharmacology framework, we identified 161 XCHD-derived compounds interacting with 37 HCoV-associated proteins. An integrated pathway analysis revealed that the mechanism of XCHD against HCoVs is related to TLR signaling pathway, RIG-I-like receptor signaling pathway, cytoplasmic DNA sensing pathway, and IL-6/STAT3 pro-inflammatory signaling pathway. Five compounds from XCHD, including betulinic acid, chrysin, isoliquiritigenin, schisandrin B, and (20R)-Ginsenoside Rh1 exerted inhibitory activity against HCoV-229E virus in Huh7 cells using in vitro CPE assay. CONCLUSION: Our work presented a comprehensive systems pharmacology approach to identify the effective molecules and explore the molecular mechanism of XCHD against HCoVs.

Whole genome sequencing and evolution analyses of Human metapneumovirus.

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.

Establishment of a neutralization assay for Nipah virus using a high-titer pseudovirus system.

OBJECTIVE: To construct a high-titer Nipah pseudovirus packaging system using the HIV lentivirus backbone vector and establish a safe neutralization assay for Nipah pseudovirus in biosafety level 2 facilities. METHODS: Nipah virus (NiV) fusion protein (F) and glycoprotein (G) recombinant expression plasmids, psPAX2, and pLenti CMV Puro LUC (w168-1) were transiently transfected into 293T cells for 72 h for the generation of a NiV pseudovirus. The neutralization ability of Nipah virus F and G protein antibodies was assessed using the pseudovirus. RESULTS: A NiV pseudovirus was constructed using 293T cells. The ideal mass ratio of plasmid psPAX2: w168-1: F: G for transfection was determined to be 4:4:1:1. The specificity of recombinant F and G protein expression was indicated by indirect immunofluorescence and western blotting. The pseudovirus particles showed obvious spikes under a transmission electron microscope. The NiV pseudovirus titer was 4.73 × 105 median tissue culture infective dose per mL, and the pseudovirus could be effectively neutralized by polyclonal antibodies specifically targeting the F and G proteins respectively. CONCLUSIONS: A NiV pseudovirus was successfully generated using HIV vector systems, and was used as a platform for a safe and reliable pseudovirus-based neutralizing assay that can be performed in biosafety level 2 facilities.

Comprehensive virome analysis of the viral spectrum in paediatric patients diagnosed with Mycoplasma pneumoniae pneumonia.

BACKGROUND: Among hospitalized children suffering from community-acquired pneumonia, Mycoplasma pneumoniae (MP) is one of the most common pathogens. MP often exists as a co-infection with bacteria or viruses, which can exacerbate the clinical symptoms. We investigated the pathogen spectrum in MP-positive and MP-negative samples from hospitalized children with respiratory tract infections in Beijing, China. METHOD: This study included 1038 samples of nasopharyngeal aspirates obtained between April, 2017 and March, 2018 from hospitalized children under 6 years of age with respiratory tract infections. To explore the impact of MP infection on the composition of the pathogen spectrum, 185 nasopharyngeal aspirates (83 MP-positive/102 MP-negative) were randomly selected for next-generation sequencing and comprehensive metagenomics analysis. Real-time PCR was used to detect and verify common respiratory viruses. RESULTS: Of the 1038 samples, 454 (43.7%) were infected with MP. In children < 6 years of age, the MP infection rate gradually increased with age, with the highest rate of 74.2% in 5-6-year-olds. The results of metagenomics analysis revealed 11 human, animal and plant virus families, and bacteriophages, including common respiratory viruses, enteroviruses and anelloviruses. The virus family with the highest number of reads in both MP-positive and MP-negative samples was the Pneumoviridae, and the number of reads for human respiratory syncytial virus (HRSV) in MP-positive samples was higher than that in MP-negative samples. Among the 83 MP-positive samples, 47 (56.63%) were co-infected with viruses, the most common of which was influenza virus (IFV). The durations of hospitalization and fever were higher in patients with MP co-infection than MP single infection, but the difference was not statistically significant. CONCLUSION: The viral family with the highest number of reads in both groups was Pneumoviridae, and the number of reads matched to HRSV in MP-positive samples was much higher than MP-negative samples. Co-infection of MP and IFV infection were the most cases.

Human metapneumovirus in hospitalized children with acute respiratory tract infections in Beijing, China.

BACKGROUND: This 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. METHODS: From 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. RESULTS: 7.9% (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). CONCLUSIONS: HMPV 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.

Human adenoviruses in paediatric patients with respiratory tract infections in Beijing, China.

BACKGROUND: Human adenoviruse (HAdV) is a major pathogen of paediatric respiratory tract infections (RTIs). Mutation or recombination of HAdV genes may cause changes in its pathogenicity and transmission. We described the epidemiology and genotypic diversity of HAdV in hospitalized children with RTIs in Beijing, China. METHODS: Nasopharyngeal aspirates were collected from hospitalized children with RTIs from April 2018 to March 2019. HAdVs were detected by a quantitative real-time PCR, and the hexon gene was used for phylogenetic analysis. RESULTS: Among 1572 samples, 90 (5.72%) were HAdV-positive. The HAdV detection rate was highest in November and July. Among HAdV-positive children, 61.11% (55/90) were co-infected with other respiratory viruses, the most common of which were human respiratory syncytial virus and human rhinovirus. The main diagnosis was bronchopneumonia, most patient have cough and fever. Children with a high viral load were more likely to have a high fever (P = 0.041) and elevated WBC count (P = 0.000). Of 55 HAdV-positive specimens, HAdV-B (63.64%), HAdV-C (27.27%), and HAdV-E (9.09%) were main epidemic species. Phylogenetic analysis indicated that hexon sequences of three samples were on the same branch with the recombinant HAdV strain (CBJ113), which was circulating in Beijing since 2016. CONCLUSION: The HAdV-B3 and HAdV-B7 are the main epidemic strains in Beijing, and the recombinant HAdV-C strain CBJ113 has formed an epidemic trend.

Epidemiology and genotypic diversity of human metapneumovirus in paediatric patients with acute respiratory infection in Beijing, China.

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 log10 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.

Isolation and characterization of WUPyV in polarized human airway epithelial cells.

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.

Discovery of Multitarget-Directed Ligands Against Influenza A Virus From Compound Yizhihao Through a Predictive System for Compound-Protein Interactions.

Influenza A virus (IAV) is a threat to public health due to its high mutation rate and resistance to existing drugs. In this investigation, 15 targets selected from an influenza virus-host interaction network were successfully constructed as a multitarget virtual screening system for new drug discovery against IAV using Naïve Bayesian, recursive partitioning, and CDOCKER methods. The predictive accuracies of the models were evaluated using training sets and test sets. The system was then used to predict active constituents of Compound Yizhihao (CYZH), a Chinese medicinal compound used to treat influenza. Twenty-eight compounds with multitarget activities were selected for subsequent in vitro evaluation. Of the four compounds predicted to be active on neuraminidase (NA), chlorogenic acid, and orientin showed inhibitory activity in vitro. Linarin, sinensetin, cedar acid, isoliquiritigenin, sinigrin, luteolin, chlorogenic acid, orientin, epigoitrin, and rupestonic acid exhibited significant effects on TNF-α expression, which is almost consistent with predicted results. Results from a cytopathic effect (CPE) reduction assay revealed acacetin, indirubin, tryptanthrin, quercetin, luteolin, emodin, and apigenin had protective effects against wild-type strains of IAV. Quercetin, luteolin, and apigenin had good efficacy against resistant IAV strains in CPE reduction assays. Finally, with the aid of Gene Ontology biological process analysis, the potential mechanisms of CYZH action were revealed. In conclusion, a compound-protein interaction-prediction system was an efficient tool for the discovery of novel compounds against influenza, and the findings from CYZH provide important information for its usage and development.

Development and biological activity of long-acting recombinant human interferon-α2b.

BACKGROUND: The type I human interferon (IFN) family consists of a group of cytokines with a multiplicity of biological activities, including antiviral, antitumor, and immunomodulatory effects. However, because the half-life of IFN is short, its clinical application is limited. Increasing the yield and biological activity of IFN while extending its half-life is currently the focus of IFN research. RESULTS: Two novel long-acting recombinant human IFN-α2b (rhIFN-α2b) proteins were designed in which the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin ß su bunit and N-linked glycosylation sequences were linked to rhIFN-α2b. They were designated IFN-1CTPON (fused at the C-terminus of rhIFN-α2b) and IFN-2CTPON (fused at both the C-terminus and N-terminus of rhIFN-α2b). Monoclonal CHO cell strains stably and efficiently expressing the IFNs were successfully selected with methotrexate (MTX), and the highest expression levels were 1468 mg/l and 1196 mg/l for IFN-1CTPON and IFN-2CTPON, respectively. The proteins were purified with affinity chromatography and molecular sieve chromatography. IFN-1CTPON and IFN-2CTPON showed antiviral and antiproliferative activities in vitro. Notably, the half-life of IFN-1CTPON and IFN-2CTPON in vivo were three-fold and two-fold longer than that of commercially available rhIFN-α2b. CONCLUSIONS: CHO cell strains stably expressing long-acting rhIFN-α2b were screened. The purified IFN-CTPON protein has biological activity and an extended half-life, and therefore potential applications.

Human adenovirus among hospitalized children with respiratory tract infections in Beijing, China, 2017-2018.

BACKGROUND: Human adenoviruses (HAdVs) cause a wide range of diseases. However, the genotype diversity and epidemiological information relating to HAdVs among hospitalized children with respiratory tract infections (RTIs) is limited. Here, we describe the epidemiology and genotype distribution of HAdVs associated with RTIs in Beijing, China. METHODS: Nasopharyngeal aspirates (NPA) were collected from hospitalized children with RTIs from April 2017 to March 2018. HAdVs were detected by a TaqMan-based quantitative real-time polymerase chain reaction (qPCR) assay, and the hexon gene was used for phylogenetic analysis. Epidemiological data were analyzed using statistical product and service solutions (SPSS) 21.0 software. RESULTS: HAdV was detected in 72 (5.64%) of the 1276 NPA specimens, with most (86.11%, 62/72) HAdV-positives cases detected among children < 6 years of age. HAdV-B3 (56.06%, 37/66) and HAdV-C2 (19.70%, 13/66) were the most frequent. Of the 72 HAdV-infected cases, 27 (37.50%) were co-infected with other respiratory viruses, most commonly parainfluenza virus (12.50%, 9/72) and rhinovirus (9.72%, 7/72). The log number of viral load ranged from 3.30 to 9.14 copies per mL of NPA, with no significant difference between the HAdV mono- and co-infection groups. The main clinical symptoms in the HAdV-infected patients were fever and cough, and 62 (86.11%, 62/72) were diagnosed with pneumonia. Additionally, HAdVs were detected throughout the year with a higher prevalence in summer. CONCLUSIONS: HAdV prevalence is related to age and season. HAdV-B and HAdV-C circulated simultaneously among the hospitalized children with RTIs in Beijing, and HAdV-B type 3 and HAdV-C type 2 were the most frequent.

Anti-H7N9 avian influenza A virus activity of interferon in pseudostratified human airway epithelium cell cultures.

BACKGROUND: Since H7N9 influenza A virus (H7N9) was first reported in 2013, five waves of outbreaks have occurred, posing a huge threat to human health. In preparation for a potential H7N9 epidemic, it is essential to evaluate the efficacy of anti-H7N9 drugs with an appropriate model. METHODS: Well-differentiated pseudostratified human airway epithelium (HAE) cells were grown at the air-liquid interface, and the H7N9 cell tropism and cytopathic effect were detected by immunostaining and hematoxylin-eosin (HE) staining. The H7N9 replication kinetics and anti-H7N9 effect of recombinant human α2b (rhIFN-α2b) and rhIFN-λ1 were compared with different cell lines. The H7N9 viral load and interferon-stimulated gene (ISG) expression were quantified by real-time PCR assays. RESULTS: H7N9 could infect both ciliated and non-ciliated cells within the three-dimensional (3D) HAE cell culture, which reduced the number of cilia and damaged the airways. The H7N9 replication kinetics differed between traditional cells and 3D HAE cells. Interferon had antiviral activity against H7N9 and alleviated epithelial cell lesions; the antiviral activity of rhIFN-α2b was slightly better than that of rhIFN-λ1. In normal cells, rhIFN-α2b induced a greater amount of ISG expression (MX1, OAS1, IFITM3, and ISG15) compared with rhIFN-λ1, but in 3D HAE cells, this trend was reversed. CONCLUSIONS: Both rhIFN-α2b and rhIFN-λ1 had antiviral activity against H7N9, and this protection was related to the induction of ISGs. The 3D cell culture model is suitable for evaluating interferon antiviral activity because it can demonstrate realistic in vivo-like effects.

The generation and biological activity of a long-lasting recombinant human interferon-λ1.

The previously generated recombinant human (rh) interferon (IFN)-λ1 protein has a short half-life, and this feature makes it challenging to conduct studies on potential clinical applications for rhIFN-λ1. In an attempt to overcome this difficulty, we constructed a 'long-life' version of rhIFN-λ1. This modified rhIFN-λ1, named rhIFN-λ1-CTPON, has a human chorionic gonadotropin ß subunit carboxyl-terminal peptide (CTP) and an N-glycosylation sequence linked to its C-terminus. We confirmed the sequence of rhIFN-λ1-CTPON by mass spectrometry and then measured its biological activities. The results show that rhIFN-λ1-CTPON had antiviral activity and anti-proliferation activity in vitro that were similar to those of rhIFN-λ1 and that it similarly promoted natural killer cell cytotoxicity. Notably, the in vivo half-life of rhIFN-λ1-CTPON was determined to be 3-fold higher than that of rhIFN-λ1. We also assessed the anti-hepatitis B virus activity of rhIFN-λ1-CTPON; it was able to inhibit the production of the antigens HBs-Ag and HBe-Ag and induce antiviral gene expression. In conclusion, rhIFN-λ1-CTPON has a longer half-life than rhIFN-λ1 and has similar biological activities, so rhIFN-λ1-CTPON is an appropriate substitute for rhIFN-λ1 in the further study of potential clinical applications for rhIFN- λ1.

IFN-λ1 in CHO cells: its expression and biological activity.

BACKGROUND: Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor. RESULTS: A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 106 IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b. CONCLUSION: The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR.

BACKGROUND: Human Malawi polyomavirus (MWPyV) was discovered in 2012, but its prevalence and clinical characteristics are largely unknown. METHODS: We used real-time TaqMan-based PCR to detect MWPyV in the feces (n = 174) of children with diarrhea, nasopharyngeal aspirates (n = 887) from children with respiratory infections, and sera (n = 200) from healthy adults, and analyzed its clinical characteristics statistically. All the MWPyV-positive specimens were also screened for other common respiratory viruses. RESULTS: Sixteen specimens were positive for MWPyV, including 13 (1.47%) respiratory samples and three (1.7%) fecal samples. The samples were all co-infected with other respiratory viruses, most commonly with influenza viruses (69.2%) and human coronaviruses (30.7%). The MWPyV-positive children were diagnosed with bronchopneumonia or viral diarrhea. They ranged in age from 12 days to 9 years, and the most frequent symptoms were cough and fever. CONCLUSIONS: Real-time PCR is an effective tool for the detection of MWPyV in different types of samples. MWPyV infection mainly occurs in young children, and fecal-oral transmission is a possible route of its transmission.

Outbreak of hand, foot, and mouth disease caused by coxsackievirus A6 in a Juku in Fengtai District, Beijing, China, 2015.

An outbreak of hand, foot, and mouth disease (HFMD) that occurred in a Juku in Fengtai District, Beijing, China, in 2015 was monitored by the China Information System for Disease Control and Prevention. Epidemiological investigation showed that 11 cases occurred from two classes in the preschool art training department in the Juku. Coxsackievirus A6 (CV-A6) was identified as the causative pathogen of the outbreak via sequences analysis of products of real-time reverse-transcription polymerase chain reaction (RT-PCR) and nested RT-PCR. Phylogenetic analysis showed that CV-A6 strains isolated in this study clustered with epidemic strains isolated in China since 2013. The outbreak ended quickly with effective measures. This event indicates that continuous surveillance of HFMD etiological agents other than enterovirus 71 and coxsackievirus A16 is necessary.

Drug Discovery of Host CLK1 Inhibitors for Influenza Treatment.

The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.

Human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in Beijing, China.

BACKGROUND: HPyV6 is a novel human polyomavirus (HPyV), and neither its natural history nor its prevalence in human disease is well known. Therefore, the epidemiology and phylogenetic status of HPyV6 must be systematically characterized. METHODS: The VP1 gene of HPyV6 was detected with an established TaqMan real-time PCR from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. The HPyV6-positive specimens were screened for other common respiratory viruses with real-time PCR assays. RESULTS: The prevalence of HPyV6 was 1.7 % (15/887), and children ≤ 5 years of age accounted for 80 % (12/15) of cases. All 15 HPyV6-positive patients were coinfected with other respiratory viruses, of which influenza virus A (IFVA) (8/15, 53.3 %) and respiratory syncytial virus (7/15, 46.7 %) were most common. All 15 HPyV6-positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from 1.38 to 182.42 copies/µl nasopharyngeal aspirate specimen. The most common symptoms were cough (100 %) and fever (86.7 %). The complete 4926-bp genome (BJ376 strain, GenBank accession number KM387421) was amplified and showed 100 % identity to HPyV6 strain 607a. CONCLUSIONS: The prevalence of HPyV6 was 1.7 % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time PCR. Because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between HPyV6 and respiratory diseases.

Identification and genetic characterization of two porcine astroviruses from domestic piglets in China.

Porcine astrovirus are divided into five genotypes. In this study, we identified two porcine astroviruses (AstV-LL-1 and AstV-LL-2) by using sequence-independent single-primer amplification (SISPA) on faecal specimens of healthy domestic piglets younger than 15 days. The detection rate for both was 2.82% (14/497). AstV-LLs were then sequenced and characterised. Phylogenetic analysis revealed that they have the characteristics of porcine astrovirus (PastV) 2 and 5 and have some unique genetic features. Our findings show that the two astroviruses are novel lineages of PAstV2 and 5. The findings may be helpful in evaluating the ecology and evolution of astroviruses in pigs.