Prevalence and genetic diversity of human rhinovirus among patients with acute respiratory infections in China, 2012-2021.

To understand the prevalence of rhinovirus (RV) among acute respiratory infection (ARI) patients, 10-year ARI surveillance in multiple provinces of China were conducted during 2012-2021. Of 15 645 ARI patients, 1180 (7.54%) were confirmed to have RV infection and 820 (69.49%) were children under 5 years of age. RV typing was performed on the 527 VP1 gene sequences, and species A, B, and C accounted for 73.24%, 4.93%, and 21.82%, respectively. Although no significant difference in the proportions of age groups or disease severity was found between RV species, RV-C was more frequently detected in children under 5 years of age, RV-A was more frequently detected in elderly individuals (≥60), and the proportions of pneumonia in RV-A and RV-C patients were higher than those in RV-B patients. The epidemic peak of RV-A was earlier than that of RV-C. A total of 57 types of RV-A, 13 types of RV-B, and 35 types of RV-C were identified in RV-infected patients, and two uncertain RV types were also detected. The findings showed a few differences in epidemiological and clinical features between RV species in ARI patients, and RV-A and RV-C were more prevalent than RV-B.

NS1 Protein N-Linked Glycosylation Site Affects the Virulence and Pathogenesis of Dengue Virus.

Live attenuated vaccine is one of the most effective vaccines against flavivirus. Recently, site-directed mutation of the flavivirus genome using reverse genetics techniques has been used for the rapid development of attenuated vaccines. However, this technique relies on basic research of critical virulence loci of the virus. To screen the attenuated sites in dengue virus, a total of eleven dengue virus type four mutant strains with deletion of N-glycosylation sites in the NS1 protein were designed and constructed. Ten of them (except for the N207-del mutant strain) were successfully rescued. Out of the ten strains, one mutant strain (N130del+207-209QQA) was found to have significantly reduced virulence through neurovirulence assay in suckling mice, but was genetically unstable. Further purification using the plaque purification assay yielded a genetically stable attenuated strain #11-puri9 with mutations of K129T, N130K, N207Q, and T209A in the NS1 protein and E99D in the NS2A protein. Identifying the virulence loci by constructing revertant mutant and chimeric viruses revealed that five amino acid adaptive mutations in the dengue virus type four non-structural proteins NS1 and NS2A dramatically affected its neurovirulence and could be used in constructing attenuated dengue chimeric viruses. Our study is the first to obtain an attenuated dengue virus strain through the deletion of amino acid residues at the N-glycosylation site, providing a theoretical basis for understanding the pathogenesis of the dengue virus and developing its live attenuated vaccines.

Comparative analysis of the clinical and epidemiological characteristics of human influenza virus versus human respiratory syncytial virus versus human metapneumovirus infection in nine provinces of China during 2009-2021.

A comparative analysis of confirmed cases of human influenza virus (HIFV), human respiratory syncytial virus (HRSV), and human metapneumovirus (HMPV) was conducted to describe their clinical and epidemiological characteristics. During 2009-2021, active surveillance of acute respiratory infections (ARIs) was performed in nine provinces of China. Clinical and epidemiological information and laboratory testing results of HIFV, HRSV, and HMPV were analyzed. Among 11591 ARI patients, the single-infection rates of HIFV, HRSV, and HMPV were 15.00%, 9.59%, and 2.24%, respectively; the coinfection rate of these three viruses was 0.64%. HIFV infection was mainly in adults aged 15-59 years, accounting for 39.10%. HRSV and HMPV infections were mainly in children under 5 years old, accounting for 87.13% and 83.46%, respectively. Patients with HRSV infection were younger than HMPV. HRSV and HMPV had high similarities in clinical manifestations, presenting with lower respiratory symptoms. HIFV mainly presented with an upper respiratory infection. The epidemic peak of HRSV was earlier than that of HIFV, and that of HMPV was later than those of HRSV and HFIV. A total of 85.14% of coinfection cases were children under 5 years old. Coinfection might increase the risk of pneumonia in HIFV cases. During 2020-2021, the positive rates and seasonal patterns of these three viruses changed due to the impact of the COVID-19 pandemic. Certain clinical and epidemiological features were observed in HIFV, HRSV, and HMPV infections, which could be beneficial for guiding clinical diagnosis, treatment, and prevention of these three viruses in China.

Airborne Transmission of Influenza Virus in a Hospital of Qinhuangdao During 2017-2018 Flu Season.

The 2017-2018 flu season is considered to be one of the most severe, with numerous influenza outbreaks worldwide. In an infectious disease hospital of Qinhuangdao, air samples were collected daily from outpatient hall, clinical laboratory, fever clinic, children's ward (Children's Ward I/Children's Ward II), and adult ward during 23-29 January 2018 (peak flu activity) and 9-15 April 2018 (low flu activity). The air samples were collected with SLC-SiOH magnetic beads using impingement samplers. Real-time PCR assay was used to detect the RNA of airborne influenza (IFVA and IFVB) in the 91 collected aerosol samples. The results indicated that the air samples collected from the children's wards, adult ward and fever clinic were detected with airborne influenza viruses. However, the samples collected from outpatient hall and clinical laboratory were absence of influenza viruses. In addition, the subtypes of pH1N1/IFVA, H3N2/IFVA, yamagata/IFVB, and victoria/IFVB were detected among the samples with positive IFVA and IFVB. Notably, a new developed subtype of pH1N1 (an epidemic in 2018) was detected in the aerosol samples. In summary, this study profiled the distribution of airborne influenza in an infectious hospital in Qinhuangdao during 2017-2018 flu season. Patients infected with influenza could release airborne particles containing the virus into their environment. Healthcare workers and visitors in those places might have frequent exposure to airborne influenza virus. Therefore, we recommend some protective measures such as air disinfection and mask wearing to prevent and control the transmission of airborne influenza in hospital.

Epidemiological and clinical characteristics of Dengue virus outbreaks in two regions of China, 2014 - 2015.

Dengue virus (DENV), a single-stranded RNA virus and Flaviviridae family member, is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. DENV causes dengue fever, which may progress to severe dengue. Hospital-based surveillance was performed in two Chinese regions, Guangzhou and Xishuangbanna, during the dengue epidemics in 2014 and 2015, respectively. Acute-phase serum was obtained from 133 patients with suspected dengue infections during the peak season for dengue cases. Viremia levels, virus sero-positivity, serotype distribution, infection type, clinical manifestations and virus phylogenetics were investigated. Of the 112 DENV-confirmed cases, 92(82.14%) were IgM antibody-positive for DENV, and 69(51.88%) were positive for DENV RNA. From these cases, 47(41.96%) were classified as primary infections, 39(34.82%) as secondary infections and 26 (23.21%) as undetermined infections. The viremia levels were negatively correlated with IgM presence, but had no relationship with the infection type. DENV-1 genotype V dominated in Guangzhou, whereas the DENV-2 Cosmopolitan genotype dominated in Xishuangbanna, where fewer DENV-1 genotype I cases occurred. DENV-2 is associated with severe dengue illness with more serious clinical issues. The strains isolated during 2014-2015 are closely related to the isolates obtained from other Chinese regions and to those isolated recently in Southeast Asian countries. Our results indicate that DENV is no longer an imported virus and is now endemic in China. An extensive seroepidemiological study of DENV and the implementation of vector control measures against it are now warranted in China.

No MERS-CoV but positive influenza viruses in returning Hajj pilgrims, China, 2013-2015.

BACKGROUND: There is global health concern that the mass movement of pilgrims to and from Mecca annually could contribute to the international spread of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). In China, about 11,000 Muslim pilgrims participate in the Hajj gathering in Mecca annually. This is the first report of MERS-CoV and respiratory virus molecular screening of returning pilgrims at points of entry in China from 2013 to 2015. METHODS AND RESULTS: A total of 847 returning Hajj pilgrims participated in this study. The test results indicated that of the travelers, 34 tested positive for influenza A virus, 14 for influenza B virus, 4 for metapneumo virus, 2 for respiratory syncytial virus, and 3 for human coronavirus. There was a significant difference in the rates of positive and negative influenza virus tests between Hajj pilgrims with symptoms and those without. The detection rates of influenza virus were not significantly different among the three years studied, at 5.3, 6.0 and 6.3% for 2013, 2014 and 2015, respectively. DISCUSSION AND CONCLUSION: The MERS-CoV and respiratory viruses detection results at points of entry in China from 2013 to 2015 indicated that there were no MERS-CoV infection but a 5.7% positive influenza viruses in returning Chinese pilgrims.

A multiplex PCR assay for the detection of five influenza viruses using a dual priming oligonucleotide system.

BACKGROUND: A cost-effective, accurate and rapid simultaneous multiplex assay is required for testing and diagnoses of conventional and emerging viruses in clinical virology laboratories. We developed and optimized a dual priming oligonucleotide (DPO) multiplex PCR assay for detecting influenza viruses including seasonal H1N1, 2009 pandemic H1N1, H3N2, influenza B and H5N1. METHODS: The optimized multiplex DPO PCR was used to detect 233 clinical human samples. The results were compared to those obtained with RT-qPCR, conventional PCR and immunochromatographic assay. RESULTS: Specificity analysis revealed that the DPO PCR assay amplified each target virus without any cross-amplification. Statistical analysis demonstrated that the multiplex DPO-PCR sensitivity was higher than for the immunochromatographic assay and lower than for qPCR, while no significant difference was observed compared with conventional PCR, when detecting influenza A and B. Additional experiments using the same sample panel indicated no significant differences between the number of positive samples detected by multiplex DPO PCR and RT-qPCR when applying a Cq with a value lower than 30. CONCLUSIONS: The five-targeted simultaneous multiplex DPO PCR assay could be easily adopted into routine practice. This approach is cost effective with a short running time, low technical requirements for the detection of influenza virus and early diagnosis in clinical laboratories.

First confirmation of imported dengue virus serotype 2 complete genome in urine from a Chinese traveler returning from India.

Dengue virus (DENV) is a mosquito-borne virus that has four serotypes. Collection of serum from patients is time- and labor- consuming, and presents a high injury risk for infants and children. The genomic and serological diagnosis of imported dengue fever from a urine sample was used as a non-invasive diagnostic method in this study. A serum sample was collected on disease day 5, and a serum and urine sample were collected on disease day 8 and 18. The results of serological tests for DENV IgM revealed that the serum samples were positive for DENV. The results of RT-qPCR assay revealed that the serum sample collected on day 5 was DENV-positive; however, the serum sample collected on day 8 and 18 were negative for DENV. The urine sample collected on day 8 and 18 were DENV-positive. We also sequenced the complete DENV genome (10723 bp) from the urine sample (GenBank KF479233). The results of phylogenetic and epidemiological analysis indicated strong confirmation that the strain was located within the DENV-2 group with a 100% bootstrap value. In this report, we (1) provided the first evidence of a DENV infection that was imported from India to a non-endemic city of China, (2) investigated the DENV genome detection having a longer timeframe for positive detection in urine sample compared to previous studies, (3) provided the sequence results for the complete DENV-2 genome from a concentrated urine sample (4) discussed how virus-typing results could be used to manage the risk of sero-specific and re-infected travel-associated dengue fever.

[Evaluation of a self-prepared anti-WNV-IgG diagnostic ELISA kit with a panel of serum samples collected from the people from areas in which West Nile fever is endemic].

In view of that there is no report of west Nile virus infection cases in our country, evaluation the self-prepared anti-WNV-IgG diagnostic ELISA kit should be employed with the establishment of the serum sample panel collected from the entry personnel. All individuals of entry personnel were traveled from epidemic area of infectious west Nile disease. In our study, the serum samples were both detected by self-prepared anti-WNV-IgG diagnostic ELISA kit and the FDA certified kits ,which are FOCUS West Nile Virus IgG Dxselect and Panbio Dengue IgG Capture ELISA kits. The self-prepared kit and FDA certified kits were compared and assessed simultaneously. Furthermore, the specificity, repeatability and stability of the kits were also evaluated. The results indicated that no significant difference of detective rates (35. 6% for self-prepared kit vs. 32.5% for FOCUS kit, χ2 = 3. 05, P > 0.05) and good consistency (Kappa = 0.8372) between the self-prepared kit and FDA certified kits. Also, the positive coincidence rate, the negative coincidence rate and the total coincidence rate were calculated as 91.18%, 95.34% and 92.66%, respectively. The laboratory self-developed kit presented similar quality as the counterpart kits with FDA certificate. The development of our self-prepared anti-WNV-IgG diagnostic ELISA kit will provide technical support for the prevention and control of west Nile virus entry.

Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae.

A total of 110 strains of Klebsiella pneumoniae were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of K. pneumoniae. For optimization of electrophoresis parameters (EPs) of XbaI-PFGE, 11 isolates were analyzed with XbaI digestion using three EPs. The EP of a switch time of 6 to 36 s for 18.5 h gave clearest patterns and was declared the optimal EP for XbaI PFGE of K. pneumoniae. By software analysis and pilot study, AvrII was chosen as another PFGE enzyme. Both XbaI- and AvrII-PFGE gave D-values higher than 0.99 for 69 K. pneumoniae isolated from different sources. Our results also showed good typeability, reproducibility of both XbaI- and AvrII-PFGE for K. pneumoniae subtyping. Furthermore, the established PFGE method also had good discriminatory power to distinguish outbreak K. pneumoniae strains and a high degree of consistency with multilocus sequence typing method. A rapid PFGE protocol was established here, which could be used for genotyping and other researches of K. pneumoniae.

Complete Genome Sequence of Wohlfahrtiimonas chitiniclastica Strain SH04, Isolated from Chrysomya megacephala Collected from Pudong International Airport in China.

Wohlfahrtiimonas chitiniclastica bacilli that live in the larvae of a parasitic fly were recently isolated and are speculated to be the cause of fulminant sepsis. Here we report and analyze the complete genome sequence of Wohlfahrtiimonas chitiniclastica strain SH04. No complete genome sequence of a Wohlfahrtiimonas chitiniclastica isolate has been documented previously.

Complete genome sequence of an amur virus isolated from Apodemus peninsulae in Northeastern China.

Amur virus was recently identified as the causative agent of hemorrhagic fever with renal syndrome. Here we report the complete genome sequence of an Amur virus isolated from Apodemus peninsulae in Northeastern China. The sequence information provided here is critical for the molecular epidemiology and evolution of Amur virus in China.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

OBJECTIVE: Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. METHODS: Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. RESULTS: We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. CONCLUSIONS: The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Complete genome sequence of Seoul virus isolated from Rattus norvegicus in the Democratic People's Republic of Korea.

Seoul virus (SEOV) is responsible for 25% of cases of hemorrhagic fever with renal syndrome in Asia. Here we report the complete genome of strain DPRK08. The sequence information provided here is useful for understanding the molecular character of SEOV in the Democratic People's Republic of Korea (DPRK) and the circulation of SEOV in East Asia.

[Removal of oral Prevotella intermedia Endotoxin by octyl phenyl polyoxyethylene ether extraction method].

OBJECTIVE: To investigate an effective purification method for removing endotoxin from Prevotella intermedia. METHODS: The main protein ingredients of bacteria prepared from ammonium sulfate precipitation were further treated with octyl phenyl polyoxyethylene ether (Triton X-114), and then processed at 4°C, 37°C and 25°C. The obtained aqueous phase after at least two more cycle repeated operations was assayed for endotoxin by Western blotting, LAL-clotting method, in vitro cell stimulation and in vivo animal experiments. RESULTS: Western blotting and LAL-clotting method demonstrated that the reduction in endotoxin level was greater than 99.99% and recovery of the proteins after endotoxin removal was greater than 90% with Triton X-114 treatment for 3 cycles. The cytokines expression level was lower in both in vitro cell stimulation and in vivo animal experiments than in untreated group (P < 0.05). CONCLUSIONS: The extraction method provides a new choice for endotoxin removal from large volumes of the oral Prevotella intermedia.

[Expression, purification and identification of St. Louis encephalitis virus-like particles in eukaryotic cells].

OBJECTIVE: To express St. Louis encephalitis virus-like particles in mammalian cells, it will provide a prerequisite for further immunological diagnostic studies. METHODS: 293T-cell were transiently transfected with recombinant PreM-E plasmid. Expression and antigenicity of the purified protein were determined by transmission electron microscope (TEM), Western-Blot, immunofluorescence assay and ELISA. RESULTS: Recombinant subviral particles, about 50 nm in diameter, were observed by TEM in the supernatant of transfected cells. The results of Western-Blot, IFA and ELISA showed the recombinant proteins retained immunoreactivity similar to those of native virus proteins. CONCLUSION: St. Louis encephalitis virus-like particles has good antigenicity and physical appearance. It will provide a prerequisite for further immune diagnostic reagent.

[Transmission electron microscopy investigation of the denudation of the envelope of influenza virus treated with Nonidet P-40 and the binding influence of antibody].

OBJECTIVE: Through observing the morphological changes of the prepared influenza viruses (H1N1) treated with the different concentration of Nonidet P-40 solutions and added with antibody against the influenza virus using transmission electron microscopy (TEM), to explore the principle of application of the internal antibody for immunoassay of influenza virus. METHODS: Through treating the virus samples with serial diluted Nonidet P-40 solutions from 0.01% to 0.2% and/or not adding antibody against the influenza virus, then investigating the samples by TEM to obtain the morphological changes of the virions. RESULTS: The serial images show that the denudation degree of the virions is proportional with the rise of NP-40 concentration, and partly denuded virion image appeared at 0.1% NP-40 treatment, also under this condition no influence was observed on antibody binding to the virus. CONCLUSION: This work demonstrated the interaction between influenza virion and its antibody under nonionic surfactants existing, which supports the advantages of sample preparation for immunoassay enveloped virus using internal antibody theoretically.