Genesis and Spread of Newly Emerged Highly Pathogenic H7N9 Avian Viruses in Mainland China.

The novel low-pathogenic avian influenza A H7N9 viruses (LPAI H7N9 viruses) have been a threat to public health since their emergence in 2013 because of the high rates of mortality and morbidity that they cause. Recently, highly pathogenic variants of these avian influenza A H7N9 viruses (HPAI H7N9 viruses) have emerged and caused human infections and outbreaks among poultry in mainland China. However, it is still unclear how the HPAI H7N9 virus was generated and how it evolved and spread in China. Here, we show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region and spread southward to the Pearl River Delta region, possibly through live poultry trade. After introduction into the Pearl River Delta region, the origin LPAI H7N9 virus acquired four amino acid insertions in the hemagglutinin (HA) protein cleavage site and mutated into the HPAI H7N9 virus in late May 2016. Afterward, the HPAI H7N9 viruses further reassorted with LPAI H7N9 or H9N2 viruses locally and generated multiple different genotypes. As of 14 July 2017, the HPAI H7N9 viruses had spread from Guangdong Province to at least 12 other provinces. The rapid geographical expansion and genetic evolution of the HPAI H7N9 viruses pose a great challenge not only to public health but also to poultry production. Effective control measures, including enhanced surveillance, are therefore urgently needed.IMPORTANCE The LPAI H7N9 virus has caused five outbreak waves in humans and was recently reported to have mutated into highly pathogenic variants. It is unknown how the HPAI H7N9 virus originated, evolved, and disseminated in China. In this study, we comprehensively analyzed the sequences of HPAI H7N9 viruses from 28 human and 21 environmental samples covering eight provinces in China that were taken from November 2016 to June 2017. The results show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region. However, the insertion of four amino acids into the HA protein cleavage site of an LPAI H7N9 virus occurred in late May 2016 in the Pearl River Delta region. The mutated HPAI H7N9 virus further reassorted with LPAI H7N9 or H9N2 viruses that were cocirculating in poultry. Considering the rapid geographical expansion of the HPAI H7N9 viruses, effective control measures are urgently needed.

Genetic variation in the hemagglutinin of A(H1N1) 09pdm and A(H3N2) influenza viruses in the Beijing area from 2009 to 2014.

BACKGROUND: A(H1N1) 09pdm and A(H3N2) influenza viruses are the main cause of occasional influenza pandemics and seasonal influenza epidemics around the world. Unfortunately, the understanding of long-term genetic variation in these viruses remains limited. METHODS: In this study, hemagglutinin genes from 90 A(H1N1) 09pdm and 48 A(H3N2) influenza viruses in the Beijing area from 2009 to 2014 were sequenced and analyzed. RESULTS: The hemagglutinin genes in A(H1N1) 09pdm and A(H3N2) shared nucleotide similarity that ranged from 93.06% - 99.88% and 98.68% - 99.29%, respectively, compared with current vaccine strains. 10 and 7 amino acid mutations in antigenic sites were identified in these two strains, respectively. In addition, a new site 177 glycosylation, which did not exist in previous circulating strains, was identified in 3 A(H1N1) 09pdm isolates. CONCLUSIONS: This study demonstrated the continued evolution of seasonal influenza viruses in the Beijing area, indicating that an update of the vaccine is needed, especially for A(H1N1) 09pdm influenza virus.

Efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers.

OBJECTIVE: We compared the efficacy of medical masks (MM) and N95 respirators (N95) in preventing bacterial colonization/infection in healthcare workers (HCWs). METHODS: A cluster randomized clinical trial (RCT) of 1441 hospital HCWs randomized to medical masks or N95 respirators, and compared to 481 control HCWs, was performed in Beijing, China, during the winter season of 2008-2009. Participants were followed for development of clinical respiratory illness (CRI). Symptomatic subjects were tested for Streptococcus pneumoniae, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae or Haemophilus influenza type B by multiplex polymerase chain reaction (PCR). RESULTS: The rate of bacterial colonization was 2.8% in the N95 group (p=0.02), 5.3% among medical mask users (p<0.01) and 7.5% among the controls (p=0.16). N95 respirators were significantly protective (adjusted RR 0.34, 95% CI: 0.21-0.56) against bacterial colonization. Co-infections of two bacteria or a virus and bacteria occurred in up to 3.7% of HCWs, and were significantly lower in the N95 arm. CONCLUSIONS: N95 respirators were significantly protective against bacterial colonization, co-colonization and viral-bacterial co-infection. We showed that dual respiratory virus or bacterial-viral co-infections can be reduced by the use of N95 respirators. This study has occupational health and safety implications for health workers.

Evaluation of the FluA-Ag rapid assay for detection of influenza A viruses of human, avian, and swine origin.

BACKGROUND: Influenza viruses which cause human disease may include viruses that originate from humans, animals, or animal/human reassortants. This study evaluated the diagnostic sensitivity and specificity of a commercial FluA-Ag rapid assay in detecting influenza A viruses, either common or reassortant strains, to meet the need of influenza A virus early screening. METHODS: The laboratory accuracy of the rapid assay was evaluated using influenza A virus isolated strains and other related respiratory virus strains. The diagnostic sensitivity and specificity were assessed using clinical specimens from hospitals, poultry and pig farms. Nucleic acid testing (real-time RT-PCR) was used as the reference method, and all samples with different results detected by the rapid assay and real-time RT-PCR were confirmed by sequencing for full identification. RESULTS: Compared with the detection results of real-time RT-PCR, the rapid assay showed high laboratory accuracy with influenza A virus strains and other respiratory virus strains, and it also showed a higher relative diagnostic sensitivity and specificity of the rapid assay with clinical samples. CONCLUSIONS: The high accuracy and the higher diagnostic sensitivity and specificity for influenza A virus detection proved that this rapid assay will be a valuable tool for the quick detection of influenza A virus in the laboratory or on-site locations.

A randomized clinical trial of three options for N95 respirators and medical masks in health workers.

RATIONALE: We compared three policy options for the use of medical masks and N95 respirators in healthcare workers (HCWs). OBJECTIVES: A cluster randomized clinical trial of 1,669 hospital-based HCWs in Beijing, China in the winter of 2009-2010. METHODS: Participants were randomized to medical masks, N95 respirators, or targeted use of N95 respirators while doing high-risk procedures or barrier nursing. Outcomes included clinical respiratory illness (CRI) and laboratory-confirmed respiratory pathogens in symptomatic subjects. MEASUREMENTS AND MAIN RESULTS: The rate of CRI was highest in the medical mask arm (98 of 572; 17%), followed by the targeted N95 arm (61 of 516; 11.8%), and the N95 arm (42 of 581; 7.2%) (P < 0.05). Bacterial respiratory tract colonization in subjects with CRI was highest in the medical mask arm (14.7%; 84 of 572), followed by the targeted N95 arm (10.1%; 52 of 516), and lowest in the N95 arm (6.2%; 36 of 581) (P = 0.02). After adjusting for confounders, only continuous use of N95 remained significant against CRI and bacterial colonization, and for just CRI compared with targeted N95 use. Targeted N95 use was not superior to medical masks. CONCLUSIONS: Continuous use of N95 respirators was more efficacious against CRI than intermittent use of N95 or medical masks. Most policies for HCWs recommend use of medical masks alone or targeted N95 respirator use. Continuous use of N95s resulted in significantly lower rates of bacterial colonization, a novel finding that points to more research on the clinical significance of bacterial infection in symptomatic HCWs. This study provides further data to inform occupational policy options for HCWs. Clinical trial registered with Australian New Zealand Clinical Trials Registry http://www.anzctr.org.au (ACTRN 12609000778280).

Characteristics of group A Streptococcus strains circulating during scarlet fever epidemic, Beijing, China, 2011.

Scarlet fever is one of a variety of diseases caused by group A Streptococcus (GAS). During 2011, a scarlet fever epidemic characterized by peak monthly incidence rates 2.9-6.7 times higher than those in 2006-2010 occurred in Beijing, China. During the epidemic, hospital-based enhanced surveillance for scarlet fever and pharyngitis was conducted to determine characteristics of circulating GAS strains. The surveillance identified 3,359 clinical cases of scarlet fever or pharyngitis. GAS was isolated from 647 of the patients; 76.4% of the strains were type emm12, and 17.1% were emm1. Almost all isolates harbored superantigens speC and ssa. All isolates were susceptible to penicillin, and resistance rates were 96.1% to erythromycin, 93.7% to tetracycline, and 79.4% to clindamycin. Because emm12 type GAS is not the predominant type in other countries, wider surveillance for the possible spread of emm12 type GAS from China to other countries is warranted.

Surveillance for avian influenza A(H7N9), Beijing, China, 2013.

During surveillance for pneumonia of unknown etiology and sentinel hospital-based surveillance in Beijing, China, we detected avian influenza A(H7N9) virus infection in 4 persons who had pneumonia, influenza-like illness, or asymptomatic infections. Samples from poultry workers, associated poultry environments, and wild birds suggest that this virus might not be present in Beijing.

[Analysis on the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing in 2009].

OBJECTIVE: To explore the characteristics of the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing. METHODS: A total of 21 samples of throat swabs were collected from surveillance-designated hospitals between June and December in 2009, including 10 severe cases (4 death cases) and 11 mild cases. RNA of the virus were extracted,and the amplified primers of the whole genome were designed.Reverse transcription and PCR were performed to the RNA and then the PCR product was sequenced by software to analyze the evolution of the viral genes and the variation of the amino acids. RESULTS: Compared with the reference vaccine strain A/California/07/2009 (H1N1), the genetic nucleotide homology in the eight segments of the pandemic H1N1 virus in Beijing in 2009 was higher than 99%, without significant variation. Among them,the genetic distance of hemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) was comparatively far, separately 0.0050, 0.0040 and 0.0040.The gene of HA, P83S, the gene of NA, N248D, the gene of polymerase (PA), P224S and the gene of NP, V100I and L122Q were found to mutate in all the samples. Genes of HA, NA, NP, PA, PB 2 and nonstructural protein (NS1) in severe cases showed obviously clustered evolution. The mutation of gene S128P and S203T of HA, gene R269R and D547E of PA, gene T588I of PB 2 and gene I123V of NS mainly happened in severe cases, separately counting 6, 9, 6, 7, 9 and 6 cases. The relevance between the mutation happened in S203T of HA, R269K and D547E of PA and the severeness of the cases showed statistical significance (P < 0.05). The mutations of HA gene were mainly on the Ca and Cb antigene domains. No drug resistant mutation was found on NA gene but happened on matrix protein 2 (M2 gene). None of the mutations were found on the virulence related genes. CONCLUSION: A high homology was found between the pandemic H1N1 virus in Beijing in 2009 and the reference vaccine strain A/California/07/2009(H1N1). Mutational sites related with the severe and fatal cases were found, but not the virulence related mutation.

[Establishment of a high-throughput respiratory virus detection technology without RNA purification and reverse transcription].

OBJECTIVE: To establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring. METHODS: We used high-throughput microsphere-based flexible multi-analyte profiling technology (xMAP) coupled with signal amplification molecules to simultaneously detect RNAs of 8 viruses including influenza viruses A and B, parainfluenza viruses type 1, 2 and 3, respiratory syncytial viruses A and B, and metapneumovirus in a 96-well plate format. The sensitivity and specificity of the method for the synthetic viral RNAs were evaluated. RESULTS: There was no cross-reactivity among the 8 respiratory viral target RNAs. The detection limits for the 8 viral in intro-transcribed RNAs ranged from 1204 to 4695 RNA copies. CONCLUSION: We establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.

Understanding the Impact of Microstructures on Reconstitution and Drying Kinetics of Lyophilized Cake Using X-ray Microscopy and Image-Based Simulation.

The drying time of lyophilization and resultant cake microstructure are dependent on each other as water and solvent leave a lyophilized cake. The drying rate affects the size, distribution, and tortuosity of the pores as these macropores evolve during the primary drying phase, which in return impact the further removal of water and solvent from the cake throughout the drying period. This interplay results in a microstructure that determines the reconstitution time for a given formulation. The current study employs advanced X-ray Microscopy (XRM) coupled with mathematical models to correlate the microstructure with the drying kinetics and the reconstitution time. The normalized diffusion coefficients, derived from the reconstructed 3D microstructure of the cake, correlate with the solid content of the pre-lyophilization solution and agree with the mass transfer coefficients from a semi-empirical drying model built with lyophilization process data. Specifically, a solution with less solid content leads to a lyophilized cake with larger pores, thinner walls, and a greater pore volume compared to a solution with more solid content. Consequently, models from the microstructure and drying experiments reveals faster mass transfer independently. While the mass transfer models from the cake structure and the lyophilization process data accurately represents the drying kinetics, both models are inadequate to describe the reconstitution process due to the significant impact from formulation ingredients that alter the mass transfer mechanism via solubility and wettability. In summary, X-ray microscopy imaging and mathematical models are powerful tools that provide insights into the lyophilization process from a new angle.

[Application of EARS in early-warning of influenza pandemic in Beijing].

OBJECTIVE: To illustrate the efficiency of cumulative sum (CUSUM) in pre-warning of the influenza peak in Beijing. METHODS: CUSUM was used to analyze the data of influenza like illness (ILI), and the results of the influenza laboratory surveillance was regarded as the gold standard to judge the approaching of the influenza peak. RESULTS: The surveillance was launched in 421 hospitals in Beijing during the 2009 to 2010 influenza season, while the influenza laboratory surveillance was launched by 7 collaborative laboratories. From Jun. 2009 to Apr. 2010, the average ILI percentage in the 421 hospitals was 2.56%. In the study, 19 262 pharyngeal swab samples were collected from the ILI cases in 11 hospitals and 5 045 of them were tested positive for the influenza virus, with the novel swine-origin influenza A H1N1 virus dominating. After analyzing of the ILI surveillance data with CUSUM, it was found that the ILI surveillance in Beijing could make a satisfactory early warning for the approaching of the influenza peak referring to the gold standard based on the influenza laboratory results. CONCLUSION: It could give the prediction and early warning for the influenza peak efficiently and precisely, by using CUSUM to analyze the influenza surveillance data of Beijing.

An Updated Review on SARS-CoV-2 Infection in Animals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has lasted for two years and caused millions of infections and deaths in humans. Although the origin of SARS-CoV-2 infection in humans remains unknown, infection in animals has been frequently reported in varieties of animals all over the world. Both experimental and natural infections of SARS-CoV-2 in different animal species provide useful information on viral host range and pathogenicity. As the pandemic continues to evolve, SARS-CoV-2 infection in animals will be expanding. In this review, we summarized SARS-CoV-2 testing and infection in animals as well as SARS-CoV-2 strains and transmission in animals. Current data showed that at least 18 different animal species tested positive for SARS-CoV-2. These 18 animal species belong to pet, captive, farmed, and wild animals. Fifteen of the eighteen animal species were known to be positive for the Delta variant and ten animal species were infected with two different types of variants. Human-to-animal, animal-to-animal, and animal-to-human transmission events were suggested in different outbreaks involved in animal infection with SARS-CoV-2. Continued testing, immunization, and surveillance are warranted.

Screening and Analysis of Serum Protein Biomarkers Infected by Coronavirus Disease 2019 (COVID-19).

Coronavirus disease 2019 (COVID-19) has spread widely around the world, and in-depth research on COVID-19 is necessary for biomarkers and target drug discovery. This analysis collected serum from six COVID-19-infected patients and six healthy people. The protein changes in the infected and healthy control serum samples were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). The differential protein signature in both groups was retrieved and analyzed by the Kyoto Encyclopedia of Gene and Genomes (KEGG), Gene ontology, COG/KOG, protein-protein interaction, and protein domain interactions tools. We shortlisted 24 differentially expressed proteins between both groups. Ten genes were significantly up-regulated in the infection group, and fourteen genes were significantly down-regulated. The GO and KEGG pathway enrichment analysis suggested that the chromosomal part and chromosome were the most enriched items. The oxytocin signaling pathway was the most enriched item of KEGG analysis. The netrin module (non-TIMP type) was the most enriched protein domain in this study. Functional analysis of S100A9, PIGR, C4B, IL-6R, IGLV3-19, IGLV3-1, and IGLV5-45 revealed that SARS-CoV-2 was closely related to immune response.

Detection by environmental surveillance and genomic characterization of H5N8 highly pathogenic avian influenza virus from a poultry meat market in Beijing, China, 2021-22.

Since 2010 the year when it was first reported in domestic ducks in China, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks in different countries. The first outbreak wave was documented in South Korea and Japan in 2014 and the second wave was reported in Asian and European countries in 2016. More importantly, zoonotic infection was first reported in poultry workers in Russia in 2021. Therefore, active surveillance on H5N8 is highly needed. Surveillance on live birds instead of environmental samples is commonly reported. In the present study, we reported detection and genomic characterization of an environmental H5N8 strain in environmental samples of Tongzhou poultry meat markets in Beijing on a monthly basis from March 2021 to February 2022. Among 600 samples screened, a total of 27 samples were positive for influenza A virus with 4 typed as H5N8, 10 H7N9, and 13 H9N2. Whole genome sequencing and analysis of one duck neck with a higher virus load showed that A/Environment sample/Beijing/TZ001/20 21 (H5N8) clade 2.3.4.4b had the highest identities (over 99%) in all eight segments with H5N8 isolates from wild birds swan and tern in Hubei and had polybasic cleavage site PLREKRRKR/G, characteristic of a HPAI virus. Overall, our data indicate that HPAI H5N8 virus is still circulating in domestic ducks in China in the study period and continued surveillance in domestic and wild birds is needed to control H5N8.

Serologic survey of pandemic influenza A (H1N1 2009) in Beijing, China.

OBJECTIVE: To examine the frequency and distribution of antibodies against pandemic influenza A (H1N1 2009) [H1N1] in populations in Beijing and elucidate influencing factors. METHODS: In January 2010, a randomized serologic survey of pandemic influenza A (H1N1 2009) was carried out. Six districts that were randomly selected with a total of 4601 participants involved in the survey have their antibody level tested by hemagglutination inhibition assay. RESULTS: Among the 4601 participants, the overall seropositive rate for pandemic influenza A (H1N1 2009) antibodies was 31.7%. The seropositivity prevalence in participants who received the pandemic H1N1 vaccination was 60.9%. Only 53.1% of the pandemic influenza A (H1N1 2009) seropositive individuals who had not received the vaccination experienced respiratory tract infection symptoms. Multivariate logistic regression revealed that factors such as age, occupation, dwelling type, whether the participant's family included students in school, and the vaccination history with pandemic influenza A (H1N1 2009) were associated with antibody titers (p<0.05). CONCLUSIONS: Our data indicated that almost 30.0% of the residents had appropriate antibody titers against pandemic influenza A (H1N1 2009) in Beijing, and these titers may provide an immune barrier.

[The significance of different sample types in study of pandemic A (H1N1) influenza diagnosis].

OBJECTIVE: To explore the value of different types of samples, including throat swabs, stools, bloods in pandemic A (H1N1) influenza diagnosis and virus shedding patterns. METHODS: From May to June in 2009, 135 samples were collected from 23 confirmed cases of pandemic influenza A (H1N1) infection, including 99 throat swabs, 14 stools, 11 bloods, 1 respiratory tract washing from 13 confirmed cases and 10 blood samples from other confirmed cases. The virus was detected by real-time RT-PCR, the antibody was detected by haemagglutination inhibition assay. RESULTS: For 99 throat swabs of 13 patients, the median time of the first positive real-time RT-PCR was 1 day (ranged from 0 to 7 days) after the onset of the symptoms of illness; the median length of time duration of positive real-time RT-PCR results from throat swabs was 3 days (ranged from 1 to 15 days). Four cases intermittently released virus. One respiratory tract washing sample was positive. In 14 stools, 8 stools were real-time RT-PCR positive, the positive rate was 57.14%. The median time of the positive real-time RT-PCR was 3 days (ranged from 1 to 4 days) after the onset of the symptoms of illness. In 21 blood samples collected at 2 to 9 days of onset, 1 blood sample was real-time RT-PCR positive, the positive rate was 4.76%. All these 21 blood samples were antibody negative. CONCLUSION: Throat swabs and stools samples can be used as A (H1N1) influenza early diagnosis. The length of time duration of positive real-time RT-PCR in throat swabs was longer than stool samples and intermittently releasing of virus were found in throat swabs. Influenza A H1N1 cases showed the presence of small amount of viremia and antibody was negative in early blood samples (< 9 days).

Review of an influenza surveillance system, Beijing, People's Republic of China.

In 2007, a surveillance system for influenza-like illness (ILI) and virologic data was established in Beijing, China. The system tracked ILI and laboratory-confirmed influenza in 153 general hospitals from September 1, 2007, through April 30, 2008. To analyze the ILI surveillance data (weekly ILI rates and counts) and the effectiveness of the system, we used the US Centers for Disease Control and Prevention Early Aberration Reporting System. The data indicated that the highest rate of influenza isolation and the highest ILI count occurred in the first week of 2008. The system enabled us to detect the onset and peak of an epidemic.

Antimicrobial susceptibility and genotyping of Mycoplasma pneumoniae isolates in Beijing, China, from 2014 to 2016.

Background: The presence of macrolide-resistant Myocplasma pneumoniae has been frequently reported in recent years, especially in China. In this study, we investigated the antimicrobial susceptibility and genotype against M. pneumoniae isolates from 2014 to 2016, Beijing. Methods: We investigated the activities of four antibiotics against 81 M. pneumoniae isolates in vitro. All isolates were amplification of domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein fragments. All isolates were genotyped with duplex real-time PCR, MLVA and VNTR detection in p1 gene. Results: The macrolide resistance rate was 65.4% (53/81). Each of the macrolide-resistant M. pneumoniae isolates was resistant to erythromycin (Minimum Inhibitory Concentration, MIC, ≥256 µg/ml) and azithromycin (MIC, 2-64 µg/ml), but susceptible to tetracycline and levofloxacin in vitro. Fifty two macrolide-resistant isolates harbored the A2063G mutation, and only 1 macrolide-resistant isolates harbored the A2064G mutation in domain V of the 23S ribosomal RNA gene. The C162A, A430G, and T279C mutations in the L4 and L22 ribosomal protein genes were not responsible for macrolide resistance, but they were related to the particular genotype of M. pneumoniae. 95.7% of type 1 isolates (45/47) were macrolide-resistance, and 23.5% of the type 2 isolates (8/34) were macrolide-resistance. Type 2 M. pneumoniae macrolide-resistance rate was 50.6% higher than that of the previous reports in China. The eight macrolide-resistant type 2 M. pneumoniae isolates were belong to 3/5/6/2 and 3/5/7/2 MLVA genotypes. Conclusion: To our knowledge, this phenomenon likely resulted from a combination of genotype shifting from type1 to type 2 and antibiotic selection pressure in M. pneumoniae in China in recent years. The increase of resistance in type 2 is not due to the spread of same clone. However, the relationship between genotype shifts and macrolide resistance in M. pneumoniae needs to be further verified with more extensive surveillance data.