Exploration of the in vitro Antiviral Effects and the Active Components of Changyanning Tablets Against Enterovirus 71.

Purpose: This study aims to investigate the in vitro antiviral effects of the aqueous solution of Changyanning (CYN) tablets on Enterovirus 71 (EV71), and to analyze its active components. Methods: The in vitro anti-EV71 effects of CYN solution and its herbal ingredients were assessed by testing the relative viral RNA (vRNA) expression level and the cell viability rates. Material basis analysis was performed using HPLC-Q-TOF-MS/MS detection. Potential targets and active components were identified by network pharmacology and molecular docking. The screened components were verified by in vitro antiviral experiments. Results: CYN solution exerted anti-EV71 activities as the vRNA is markedly reduced after treatment, with a half maximal inhibitory concentration (IC50) of 996.85 µg/mL. Of its five herbal ingredients, aqueous extract of Mosla chinensis (AEMC) and leaves of Liquidambar formosana Hance (AELLF) significantly inhibited the intracellular replication of EV71, and the IC50 was tested as 202.57 µg/mL and 174.77 µg/mL, respectively. Based on HPLC-Q-TOF-MS/MS results, as well as the comparison with the material basis of CYN solution, a total of 44 components were identified from AEMC and AELLF. Through network pharmacology, AKT1, ALB, and SRC were identified as core targets. Molecular docking performed between core targets and the components indicated that 21 components may have anti-EV71 effects. Of these, nine were selected for in vitro pharmacodynamic verification, and only rosmarinic acid manifested in vitro anti-EV71 activity, with an IC50 of 11.90 µg/mL. Moreover, rosmarinic acid can stably bind with three core targets by forming hydrogen bonds. Conclusion: CYN solution has inhibitory effects on EV71 replication in vitro, and its active component was identified as rosmarinic acid. Our study provides a new approach for screening and confirmation of the effective components in Chinese herbal preparation.

Development of a universal real-time RT-PCR assay for detection of pan-SARS-coronaviruses with an RNA-based internal control.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for rapid diagnostic assays to prompt intensified virological monitoring both in human and wild animal populations. To date, there are no clinical validated assays for pan-SARS-coronavirus (pan-SARS-CoV) detection. Here, we suggest an innovative primer design strategy for the diagnosis of pan-SARS-CoVs targeting the envelope (E) gene using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, we developed a new primer-probe set targeting human ß2-microglobulin (B2M) as an RNA-based internal control for process efficacy. The universal RT-qPCR assay demonstrated no false-positive amplifications with other human coronaviruses or 20 common respiratory viruses, and its limit of detection (LOD) was 159.16 copies/ml at 95% detection probability. In clinical validation, the assay delivered 100% sensitive results in the detection of SARS-CoV-2-positive oropharyngeal samples (n = 120), including three variants of concern (Wuhan, Delta, and Omicron). Taken together, this universal RT-qPCR assay provides a highly sensitive, robust, and rapid detection of SARS-CoV-1, SARS-CoV-2, and animal-derived SARS-related CoVs.

Disinfection of influenza a viruses by Hypocrellin a-mediated photodynamic inactivation.

BACKGROUND: Influenza A viruses can be transmitted indirectly by surviving on the surface of an object. Photodynamic inactivation (PDI) is a promising approach for disinfection of pathogens. METHODS: PDI was generated using Hypocrellin A (HA) and red light emitting diode (625-635 nm, 280 W/m2). Effects of the HA-mediated PDI on influenza viruses H1N1 and H3N2 were evaluated by the reduction of viral titers compared to virus control. After selection of the HA concentrations and illumination times, the applicability of PDI was assessed on surgical masks. Reactive oxygen species (ROS) were determined using a 2'-7'-dichlorodihydrofluorescein diacetate fluorescence probe. RESULTS: In solution, 10 µM HA inactivated up to 5.11 ± 0.19 log10 TCID50 of H1N1 and 4.89 ± 0.38 log10 TCID50 of H3N2 by illumination for 5 and 30 min, respectively. When surgical masks were contaminated by virus before HA addition, PDI inactivated 99.99% (4.33 ± 0.34 log reduction) of H1N1 and 99.40% (2.22 ± 0.39 log reduction) of H3N2 under the selected condition. When the masks were pretreated with HA before virus addition, PDI decontaminated 99.92% (3.11 ± 0.19 log reduction) of H1N1 and 98.71% (1.89 ± 0.20 log reduction) of H3N2 virus. The fluorescence intensity of 2',7'-dichlorofluorescein in photoactivated HA was significantly higher than the cell control (P > 0.05), indicating that HA efficiently generated ROS. CONCLUSIONS: HA-mediated PDI is effective for the disinfection of influenza viruses H1N1 and H3N2. The approach could be an alternative to decontaminating influenza A viruses on the surfaces of objects.

Transmission Pattern of Measles Virus Circulating in China During 1993-2021: Genotyping Evidence Supports That China Is Approaching Measles Elimination.

BACKGROUND: To provide useful insights into measles elimination progress in China, measles surveillance data were reviewed, and the transmission patterns of measles viruses circulating in China during 1993-2021 were analyzed. METHODS: Measles incidence data from the National Notifiable Disease Reporting System of the China Center for Disease Control and Prevention were analyzed. A total of 17 570 strains were obtained from 30 of 31 provinces in mainland China during 1993-2021. The recommended genotyping window was amplified. Genotyping analysis was conducted for comparison with the reference strains. Phylogenetic analyses were performed to identify genetic relationships among different lineages within the genotypes. RESULTS: With high coverage of routine immunization and intensive supplementary immunization activities, measles incidence has shown a downward trend since 1993, despite 2 resurgences, reaching a historic low level in 2020-2021 (average 0.5 per million). During 1993-2021, 9 genotypes including domestic genotype H1; imported genotypes B3, D4, D8, D9, D11, G3, and H2; and vaccine-associated genotype A were identified. Among them, the genotype H1 strain circulated endemically in China for more than 25 years; the last strain was detected in Yunnan Province in September 2019. Multiple imported genotypes have been identified since 2009 showing different transmission patterns. Since April 2020, no imported strains have been detected, while vaccine-associated genotype A continues to be detected. CONCLUSIONS: The evidence of low incidence during 2020-2021 and virological surveillance data in this study confirm that China is currently approaching measles elimination.

A single-domain antibody inhibits SFTSV and mitigates virus-induced pathogenesis in vivo.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus that recently emerged in East Asian countries. SFTS is characterized by high fever, thrombocytopenia, leukopenia, multiorgan failure, and hemorrhage with case fatality rates of 6.3% to 30%. Neither antivirals nor vaccines are available at present. We previously demonstrated that neutralizing antibodies specific for SFTSV glycoprotein (Gn) played a vital role in the survival of patients with SFTS. Nanobodies from camels present unique properties, such as thermostability, high affinity, and low immunogenicity. In the current study, mammalian expressed SFTSV Gn was used to immunize a camel, and functional nanobodies were isolated from the B cell nanobody library constructed from the immunized animal. Clone SNB02 was selected for in-depth analysis for its inhibition of SFTSV replication both in vitro and in vivo. We showed that SNB02 potently inhibited SFTSV infection and prevented thrombocytopenia in a humanized mouse model and is a potential candidate for therapeutics.

High resolution metagenomic characterization of complex infectomes in paediatric acute respiratory infection.

The diversity of pathogens associated with acute respiratory infection (ARI) makes diagnosis challenging. Traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing ("meta-transcriptomics") to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARI. This provided comprehensive and clinically informative information relevant to understanding respiratory disease.

Establishment and characterization of an oral gerbil model for a non-mouse-adapted enterovirus 71 strain.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease (HFMD) with neurological and systemic complications worldwide, and it is mostly discovered in infants and young children. It is of great significance to establish suitable animal models of EV71 infection on research of distribution and pathogenesis of the virus. In this study, we established a successful infection of a non-mouse-adapted isolate of EV71 via oral route in 7-day-old Mongolian gerbil (Meriones unguiculatus), all of which were paralyzed and died within 10 days post infection. Analysis of virus loads in twelve tissues showed that virus was first detected in intestine, blood, heart, lung, and muscle one day post-infection, and then in the rest of the tissues/organs within the next few days, among which thymus, spleen, spinal cord and muscles had the highest virus titer at 5 days post infection. Pathological examination showed that severe necrosis was observed in skeletal muscle and spinal cord, and edema was observed in both heart and lung. Comparisons of host gene expression of various tissues from infected and non-infected gerbils revealed a general up-regulation of genes related to anti-viral response and a viral receptor gene (sialic acid-linked glycans), as well as a tissue(gut)-specific up-regulation of genes related to neuronal communication. Collectively, our results showed that EV71 could induce severe neurological complications as well as massive tissue damage all over the body, which indicates that oral infection of 7-day gerbils can be a suitable animal model to study the infection of EV71 in human.

Replication capacity and adaptability of a severe fever with thrombocytopenia syndrome virus at different temperatures.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease caused by the SFTS virus (SFTSV). Although fever and thrombocytopenia are the typical manifestations of SFTS, a specific SFTS case with no fever was observed in Zhejiang, China. In this report, we aimed to explore the probable reason for the absence of fever by analyzing the genetic characteristics and temperature sensitivity (ts) of the SFTSV strain ZJ2013-06, which was isolated from the specific case. Phylogenetically, different clusters of SFTSV strains circulated in Zhejiang. ZJ2013-06 was farthest from ZJ2014-02, an isolate belonging to a Chinese dominant cluster, and nearest to the coastal strain NB24/CHN/2013. Ts tests, performed on Vero cells at 37°C and 39°C, indicated that ZJ2013-06 had restricted replication at 39°C. Its viral loads were substantially reduced at 39°C compared with that at 37°C (approximately 100-fold reduction) and were significantly lower than that of ZJ2014-02 at 39°C (P < 0.01). By adaptive culture at 39°C, the induced strain ZJ2013-06-P7 was obtained. Owing to a reverse mutation (S1616), ZJ2013-06-P7 lost the ts of the original strain, displaying similar replication processes with NB24/CHN/2013. The results indicated that the amino acid residue 1616 was related to the ts characteristics of ZJ2013-06. Our study revealed that ZJ2013-06 was temperature-sensitive and may be related to the absence of fever in our case.

Molecular Epidemiology and Prevalence of Echovirus 30 in Zhejiang Province, China, from 2002 to 2015.

Echovirus serotype 30 (ECHO30) has been responsible for several recent worldwide outbreaks of viral meningitis. In Zhejiang Province, China, ECHO30 has been one of the main causes of viral meningitis for years. This study, using phylogenetic analysis of the VP1 gene, was performed to investigate the general molecular epidemiology and genetic patterns of ECHO30 circulating in Zhejiang Province between the years 2002 and 2015. The nucleotide sequences of ECHO30 VP1 showed that they were 64.8% identical with the prototype strain, Bastianni, while the amino acids were 84.9% identical. Phylogenetic analyses showed that ECHO30 in the Zhejiang area has diverged into two genotypes. Genotype I consists of strains isolated since 2002, whereas genotype II includes strains that were mainly isolated during the 2002 to 2004 outbreak. ECHO30 has been endemically circulating in both humans and the environment for a long period of time. Additionally, we evaluated the significance of recombination presented during the years 2005 to 2007 to demonstrate that recombination plays an important role in the prevalence of ECHO30 in the Zhejiang area.

Laboratory diagnosis of vaccine-associated measles in Zhejiang Province, China.

BACKGROUND/PURPOSE: Along with the improving vaccine coverage, suspected vaccine-associated measles has been reported in Zhejiang Province, China. In order to maintain the accuracy of the measles surveillance system, it is critical to discriminate between measles vaccine and wild-type virus. METHODS: Eight suspected cases of vaccine-associated measles were reported in Zhejiang Province during 2011 and 2014. Sera collected within 4 days and throat swabs collected within 6 days after rash onset were tested with immunoglobulin M and measles virus (MeV) RNA to confirm MeV infection. In order to further identify the vaccine-associated cases, throat swabs with positive MeV RNA were tested using an allelic discrimination real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay developed in this study, RT-PCR-restriction fragment length polymorphism (RFLP) recommended by the National Measles Laboratory, and RT-PCR followed by sequencing and genotyping. RESULTS: Combining anti-measles immunoglobulin M and RNA testing, eight cases were confirmed as MeV infection. Of the eight, two were identified as vaccine-associated cases by the allelic discrimination rRT-PCR assay, and one was identified by RT-PCR-RFLP. Subsequent sequencing and genotyping confirmed that the sequences of the two cases were identical to that of the Chinese vaccine strain. The developed allelic discrimination rRT-PCR was 10 times more sensitive than the RT-PCR-RFLP assay when RNA standards generated from three genotypes of MeV were tested. CONCLUSION: Vaccine-associated measles has been identified in Zhejiang. The developed allelic discrimination rRT-PCR assay is rapid and sensitive, which will facilitate the surveillance for vaccine-associated measles.

Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip.

West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 10(2) copies/µl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 10(1.5) TCID50/ml and 10(1.33) TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

Rapid detection of measles virus using reverse transcription loop-mediated isothermal amplification coupled with a disposable lateral flow device.

The measles virus (MeV) causes a highly contagious disease and efforts to reduce its spread are critical. A reverse transcription loop-mediated isothermal amplification assay coupled with a disposable lateral flow device (RT-LAMP-LFD) was developed for the rapid detection of MeV. The assay was performed in 40 min at an optimal temperature of 58 °C, with endpoint results visualized directly. A probe that was complementary to the RT-LAMP amplicon was designed to enhance assay specificity. Detection limit of the assay was 8.8 copies/µL synthetic RNA, which equals the sensitivity of real-time RT-PCR. Clinical specimens were used to validate the RT-LAMP-LFD in provincial Center for Disease Control and Prevention (CDC) (n = 245) and six municipal CDCs (n = 249). The results obtained using RT-LAMP-LFD and real-time RT-PCR were highly concordant. The RT-LAMP-LFD is rapid, stable, and does not require expensive equipment, which can be used for routine MeV monitoring in CDC laboratories.

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

Fast throughput determination of 21 allergenic disperse dyes from river water using reusable three-dimensional interconnected magnetic chemically modified graphene oxide followed by liquid chromatography-tandem quadrupole mass spectrometry.

We report the template-free fabrication of three-dimensional hierarchical nanostructures, i.e., three-dimensional interconnected magnetic chemically modified graphene oxide (3D-Mag-CMGO), through a simple and low-cost self-assembly process using one-pot reaction based on solvothermal method. The excellent properties of the 3D-Mag-CMGO are characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), vibrating sample magnetometer (VSM), FTIR, elementary analyzer (EA) and X-ray photoelectron spectroscopy (XPS). The easiness-to-handle of the magnetic dispersive solid phase extraction (Mag-dSPE) procedure is developed for preconcentration of 21 allergenic disperse dyes from river water. The obtained results show the higher extraction capacity of 3D-Mag-CMGO with recoveries between 80.0-112.0%. Furthermore, an ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) method for determination of 21 allergenic disperse dyes in river at sub-ppt levels has been developed with pretreatment of the samples by Mag-dSPE. The limits of quantification (LOQs) for the allergenic disperse dyes are between 0.57-34.05ng/L. Validation results on linearity, specificity, trueness and precision, as well as on application to the analysis of 21 allergenic disperse dyes in fifty real samples demonstrate the applicability to environment monitoring analysis.

[Prediction and analysis of epitopes of hemagglutinin of measles virus].

OBJECTIVE: To discuss the antigenic change caused by the mutation of amino acid on the epitopes of the hemagglutinin of measles virus. METHODS: The B cell linear epitopes in the hemagglutinin were predicted with bioinformatics software. Peptide pairs, which located on the same region but originated from measles vaccine and wild-type virus respectively, were designed and synthesized. After detecting the immunogenicity of peptides with indirect ELISA assay, sera against each peptide was prepared. Antigenic specificity between the two peptides within each peptide pair were tested by using cross ELISA assay, and then antigen ratios were calculated. RESULTS: All the synthesized peptides could bind with immune sera against measles virus, of which the peptide pair CW23/CW22 designed on the epitope region (273-282 aa) possessed the highest binding ability, while the peptide pair CW150/CW151 designed on the non-epitope region (418-427 aa) showed the lowest binding ability. The difference in antigenic specificity between the two peptides from different sources was significant. The antigenic ratio was up to 16 between CW23 (vaccine-originated) and CW22 (wild-type originated) , and 2.877±0.583 between CW123 (vaccine-originated) and CW124 (wild-type originated) (236-246 aa) . On the non-epitope regions, the antigenic ratios was only 1.631±0.481 between peptide pair CW125 and CW126 (356-364 aa) , but reached to 10.367±1.617 between CW150 and CW151. CONCLUSION: Although there were several conservative epitopes, specific amino acid mutation on the predicted epitope or non-epitope regions might cause the antigenic change of wild-type measles virus.

[Vein Scanning Projection Instrument Based on Two-Dimensional Scanning Mirror].

With the development of science and technology, new medical equipments is toward the direction of intelligent and portable. In order to assist medical personnel to patients with blood, developing from previous devices, a new kind of vein locating projection instrument based on two-dimensional scanning mirror is put forward. It can scan and project vein image using a scanning mirror. The related algorithm is also be improved, make vein scan projection more practical. The system finally set up can perform well in vein scan projection.

Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China.

In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E-3 (95% Highest posterior density: 3.8218E-3 to 7.7873E-3) while the NA gene showed 2.243E-3 (4.378E-4 to 3.79E-3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation.

[Variations on hemagglutinin gene of Zhejiang measles virus strains and differences with measles strains circulated both at home and abroad].

OBJECTIVE: To investigate the variations on hemagglutinin (H) gene of measles virus (MV) in Zhejiang province, and to analyze the differences with strains circulated both at home and abroad. METHODS: In total, 33 MV strains isolated in Zhejiang province between 1999 and 2011 were collected.RNA of the isolated MV strains was extracted and the complete sequences on H gene were amplified using RT-PCR assay. The products were compared with the Chinese vaccine strain Shanghai-191, which was downloaded from GenBank, and other 95 different MV strains from all over the world. RESULTS: 33 MV strains, isolated from the throat swab specimens collected from MV patients in Zhejiang province during 1999 to 2001, were used to conduct phylogenetic analysis with MV strains circulated in other areas of China during 1993 to 2007. The phylogenetic tree based on H gene sequences showed that all the Zhejiang MV strains located in H1a cluster, and no apparent time series and geographic restrictions were observed. Compared to the referenced vaccine strain Shanghai-191, the average variation rate on nucleotides and amino acids, and the evolutionary rate of H1a viruses from China during 2003 to 2011 were separately 5.15%, 4.44% and 5.81%, which were higher than the rates of H1a viruses during 1965 to 1993 (4.75%, 3.86% and 5.30%), and the rates of viruses during 1994 to 2002 (4.80%, 4.08% and 5.37%).However, the dn/ds ratios of strains within the three time periods were 0.19,0.21 and 0.23 respectively, which indicated that no evidence of positive selection was found on H1a MV strains during 1993 to 2011. A 24 stable amino acid variation sites on H gene was found between H1a viruses during 2003 to 2011 and the vaccine strain Shanghai-191. The largest variation occurred between vaccine and H1a strains, with 0.053 of the p-distance and 26-28 of amino acid mutations.However, only 15 stable amino acid variations were found between vaccine strain and genotype B3 or D4 strains.In addition, significant differences were found between H1a viruses and genotype B or D viruses, with 0.074 and 0.071 of p-distance and 27-33 of amino acid differences. CONCLUSION: Significant differences were found on H gene between MV strains subtype H1a and vaccine strains and other genotype strains. The variations were enlarged with the time coursing; therefore, the surveillance on variation of Chinese MV strains should be taken into account.

Origin and characteristics of internal genes affect infectivity of the novel avian-origin influenza A (H7N9) virus.

BACKGROUND: Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus. METHODOLOGY AND PRINCIPAL FINDINGS: We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. CONCLUSIONS: These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.

[Genomic sequences of human infection of avian-origin influenza A(H7N9)virus in Zhejiang province].

OBJECTIVE: To analyze the etiology and genomic sequences of human infection of avian-origin influenza A(H7N9)virus from Zhejiang province. METHODS: Viral RNA was extracted from patients of suspected H7N9 influenza virus infection and real-time RT-PCR was conducted for detection of viral RNA. All 8 segments of influenza virus were amplified by one-step RT-PCR and genomic sequences were assembled using the sequencing data. All the currently available HA and NA genes of the novel H7N9 virus, some other HAs from H7 subtype and NAs from N9 subtype were downloaded from public database for phylogenetic analysis, using the Mega 5.1 software. Mutations and variations were analyzed, using the genomic sequence data. RESULTS: Reactions for influenza type A, subtype H7 and subtype N9 were all positive and all the genomic fragments were amplified for sequencing. After alignment, sequences were subjected for phylogenetic analysis. The results revealed highest homology with A/duck/Zhejiang/12/2011(H7N3)in HA gene and with A/wild bird/Korea/ A14/2011(H7N9)in NA gene of the H7N9 influenza virus. All 6 genes coding for internal proteins shared highest identities with H9N2 avian influenza which were circulated in the Chinese mainland, in the last two years. The sequenced virus showed Q226L mutation in HA protein, but E627K was not presented in PB2 protein of this virus. The E627K mutation was shared by all the other novel H7N9 viruses resulted in human infections through analysis on the currently available sequences. CONCLUSION: Using the clinical samples, both detection of the viral genes and amplification of all 8 segments of the novel H7N9 influenza virus were accomplished. High homology of the novel H7N9 influenza viruses was observed by phylogenetic test, using the currently available sequences. The virus showed Q226L mutation on HA protein but E627K did not present on PB2 protein of this virus.