CqsA-introduced quorum sensing inhibits type VI secretion system 2 through an OpaR-dependent pathway in Vibrio parahaemolyticus.

The well-known food-borne pathogen Vibrio parahaemolyticus employs at least three quorum sensing signals to maintain its high environmental adaptability. V. parahaemolyticus CqsA, the synthase involved in 3-hydroxyundecan-4-one quorum sensing signal, introduces a quorum sensing network. The V. parahaemolyticus virulent factor type VI secretion system 2 (T6SS2), which is associated with adhesion to host cells, was previously reported to be regulated by a quorum sensing system. Herein, we set out to determine the role of CqsA-introduced quorum sensing (CIQS) in T6SS2-associated virulent regulation. Using a tandem mass tag (TMT)-based quantitative proteomics assay, 17 T6SS2 proteins were found having significantly higher abundances in the ΔcqsA strain than in the wild type strain. TMT proteomics assay results were confirmed by a parallel reaction-monitoring (PRM)-based proteomics assay. Two T6SS2 up-regulators, OpaR and CalR, were found under control of CIQS in the TMT proteomics assay, while OpaR was down-regulated and CalR was up-regulated by CIQS. Thus, it was hypothesized that CIQS would inhibit T6SS2 with an OpaR-dependent mechanism. Epistasis experiment with quantitative PCR was designed to analyze the role of OpaR in the process of CIQS inhibiting T6SS2 production. The mRNA levels of T6SS2 genes were up-regulated in the ΔcqsA strain while down-regulated in the ΔopaR strain and in the ΔcqsAΔopaR mutant, indicating that OpaR plays a predominant role in the regulation of T6SS2 by CIQS. Using a cell adhesion assay, we further found that the T6SS2-dependent adhesion activity of V. parahaemolyticus to Hela cells was also inhibited by CIQS and the inhibition was OpaR-dependent. In this study, we confirmed that V. parahaemolyticus CIQS inhibited T6SS2 through an OpaR-dependent pathway. It enriches the knowledge of how V. parahaemolyticus quorum sensing regulates its virulence.

Pathogen change of avian influenza virus in the live poultry market before and after vaccination of poultry in southern China.

BACKGROUND: The fifth wave of H7N9 avian influenza virus caused a large number of human infections and a large number of poultry deaths in China. Since September 2017, mainland China has begun to vaccinate poultry with H5 + H7 avian influenza vaccine. We investigated the avian influenza virus infections in different types of live poultry markets and samples before and after genotype H5 + H7 vaccination in Nanchang, and analyzed the changes of the HA subtypes of AIVs. METHODS: From 2016 to 2019, we monitored different live poultry markets and collected specimens, using real-time reverse transcription polymerase chain reaction (RT-PCR) technology to detect the nucleic acid of type A avian influenza virus in the samples. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. The χ2 test was used to compare the differences in the separation rates of different avian influenza subtypes. RESULTS: We analyzed 5,196 samples collected before and after vaccination and found that the infection rate of AIV in wholesale market (21.73%) was lower than that in retail market (24.74%) (P < 0.05). Among all the samples, the positive rate of sewage samples (33.90%) was the highest (P < 0.001). After vaccination, the positive rate of H5 and H7 subtypes decreased, and the positive rate of H9 subtype and untypable HA type increased significantly (P < 0.001). The positive rates of H9 subtype in different types of LPMs and different types of samples increased significantly (P < 0.01), and the positive rates of untypable HA type increased significantly in all environmental samples (P < 0.05). CONCLUSIONS: Since vaccination, the positive rates of H5 and H7 subtypes have decreased, but the positive rates of H9 subtypes have increased to varying degrees in different testing locations and all samples. This results show that the government should establish more complete measures to achieve long-term control of the avian influenza virus.

Corynebacterium anserum sp. nov., isolated from the faeces of greater white-fronted geese (Anser albifrons) at Poyang Lake, PR China.

Two Gram-stain-positive, facultatively aerobic, non-motile and rod- to coccoid-shaped bacterial strains, 23H37-10T and 4HC-13, were isolated from the faeces of greater white-fronted geese (Anser albifrons) at Poyang Lake, Jiangxi Province, PR China. Optimal growth was observed at 35-37 °C, pH 7.0-8.0 and with 0.5-1.5 % (w/v) NaCl. The 16S rRNA gene sequences of strains 23H37-10T and 4HC-13 were identical. Phylogenetic and phylogenomic analyses indicated that strains 23H37-10T and 4HC-13 formed an independent cluster within the genus Corynebacterium and showed 98.8, 97.4, 97.4 and 97.2 % 16S rRNA gene sequence similarity to Corynebacterium urogenitale LMM 1652T, Corynebacterium urealyticum DSM 7109T, Corynebacterium falsenii DSM 44353T and Corynebacterium jeikeium NCTC 11913T, respectively. Cells contained C18 :1 ω9c, C18 : 0 and C16 : 0 as the major cellular fatty acids and MK-9 (H2) as the predominant respiratory quinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol mannosides, two unidentified phospholipids, four unidentified glycolipids and one unidentified lipid. Strain 23H37-10T contained mycolic acids, with meso-diaminopimelic acid and arabinose as the major whole-cell hydrolysates. The genome G+C content of strains 23H37-10T and 4HC-13 was 55.2 mol%. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains 23H37-10T and 4HC-13 were 94.4 and 99.6 %, respectively. Strains 23H37-10T and 4HC-13 had dDDH and ANI values of less than 70 and 96 % with all available genomes of the genus Corynebacterium, respectively. The differential genotypic inferences, together with phenotypic and biochemical characteristics, suggested that strains 23H37-10T and 4HC-13 represent a novel species within the genus Corynebacterium, for which the name Corynebacterium anserum sp. nov. is proposed. The type strain is 23H37-10T (=GDMCC 1.1737T=KACC 21672T).

Nanchangia anserum gen. nov., sp. nov., isolated from feces of greater white-fronted geese (Anser albifrons).

Two rod-shaped and Gram-stain-positive bacteria (strains C64T and C62) were isolated in 2020 from faeces of greater white-fronted geese (Anser albifrons) from Poyang Lake, PR China. Their optimal growth conditions were at 37 °C, pH 7.0 and with 0.5 % (w/v) NaCl. The two isolates showed a highest 16S rRNA gene sequence similarity to Bowdeniella nasicola DSM 19116T (92.1 %). Phylogenetic/phylogenomic analyses indicated that strains C64T and C62 clustered independently in the vicinity of the genera Varibaculum, Winkia and Mobiluncus within the family Actinomycetaceae, but could not be classified clearly as members of any of these known genera. The average amino acid identity values between our isolates and available genomes of members of the family Actinomycetaceae were around the genus threshold value (45-65 %). The major cellular fatty acids of the strains were C18 : 1ω9c and C16 : 0. The predominant polar lipids were phosphatidylinositol, phosphatidylglycerol, phosphatidylcholine, diacylglycerol, triacylglycerol and cardiolipin. The amino acid composition of peptidoglycan contained alanine, glutamic acid and glycine. The major respiratory menaquinones were MK-8(H4) and MK-9(H4). The whole cell sugars included galactose, arabinose and glucose. On the basis of the results of the 16S rRNA gene sequences comparison, whole-genome phylogenomic analysis, phenotypic and chemotaxonomic characteristics, we propose that strains C64T and C62 represent a novel species belonging to a novel genus within the family Actinomycetaceae, for which the name Nanchangia anserum gen. nov., sp. nov. is proposed. The type strain is Nanchangia anserum C64T (=CGMCC 1.18410T=GDMCC 1.1969T=KCTC 49511T=KACC 22143T).

Molecular epidemiology of an outbreak of hand, foot, and mouth disease associated with subgenotype C4a of enterovirus A71 in Nanchang, China in 2014.

An outbreak of hand, foot, and mouth disease was reported through hospital-based surveillance in Nanchang, China in 2014. A total of 244 cases were reported, 176 (72.1%) cases were tested positive for enteroviruses by direct reverse transcription-polymerase chain reaction, in which enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), and untyped enteroviruses (UEV) accounted for 84.1%, 3.4%, and 12.5%, respectively. In this outbreak, children under 5 years old constituted more than 98% of the positive cases, and the ratio of male to female cases was 2.6 to 1 (P < 0.01). Phylogenetic analysis indicated that the Nanchang EV-A71 strains belonged to subgenotype C4a undergoing continuously evolutionary changes.

[Inhibitors of DNA-dependent protein kinase promote p53-independent apoptosis induced by 1, 4-benzoquinone in HL60 cells].

OBJECTIVE: To investigate the impact of NU7026 and Wortmannin, inhibitors of DNA-dependent protein kinase (DNA-PK), in HL60 cells apoptosis induced by 1, 4-benzoquinone (1, 4-BQ). METHODS: HL60 cells were divided into three groups according to the exposures: the poisoned groups which were treated with 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ for 24 h, respectively, the NU7026 groups which were preincubated with 10 µmol/L NU7026 for 1h prior to the 24h treatment of 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ and the Wortmannin groups which were preincubated with 25 µmol/L Wortmannin for 1h prior to the 24 h treatment of 0, 5, 10, 25 and 50 µmol/L 1, 4-BQ. Then we detected the apoptosis via flowcytometry Annexin V-FITC/PI and the DNA Ladder, respectively. We also tested the expressions of Bax mRNA with Real-Time PCR in HL60 cells which were exposed to 10 µmol/L NU7026 for 24 h, 25 µmol/L Wortmannin 24 h, 10 µmol/L 1, 4-BQ 24 h, 10 µmol/L NU7026 1h+10 µmol/L 1, 4-BQ 24 h and 25 µmol/L Wortmannin 1 h+10 µmol/L 1, 4-BQ 24 h, as well as null (control). We also examed the expressions of p53 in HL60 cells with Western blot. RESULTS: Annexin V-FITC/PI apoptosis tests suggested that apoptosis rates of NU7026+10 µmol/L 1, 4-BQ group and Wortmannin +10 µmol/L 1, 4-BQ were 17.6±1.19% and 15.2±1.22%, respectively. Both of results were higher than that of 10 µmol/L 1, 4-BQ group (6.3±1.04%); Apoptosis of NU7026+25 µmol/L 1, 4-BQ group was 46.2±3.55%, and Wortmannin +25 µmol/L 1, 4-BQ group 26.9±2.62%. Both of results were also higher than that of 25 µmol/L 1, 4-BQ group (14.1±1.54%); Apoptosis of NU7026+50 µmol/L 1, 4-BQ group (61.8±1.78%) was higher than that of 50 µmol/L 1, 4-BQ group (35.9±4.51%). The above results were all statistically significant (P < 0.05). RESULTS: of DNA-Ladder were basically consistent with those of Annexin V-FITC/PI apoptosis tests. In addition, both NU7026 and Wortmannin pretreatment elicited the higher expression of Bax mRNA in HL60 treated by 1, 4-benzoquinone with statistically significance (P < 0.05). However, p53 protein was not detected in HL60 cells as the western blot indicated. CONCLUSION: Inhibitors of DNA-PK, NU7026 and Wortmannin, promote p53-independent apoptosis induced by 1, 4-benzoquinone in HL60 cells.

Severe Rhipicephalus sanguineus tick infestation in a home from rural area of Nanchang in China.

On October 13, 2021, a tick infestation occurred in a home in rural area of Nanchang city, China, and we were asked to inspect the tick infestation. Ticks were collected in the largest number on courtyard door jambs, followed by living room and bedroom door jambs. Ticks were identified morphologically as Rhipicephalus sanguineus adults. The 16S rRNA analysis effectively distinguished the ticks in this study from other Rhipicephalus species, including R. sanguineus south-east, temperate and tropical lineages and identified genetically as R. sanguineus south China lineage. Tick samples were subjected to conventional PCR analysis and detected negative for the presence of tick-borne pathogens. Our findings indicate that there was low transmission risk of tick-borne pathogens to humans in the tick-infested home. Further studies are needed to proactively investigate the tick species in Nanchang, and determine the presence of tick-borne pathogens for assessing their threat to human health in the region.

Description of Corynebacterium poyangense sp. nov., isolated from the feces of the greater white-fronted geese (Anser albifrons).

Two novel Gram-positive, non-spore-forming, facultatively anaerobic, non-motile, and short rods to coccoid strains were isolated from the feces of the greater white-fronted geese (Anser albifrons) at Poyang Lake. The 16S rRNA gene sequences of strains 4H37-19T and 3HC-13 shared highest identity to that of Corynebacterium uropygiale Iso10T (97.8%). Phylogenetic and phylogenomic analyses indicated that strains 4H37-19T and 3HC-13 formed an independent clade within genus Corynebacterium and clustered with Corynebacterium uropygiale Iso10T. The average nucleotide identity and digital DNA-DNA hybridization value between strains 4H37-19T and 3HC-13 and members within genus Corynebacterium were all below 95% and 70%, respectively. The genomic G + C content of strains 4H37-19T and 3HC-13 was 52.5%. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine, and phosphatidyl inositol mannosides (PIM) were the major polar lipids, with C18:1ω9c, C16:0, and C18:0 as the major fatty acids, and MK-8 (H4), MK-8(H2), and MK-9(H2) as the predominant respiratory quinones. The major whole cell sugar was arabinose, and the cell wall included mycolic acids. The cell wall peptidoglycan contained meso-diaminopimelic acid (meso-DAP). The polyphasic taxonomic data shows that these two strains represent a novel species of the genus Corynebacterium, for which the name Corynebacterium poyangense sp. nov. is proposed. The type strain of Corynebacterium poyangense is 4H37-19T (=GDMCC 1.1738T = KACC 21671T).

Haemaphysalis longicornis calreticulin is not an effective molecular tool for tick bite diagnosis and disruption of tick infestations.

BACKGROUND: Tick calreticulin (CRT) is a calcium-binding protein secreted into the host during blood feeding. It has been used as a biomarker of tick exposure and has potential as an anti-tick vaccine, but there is no information about these uses for Haemaphysalis longicornis CRT (HlCRT). We synthesized recombinant H. longicornis CRT (rHlCRT) and evaluated its potential for tick bite diagnosis and for disrupting tick infestations. METHODS: The responses of mice and rabbits exposed to H. longicornis ticks were measured with ELISA to determine the antibody level against rHlCRT. To evaluate the effects of rHlCRT-induced anti-tick immunity, engorgement weight, tick engorgement index (TEI), feeding duration, ecdysis rate, and egg weight per engorged tick were compared between ticks fed on immunized and normal mice. RESULTS: Mean anti-tick CRT antibody levels in sera collected from mice at 1 and 15 days after primary tick exposure were not significantly different from the mean antibody levels in negative control mice that were not bitten by ticks (all P values > 0.05). No significant anti-HlCRT IgG responses developed in mice after second exposure to tick bites compared with the level of anti-HlCRT antibody response in negative control mice (all P values > 0.25). For rabbits, no significant differences in the antibody levels were observed in animals before challenge infestation and after tick exposures, and in animals after two tick exposures (all P values > 0.10). There were no significant differences in the body weight of ticks fed on immunized and normal mice (all P values > 0.15). No significant differences in TEI were observed between ticks fed on immunized mice and normal control mice (all P values > 0.50). There were no significant differences in feeding duration for female ticks, and feeding duration and ecdysis rate for nymphs in the experimental and control groups (all P values > 0.10 for feeding duration and P value = 0.19 for ecdysis rate). We did not observe a significant difference in egg weight per tick in the rHlCRT-immunized and the control groups (P = 0.88). CONCLUSIONS: HlCRT in H. longicornis tick saliva proteins appears to be nonimmunogenic to mammalian hosts like mice and rabbits. Vaccination with rHlCRT did not generate effective immunity against parthenogenetic and bisexual H. longicornis nymphs or female ticks. These results indicate that HlCRT is not a suitable molecular candidate for H. longicornis tick bite diagnosis and not effective for the disruption of tick infestations.

Identification and Genomic Characterization of Escherichia albertii in Migratory Birds from Poyang Lake, China.

Escherichia albertii is an emerging zoonotic foodborne enteropathogen leading to human gastroenteritis outbreaks. Although E. albertii has been isolated from birds which have been considered as the potential reservoirs of this bacterium, its prevalence in migratory birds has rarely been described. In this study, E. albertii in migratory birds from Poyang Lake was investigated and characterized using whole genome sequencing. Eighty-one fecal samples from nine species of migratory birds were collected and 24/81 (29.6%) tested PCR-positive for E. albertii-specific genes. A total of 47 isolates was recovered from 18 out of 24 PCR-positive samples. All isolates carried eae and cdtB genes. These isolates were classified into eight E. albertii O-genotypes (EAOgs) (including three novel EAOgs) and three E. albertii H-genotypes (EAHgs). Whole genome phylogeny separated migratory bird-derived isolates into different lineages, some isolates in this study were phylogenetically closely grouped with poultry-derived or patient-derived strains. Our findings showed that migratory birds may serve as an important reservoir for heterogeneous E. albertii, thereby acting as potential transmission vehicles of E. albertii to humans.

Changes of avian influenza virus subtypes before and after vaccination in live poultry in Nanchang, China from 2016 to 2019.

We investigated fluctuations in the detection rates of avian influenza virus (AIV) subtypes H5, H7, and H9 in live poultry in Nanchang city, Chinese province Jiangxi, before and after the Chinese nationwide AIV vaccination campaign against highly pathogenic (HP) AIV subtype H5 and H7. Samples were tested for nucleic acid of type A avian influenza virus by real-time reverse transcription polymerase chain reaction technology. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. Based on the analysis of 2119 samples collected from February 2016 to December 2019, we found that AIV subtypes H5, H7, H9 showed a seasonal pattern, and the positive rate of avian influenza tended to reach its peak in the colder season. The detection rate of AIV subtypes H5, H7, H9 of chickens (39.26%) was significantly higher than that of ducks (5.78%) and pigeons (4.31%). After vaccination, the positive rates of the H5 subtype (0.27%) and the H7 subtype (0.00%) decreased significantly, while the positive rate of the H9 subtype (29.95%) increased significantly. The H9 subtype has become the dominant subtype detected in live poultry and occupies a dominant position in the live bird market. This study showed that the government of China should establish measures for the long-term control of avian influenza.

The Impact of the Closure of the Live Poultry Market due to COVID-19 on the Avian Influenza Virus in Nanchang, Jiangxi Province, China.

This article aims to understand the changes in the detection rates of H5, H7, and H9 subtypes of avian influenza viruses (AIVs) in the live poultry markets (LPMs) in Nanchang City, Jiangxi Province, before and after the outbreak of the COVID-19. From 2019 to 2020, we monitored the LPM and collected specimens, using real-time reverse transcription polymerase chain reaction technology to detect the nucleic acid of type A AIV in the samples. The H5, H7, and H9 subtypes of influenza viruses were further classified for positive results. We analyzed 1,959 samples before and after the outbreak and found that the positive rates of avian influenza A virus (39.69%) and H9 subtype (30.66%) after the outbreak were significantly higher than before the outbreak (26.84% and 20.90%, respectively; P < 0.001). In various LPMs, the positive rate of H9 subtypes has increased significantly (P ≤ 0.001). Positive rates of the H9 subtype in duck, fecal, daub, and sewage samples, but not chicken samples, have increased to varying degrees. This study shows that additional measures are needed to strengthen the control of AIVs now that LPMs have reopened after the relaxing of COVID-19-related restrictions.

Surveillance, Epidemiology and Impact of EV-A71 Vaccination on Hand, Foot, and Mouth Disease in Nanchang, China, 2010-2019.

After the first national-scale outbreak of Hand, foot, and mouth disease (HFMD) in China, a national surveillance network was established. Here we described the epidemiology and pathogenic profile of HFMD and the impact of EV-A71 vaccination on pathogen spectrum of enteroviruses in the southeastern Chinese city of Nanchang during 2010-2019. A total of 7,951 HFMD cases from sentinel hospitals were included, of which 4,800 EV-positive cases (60.4%) were identified by real-time RT-PCR. During 2010-2012, enterovirus 71 (EV-A71) was the main causative agent of HFMD, causing 63.1% of cases, followed by 19.3% cases associated with coxsackievirus A16 (CV-A16). Since 2013, the proportion of other enteroviruses has increased dramatically, with the sub genotype D3 strain of Coxsackievirus A6 (CV-A6) replacing the dominance of EV-A71. These genetically diverse native strains of CV-A6 have co-transmitted and co-evolved in Nanchang. Unlike EV-A71 and CV-A16, most CV-A6 infections were concentrated in autumn and winter. The incidence of EV-A71 infection negatively correlated with EV-A71 vaccination (r = -0.990, p = 0.01). And severe cases sharply declined as the promotion of EV-A71 vaccines. After 2-year implementation of EV-A71 vaccination, EV-A71 is no longer detected from the reported HFMD cases in Nanchang. In conclusion, EV-A71 vaccination changed the pattern of HFMD epidemic, and CV-A6 replaced the dominance of EV-A71 over time.

Spatiotemporal dynamics of hemorrhagic fever with renal syndrome in Jiangxi province, China.

Historically, Jiangxi province has had the largest HFRS burden in China. However, thus far, the comprehensive understanding of the spatiotemporal distributions of HFRS is limited in Jiangxi. In this study, seasonal decomposition analysis, spatial autocorrelation analysis, and space-time scan statistic analyses were performed to detect the spatiotemporal dynamics distribution of HFRS cases from 2005 to 2018 in Jiangxi at the county scale. The epidemic of HFRS showed the characteristic of bi-peak seasonality, the primary peak in winter (November to January) and the second peak in early summer (May to June), and the amplitude and the magnitude of HFRS outbreaks have been increasing. The results of global and local spatial autocorrelation analysis showed that the HFRS epidemic exhibited the characteristic of highly spatially heterogeneous, and Anyi, Fengxin, Yifeng, Shanggao, Jing'an and Gao'an county were hot spots areas. A most likely cluster, and two secondary likely clusters were detected in 14-years duration. The higher risk areas of the HFRS outbreak were mainly located in Jiangxi northern hilly state, spreading to Wuyi mountain hilly state as time advanced. This study provided valuable information for local public health authorities to design and implement effective measures for the control and prevention of HFRS.

Identification of Haemaphysalis longicornis Genes Differentially Expressed in Response to Babesia microti Infection.

Haemaphysalis longicornis is a tick and a vector of various pathogens, including the human pathogenetic Babesia microti. The objective of this study was to identify female H. longicornis genes differentially expressed in response to infection with B. microti Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to Babesia infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including ola1, crt and Vg2. Functional analysis of up-regulated genes in fed or Babesia-infected ticks by RNA interference showed that knockdown of crt and Vg2 in infected ticks and knockdown of ola1 in uninfected ticks accelerated engorgement. In contrast, Vg1 knockdown in infected ticks had delayed engorgement. Knockdown of crt and Vg1 in infected ticks decreased engorged female weight. Vg2 knockdown reduced B. microti infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of H. longicornis genes in blood feeding and B. microti infection.

Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City, China.

Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response.

Knowledge, Attitudes, and Practices (KAP) Relating to Avian Influenza (H10N8) among Farmers' Markets Workers in Nanchang, China.

Three cases of avian influenza virus H10N8 were reported in Nanchang, China, as of April 2014. To identify the knowledge, attitudes, and practices (KAP) related to H10N8 among farmers' market workers, a cross-sectional survey was conducted in 63 farmers' markets in Nanchang. Using the resulting data, characteristics of poultry and non-poultry workers' knowledge, attitudes, and practice were described. Results suggest that interventions targeting high-risk workers should be developed and implemented by public health agencies to prevent the spread of H10N8. Additionally policies that encourage farmers' market workers to receive influenza vaccine should be developed, adopted, and enforced.

Coexistence of Avian Influenza Virus H10 and H9 Subtypes among Chickens in Live Poultry Markets during an Outbreak of Infection with a Novel H10N8 Virus in Humans in Nanchang, China.

Infection with the novel H10N8 virus in humans has raised concerns about its pandemic potential worldwide. We report the results of a cross-sectional study of avian influenza viruses (AIVs) in live poultry markets (LPMs) in Nanchang, China, after the first human case of H10N8 virus infection was reported in the city. A total of 201 specimens tested positive for AIVs among 618 samples collected from 24 LPMs in Nanchang from December 2013 to January 2014. We found that the LPMs were heavily contaminated by AIVs, with H9, H10, and H5 being the predominant subtypes and more than half of the LPMs providing samples that were positive for the H10 subtype. Moreover, the coexistence of different subtypes was common in LPMs. Of the 201 positive samples, 20.9% (42/201) had mixed infections with AIVs of different HA subtypes. Of the 42 mixed infections, 50% (21/42) showed the coexistence of the H9 and H10 subtypes, with or without H5, and were from chicken samples. This indicated that the H10N8 virus probably originated from segment reassortment of the H9 and H10 subtypes.

Genetic diversity of avian influenza A (H10N8) virus in live poultry markets and its association with human infections in China.

Following the first human infection with the influenza A (H10N8) virus in Nanchang, China in December 2013, we identified two additional patients on January 19 and February 9, 2014. The epidemiologic, clinical, and virological data from the patients and the environmental specimen collected from 23 local live poultry markets (LPMs) were analyzed. The three H10N8 cases had a history of poultry exposure and presented with high fever (>38°C), rapidly progressive pneumonia and lymphopenia. Substantial high levels of cytokines and chemokines were observed. The sequences from an isolate (A/Environment/Jiangxi/03489/2013 [H10N8]) in an epidemiologically linked LPM showed highly identity with human H10N8 virus, evidencing LPM as the source of human infection. The HA and NA of human and environmental H10N8 isolates showed high identity (99.1-99.9%) while six genotypes with internal genes derived from H9N2, H7N3 and H7N9 subtype viruses were detected in environmental H10N8 isolates. The genotype of the virus causing human infection, Jiangxi/346, possessed a whole internal gene set of the A/Environment/Jiangxi/10618/2014(H9N2)-like virus. Thus, our findings support the notion that LPMs can act as both a gene pool for the generation of novel reassortants and a source for human infection, and intensive surveillance and management should therefore be conducted.