bTo gain insight into the ecology of avian influenza viruses (AIV), we conducted active influenza virus surveillance in domestic ducks on farms located on the flyway of migratory birds in the Dongting Lake region of Hunan Province, China, from winter 2011 until spring 2012. Specimens comprising 3,030 duck swab samples and 1,010 environmental samples were collected from 101 duck farms. We isolated AIV of various HA subtypes, including H3, H4, H5, H6, H9, H10, H11, and H12. We sequenced the entire coding sequences of the genomes of 28 representative isolates constituting 13 specific subtypes. When the phylogenetic relationships among these isolates were examined, we observed that extensive reassortment events had occurred. Among the 28 Dongting Lake viruses, 21 genotypes involving the six internal genes were identified. Furthermore, we identified viruses or viral genes introduced from other countries, viral gene segments of unknown origin, and a novel HA/NA combination. Our findings emphasize the importance of farmed domestic ducks in the Dongting Lake region to the genesis and evolution of AIV and highlight the need for continued surveillance of domestic ducks in this region.

bWe analyzed eight H10N8 viruses isolated from ducks and chickens in live poultry markets from 2009 to 2013 in China. These viruses showed distinct genetic diversity and formed five genotypes: the four duck isolates formed four different genotypes, whereas the four chicken viruses belong to a single genotype. The viruses bound to both human-and avian-type receptors, and four of the viruses caused 12.7% to 22.5% body weight loss in mice. Influenza viruses bearing the H10 subtype hemagglutinin (HA) have been detected in avian species across wide geographic areas. The first H10 isolate, an H10N7 virus, was detected in chickens in Germany in 1949 (1, 2). Since then, viruses bearing H10 HA and different neuraminidase (NA) subtypes have been widely detected in wild birds and domestic poultry around the world (3-19). Moreover, an H10N4 virus caused an outbreak of a respiratory disease in mink in Sweden in 1984 (20), and more recently, several U.S. turkey workers tested seropositive for H10 influenza virus (15). In March 2010, an H10N7 virus caused an outbreak on a chicken farm in Australia; after processing clinically normal birds from the farm, seven abattoir workers reported conjunctivitis and minor upper respiratory tract symptoms and H10 virus infection was detected in two of the seven workers (11). In 2013, H10N8 virus caused three human infections in China, two of which were fatal (21, 22). Although sequence information about the H10 viruses is increasing (23-26), the biologic properties of these viruses remain largely unknown.A total of eight H10N8 influenza viruses were isolated from ducks and chickens during our routine surveillance from To investigate the genetic relationships among these viruses, we sequenced the genomes of all eight viruses. The amino acid motif at the HA cleavage site of these isolates is -R-, which is a characteristic of viruses of low pathogenicity in chickens. The eight genes of the viruses showed distinct diversity, with the HA, NA, PB2, PB1, PA, NP, M, and NS genes of the eight viruses sharing 94.7 to 100, 78.4 to 99.9, 89.9 to 100, 90.1 to 100, 89.7 to 100, 90.0 to 99.9, 90.5 to 100, and 89.3 to 99.9% identity, respectively, at the nucleotide level. The HA, PA, and NS genes each formed two branches in their phylogenetic trees (Fig. 1A, E, and H), whereas the NA, PB2, PB1, and M genes formed three branches each in their phylogenetic trees (Fig. 1B, C, D, and G) and the NP gene formed four branches in its phylogenetic tree (Fig. 1F). Of note, the six internal genes of the H10N8 viruses in branch 1 were clustered with the corresponding genes of H9N2 influenza viruses (Fig. 1C to H), and the NA gene in group 1 belongs to the North American lineage (Fig. 1B).On the basis of this genomic diversity, we divided the viruses into five genotypes (Table 1). Of note, the four duck viruses belong to four different genotypes, whereas the four chicken viruses belong to a single genotype, and the eight gene segments of the CK/JX/S3581/13 virus shared 99.2% to 99.9% identity with the human H10N8 virus...

This study aimed to determine the mechanism by which H9N2 avian influenza virus (AIV) affects eggshell quality. Thirty-week-old specific pathogen free egg-laying hens were inoculated with the chicken-origin H9N2 AIV strain (A/Chicken/shaanxi/01/2011) or with inoculating media without virus by combined intraocular and intranasal routes. The time course for the appearance of viral antigen and tissue lesions in the oviduct was coincident with the adverse changes in egg production in the infected hens. The viral loads of AIV have a close correlation with the changes in the uterus CaBP-D28k mRNA expression as well as the Ca concentrations in the eggshells in the infected hens from 1 to 7 days post inoculation (dpi). Ultrastructural examination of eggshells showed significantly decreased shell thickness in the infected hens from 1 to 5 dpi (P < 0.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens from 1 to 5 dpi as compared with the control hens. In conclusion, this study confirmed that H9N2 AIV strain (A/Chicken/shaanxi/01/2011) infection is associated with severe lesions of the uterus and abnormal expression of CaBP-D28k mRNA in the uteri of the infected hens. The change of CaBP-D28k mRNA expression may contribute to the deterioration of the eggshell quality of the laying hens infected with AIV. It is noteworthy that the pathogenicity of H9N2 AIV strains may vary depending on the virus strain and host preference.

Background: A series of pyrimidine amine derivatives has been synthesized by modifying the pyrimidine ring group of diflumetorim-a mitochondrial complex I inhibiting fungicide. One derivative, code number SYP-34773, is investigated in this study involving Magnaporthe oryzae, the causal agent of rice blast, which is the most devastating disease in rice. The response, resistance profile and mechanism of M. oryzae to SYP-34773 were investigated, which provides or provide?? important data for the registration and rational use of pyrimidine amines. Results: SYP-34773 showed greater control efficacy than fungicide isoprothiolane in the field. The baseline sensitivity was established at a mean 50% effective concentration (EC 50) of 0.08 ∼g ml −1. Four stable SYP-34773-resistant isolates with reduced sensitivity were generated from one (S118) of ten sensitive isolates with a resistance factor of EC 50 ranging from 7.00 to 15.00. Conidia production and pathogenicity were similar to that of S118, although there was a significant decrease in mycelial growth and conidial germination in resistant isolates. Positive cross-resistance was observed between SYP-34773 and diflumetorim; and the SYP-34773-resistant isolates were still sensitive to isoprothiolane, carbendazim, fluazinam, azoxystrobin, or prochloraz. RNA-Seq analyses revealed three cytochrome P450 genes were upregulated in the resistant isolate under the treatment with SYP-34773, as confirmed by quantitative real-time PCR. The SYP-34773 content was significantly reduced in the resistant isolate when compared with the parental isolate. Conclusion: The study demonstrated that SYP-34773 exhibits high activity against M. oryzae. Overexpression of three cytochrome P450 genes has an important role in the resistance of M. oryzae to novel pyrimidine amines.

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BackgroundNewcastle disease virus (NDV) can cause serious damage to the reproductive tracts of egg-laying hens and leads to egg production and quality reduction. However, the mechanism of severe pathological damage in the oviducts of egg-laying hens after NDV infection has not been fully elucidated. In this study, the correlation between the primary pathological lesions and viral load in the oviducts of egg-laying hens infected with the velogenic genotype VIId NDV strain was evaluated by pathological observation and virus detection. Subsequently, apoptosis, the expression of immune-related genes and lymphocyte infiltration into the infected oviducts were determined to explore the potential causes of the pathological changes.ResultsA higher viral load and severe tissue lesions and apoptotic bodies were observed in the oviduct of NDV-infected hens compared with the control. Immune-related genes, including TLR3/7/21, MDA5, IL-2/6/1β, IFN-β, CXCLi1/2, and CCR5, were significantly upregulated in the magnum and uterus. IL-2 presented the highest mRNA level change (137-fold) at 5 days post infection (dpi) in the magnum. Infection led to CD3+CD4+ and CD3+CD8α+ lymphocyte infiltration into the magnum of the oviduct. A higher viral load was found to be associated with pathological changes and the elevated expression of proinflammatory cytokines in the NDV-infected hens.ConclusionsOur results indicate that the severe lesions and apoptosis in the oviducts of egg-laying hens caused by genotype VIId NDV strains are associated with the excessive release of inflammatory cytokines, chemokines and lymphocyte infiltration, which contribute to the dysfunction of the oviducts and the decrease of egg production in hens.

Various virulence-associated genes or pathogenicity island are responsible for determining the pathogenicity of Escherichia coli strains. However, the correlation of the number and combination patterns of virulence-associated genes in Escherichia coli strains with their pathogenicity remains largely unknown. In this work, 581 chicken Escherichia coli strains were isolated from 1045 liver samples of dead chickens from 50 chicken farms at four provinces in China during 2007-2012. Based on the pathogenic test of SPF chickens, 320 chickens pathogenic Escherichia coli isolates were identified as highly (n = 193), intermediate (n = 98) and low pathogenic (n = 29) strains, respectively. Furthermore, the number of virulence genes in the 320 chicken pathogenic and 50 non-pathogenic Escherichia coli strains was examined. Our results reveal that thirteen virulence genes in Escherichia coli strains were detected, and all strains carried at least two or more than two virulence-associated genes. This study also suggests that highly pathogenic E. coli strains simultaneously carried at least 8 to13 virulence genes while intermediate pathogenic strains carried at least 5 to 8 virulence genes. The number of virulence-associated genes detected in highly pathogenic strains showed there were more significant differences than that in low pathogenic strains (P < 0.01). The detection rate of genes irp2, fyuA, and colV in high pathogenic strains was significantly higher than that in low and non-pathogenic strains (P < 0.01). Nine virulence-asso-* Corresponding authors. J. Y. Wang et al. 244 ciated genes irp2, fyuA, iucA, iucD, iutA, papC, iss, tsh, and colV were more often detected in highly and intermediate pathogenic E. coli strains. Taken together, our results provide evidences demonstrating that the pathogenicity of Escherichia coli strains is closely associated with the number and combination patterns of virulence-associated genes.

Background: The widespread occurrence of fungicide resistance in fungal plant pathogens requires the development of new compounds with different mode(s) of action (MOA) to avoid cross resistance. This will require a rapid method to identify MOAs. Results: Here, gas chromatography-mass spectrometry (GC-MS) based metabolic fingerprinting was used to elucidate the MOAs of fungicides. Botrytis cinerea, an important pathogen of vegetables and flowers, can be inhibited by a wide range of chemical fungicides with different MOAs. A sensitive strain of B. cinerea was exposed to EC 50 concentrations of 13 fungicides with different known MOAs and one with unknown MOA. The mycelial extracts were analyzed for their "metabolic fingerprint" using GC-MS. A comparison among the GC-MS vector' profiles of cultures treated with fungicides were performeded. A model based on hierarchical clustering was established which allowed these antifungal compounds to be distinguished and classified coinciding with their MOAs. Thus, metabolic fingerprinting represents a rapid, convenient, and information-rich method for classifying the MOAs of antifungal substances. The biomarkers of fungicide MOAs were also established by an analysis of variance and included succinate for succinate dehydrogenase inhibitors and cystathionine for methionine synthesis inhibitors. Using the metabolic model and the common perturbation of metabolites, the new fungicide SYP-14288 was identified as having the same MOA as fluazinam. Conclusion: This study provides a comprehensive database of the metabolic perturbations of B. cinerea induced by diverse MOA inhibitors and highlights the utility of metabolic fingerprinting for defining MOAs, which will assist in the development and optimization of new fungicides.