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Background: The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. Methods and results: Based on multiple 5’ noncoding region (5’-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. Conclusions and significance: This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening. [ABSTRACT FROM AUTHOR]

Hand, foot, and mouth disease (HFMD) is well recognized as one of the major threats to children's health globally. The increasing complexity of the etiology of HFMD still challenges disease control in China. There is little surveillance of the molecular epidemiological characteristics of the enteroviruses (EVs) that cause HFMD in Neijiang city or the Sichuan Basin area in Southwest China. In this study, demographic and epidemiological information for 14,928 probable HFMD cases was extracted and analyzed to describe the epidemic features of HFMD in Neijiang city from Jan 2010 to Dec 2017. The swab samples of select probable HFMD cases from 2012 to 2017 were tested by reverse transcription (RT) real-time PCR to identify the serotype distribution of EVs, and 110 randomly selected RT-real-time PCR positive samples were then amplified and analyzed for the VP1 or VP4 regions of EVs to further analyze the phylogenetic characteristics of the circulating strains in this area. The eight-year average annual incidence was 49.82 per 100,000 in Neijiang. The incidence rates varied between 19.51 and 70.73 per 100,000, demonstrating peaks of incidence in even-number years (2012, 2014 and 2016). The median age of the probable cases was 27 months and the interquartile range (25th to 75th percentile) of ages for the probable HFMD cases was between 14 and 42 months. The male-to-female ratio of the probable HFMD cases was 1.47:1, and scattered children were the major population classification (81.7%). Two epidemic peaks were observed: one major peak between April and July and the other lesser peak between October and December. Of 6513 probable cases tested with RT-real-time PCR, 4015 (61.6%) were positive for enterovirus with the serotype distribution as follows: EV71+, 30.1% (n = 1210); CV-A16+, 28.7% (n = 1154) and a sole pan-enterovirus+, 41.1% (n = 1651). A total of 91 cases (82.7%, 91/110) were successfully amplified and underwent phylogenetic analysis: all EV71+ cases were C4a serotype (n = 23/30); all CV-A16+ cases were B2b serotype (n = 24/30); of 42 sole pan-enterovirus+ samples, 20 were CV-A6, 14 were CV-A10 and the rest within this group were CV-A4 (n = 4), CV-A8 (n = 2), CV-A9 (n = 1) and CV-B3 (n = 1). Our findings provide important evidence that aids the improvement of strategies for vaccination against HFMD and comprehensive disease control in China. [ABSTRACT FROM AUTHOR]

Adzuki bean (Vigna angularis) is one of the most important legume crops in Asian countries like China, Japan and Korea due to its nutritious protein and starch contents. In spite of its economic importance, gene expression analysis system for gene function verification of adzuki bean is still absent. Therefore, reference genes for gene expression analysis based on the quantitative real time PCR (qRT-PCR) were screened in current study. A total of nine general housekeeping genes, including ACT, Fbox, ZMPP, GAPDH, EF, PP2A, UBC, UBN and PTB were evaluated for their expression stability by qRT-PCR in four adzuki bean cultivars, three different tissues, four abiotic stress and one biotic stress. The best group of candidates as reference genes were as follows: PTB and ACT for different cultivars; EF and UBN for different tissues; ACT and ZMPP for biotic stress and waterlogging stress; Fbox and UBC for salinity-alkalinity stress; Fbox and PTB for drought stress. Our results will provide a more accurate and reliable normalization of qRT-PCR data in adzuki bean. [ABSTRACT FROM AUTHOR]

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is a biological hazard that must be controlled and is a recognized etiological agent in viral hepatitis. This is a zoonotic virus and can be transmitted through the fecal-oral route. The pig is an important reservoir host of HEV, and is a source of contamination for the consumer after the consumption of raw or undercooked pork products. When detected, the most prevalent genotype of HEV in China is genotype 4 (denoted as HEV-4). To ensure the safety of this food of animal origin, we undertook a survey of HEV contamination in pig livers and pork samples available for sale, in retail outlets in selected cities in China. Viral RNA was purified from samples collected by lysing in Trizol followed by purification using trichloromethane and virus RNA extract kit. An additional step was applied to improve the recovery rate by adding RNase OUT when extracting virus RNA from pig livers, and the RNA productions were washed in 75% (v/v) ethanol to remove inhibitors. In total, 158 pig livers and 80 pork samples were procured and analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After purification of total RNA from all samples taken and analyzed by RT-qPCR, a single pig liver was positive by this method for HEV. The positive rate was calculated as 0.63%. In this study, a single positive sample was detected. Considering the dietary habits of Chinese people, pork is a popular food that on occasion may be contaminated with HEV, thereby posing a threat to consumer health. Ongoing surveillance is required to assess the risk to human health arising from HEV-contaminated pork being offered for sale, at retail outlets, especially in the areas of China where pig production is practiced.

Background: Severe fever with thrombocytopenia syndrome bunyavirus (sftsv) is an emerging tick-borne rna virus recently identified as the pathogen that causes severe fever with thrombocytopenia syndrome (sfts) in china. the existing commercial nucleic acid testing (comnat) assay with a relatively high claimed limit of quantitative detection (loqd) is not capable of sensitive detection and quantitation of sftsv. Thus, a new real-time reverse transcriptase (rt)-pcr assay with improved sensitivity is needed for clinical diagnosis; it could also be used to screen blood donors if necessary., Materials and Methods: We developed a new sftsv rt-pcr nat assay (newnat). About 129 plasma samples from 93 suspected sfts patients with typical clinical symptoms were tested using an anti-sftsv total antibody elisa and both comnat and newnat. The test performance of the two nat assays was evaluated and compared., Results: The newnat had a lower limit for quantitative testing compared to comnat. Twelve samples were comnat negative but newnat positive. Out of 35 suspected sfts patients who were comnat negative and anti-sftsv total antibody negative, four tested positive by the newnat assay and one of these four seroconverted within 2-4 days after testing newnat positive. A high correlation was observed between the cts of the newnat and comnat assays., Conclusion: The newnat assay was sensitive for quantitative detection of sftsv and may be applicable to clinical diagnosis and studies of the need for blood donor screening., (© 2017 Wiley Periodicals, Inc.)