Background: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. Results: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion: The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

Since mid-2015, numerous outbreaks of hydropericardium-hepatitis syndrome (HHS), which is caused by a novel fowl adenovirus serotype 4 (FAdV-4), have been reported in chickens in parts of China, thereby causing huge economic losses to the poultry industry. Thus, an effective vaccine to control the further spread of infections with this hyper-virulent FAdV-4 is imperative. In this study, we isolated a novel FAdV-4 strain SDJN0105 from a broiler farm with HHS disease in Shandong Province. Pathogenicity was evaluated by the observation of clinical symptoms, necropsy changes, and pathological tissue sections after oral and intramuscular (IM) infection of Specific pathogen free (SPF) chickens. The chickens infected by IM injection all died within three days, and chickens infected via the oculonasal route died within five days post-infection (dpi). Histopathological examination revealed that the pathology was confined to heart, liver, spleen, lung, kidney, and particularly the liver. Irrespective of the inoculation route, the highest viral DNA copy numbers were detected in the livers of infected chickens. The mRNA expression levels of IL-1β, IL-6, IL-8, IFNs, TNF-α, Mx, and OASL were significantly upregulated during the viral infection. In addition, an inactivated oil-emulsion FAdV-4 vaccine was developed. The vaccine could provide full protection for SPF chickens against a lethal dose of the FAdV-4 strain SDJN0105 and a high level of antibodies. These results improve our understanding of the innate immune responses in chickens infected with FAdV-4 and the pathogenesis of FAdV-4 caused by host factors, and the developed FAdV-4 vaccine is promising as a drug candidate for the prevention and reduction of the spread of HHS in poultry in China.

Fowl adenovirus (FAdV) is an infectious agent that can cause jaundice, severe anaemia, dyspnoea and reproductive disorders in fowls. Since 2015, FAdV disease has been rapidly spreading among broiler chickens in China, where it has caused significant economic losses. In this study, a loop-mediated isothermal amplification (LAMP) real-time turbidity method with strong specificity to FAdV was established. The established assay was specific to FAdV-4, and the lower limit of detection was 75 copies/μL of extracted DNA. The positive detection rate for the suspected tissue samples was 33.3% (14/42), which was consistent with that of the real-time PCR. The proposed LAMP method can quickly and accurately detect prevalent FAdV via real-time turbidity assay, thereby providing a diagnostic platform for the prevention and control of the FAdV disease.

Goose-origin astrovirus (GoAstV), as a newly emerging virus in 2017, is different from previously known astroviruses in the genus Avastrovirus . So far, few studies have focused on the novel virus. Considering the infectious development of astrovirus (AstV), we established a reverse transcription-quantitative PCR (RT-qPCR) assay with a strong specificity to quickly and accurately diagnose GoAstV. Confirmed by clinical application, this method can quickly and accurately detect prevalent GoAstV. The assay is thus convenient for clinical operation and is applicable to the monitoring of GoAstV disease.

Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA) and matrix (M) genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL) is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.