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Low pathogenic avian influenza viruses (LPAIVs) have been widespread in poultry and wild birds throughout the world for many decades. LPAIV infections are usually asymptomatic or cause subclinical symptoms. However, the genetic reassortment of LPAIVs may generate novel viruses with increased virulence and cross-species transmission, posing potential risks to public health. To evaluate the epidemic potential and infection landscape of LPAIVs in Guangxi Province, China, we collected and analyzed throat and cloacal swab samples from chickens, ducks and geese from the live poultry markets on a regular basis from 2016 to 2019. Among the 7,567 samples, 974 (12.87%) were LPAIVs-positive, with 890 single and 84 mixed infections. Higher yearly isolation rates were observed in 2017 and 2018. Additionally, geese had the highest isolation rate, followed by ducks and chickens. Seasonally, spring had the highest isolation rate. Subtype H3, H4, H6 and H9 viruses were detected over prolonged periods, while H1 and H11 viruses were detected transiently. The predominant subtypes in chickens, ducks and geese were H9, H3, and H6, respectively. The 84 mixed infection samples contained 22 combinations. Most mixed infections involved two subtypes, with H3 + H4 as the most common combination. Our study provides important epidemiological data regarding the isolation rates, distributions of prevalent subtypes and mixed infections of LPAIVs. These results will improve our knowledge and ability to control epidemics, guide disease management strategies and provide early awareness of newly emerged AIV reassortants with pandemic potential.

Additional file 1. Appendix A. Supplementary data.

Our present study aimed to identify host cell proteins that may interact with avian reovirus (ARV) σA protein and their potential effect on ARV replication. The ARV structural protein σA has been demonstrated to suppress interferon production and confirmed to activate the PI3K/Akt pathway. However, host cell factors interacting with σA to affect ARV replication remain unknown. In current study, a cDNA library of chicken embryo fibroblasts (CEFs) was constructed, and host cell proteins interacting with σA were screened by a yeast two-hybrid system. We identified four candidate cellular proteins that interact with ARV σA protein. Among them, Gallus NME/NM23 nucleoside diphosphate kinase 2 (NME2) was further validated as a σA-binding protein through co-immunoprecipitation. The key interaction domain was identified at amino acids (aa) 121-416 in NME2 and at aa 71-139 in σA, respectively. We demonstrated that overexpression of NME2 substantially inhibited ARV replication. In addition silencing NME2 by small interfering RNAs (siRNAs) resulted in marked enhancement of ARV replication. Our work has demonstrated that NME2 is a σA-binding protein that may affect ARV replication in CEF cells.

A previously unidentified chicken parvovirus (ChPV) and turkey parvovirus (TuPV) strain, associated with runting-stunting syndrome (RSS) and poultry enteritis and mortality syndrome (PEMS) in turkeys, is now prevalent among chickens in China. In this study, a large-scale surveillance of parvoviruses in chickens and turkeys using conserved PCR assays was performed. We assessed the prevalence of ChPV/TuPV in commercial chicken and turkey farms in China between 2014 and 2019. Parvoviruses were prevalent in 51.73% (1,795/3,470) of commercial chicken and turkey farms in Guangxi, China. The highest frequency of ChPV positive samples tested by PCR occurred in chickens that were broiler chickens 64.18% (1,041/1,622) compared with breeder chickens 38.75% (572/1,476) and layer hens 38.89% (112/288), and TuPV was detected in 70/84 (83.33%). Native and exotic chicken species were both prevalent in commercial farms in southern China, and exotic broiler chickens had a higher positive rate with 88.10% (148/168), while native chickens were 50.00% (1,465/2,930). The environmental samples from poultry houses tested positive for ChPV and TuPV were 47.05% (415/874). Samples from open house flocks had higher prevalence rates of ChPV than those of closed house flocks (Table 5), among which those from the open house showed 84.16% (85/101) positivity, those from litter showed 62.86% (44/70) positivity, and those from drinking water showed 50.00% (56/112) positivity, whereas those from the closed house litter were 53.57% (60/112), those from swabs were 50.18% (138/275), and those from drinking water were 15.69% (32/204). Samples collected during spring were more frequently ChPV/ TuPV positive than those collected during other seasons. This study is the first report regarding the epidemiological surveillance of ChPV and TuPV in chicken/turkey flocks in Guangxi, China. Our results suggest that ChPV and TuPV are widely distributed in commercial fowl in Guangxi. These findings highlight the need for further epidemiological and genetic research on ChPV and TuPV in this area.

An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Graphene (Gra) was used for both the conjugation of an anti-Newcastle disease virus monoclonal antibody (MAb/NDV) and the immobilization of anti-Newcastle disease virus polyclonal antibodies (PAb/NDV). Cu(I)/Cu(II) was selected as an electroactive probe, immobilized on a chitosan-graphene (Chi-Gra) hybrid material, and detected by differential pulse voltammetry (DPV) after a sandwich-type immune response. Because Gra had a large surface area, many antibodies were loaded onto the electrochemical immunosensor to effectively increase the electrical signal. Additionally, the introduction of Gra significantly increased the loading amount of electroactive probes (Cu(I)/Cu(II)), and the electrical signal was further amplified. Cu(I)/Cu(II) and Cu(I)/Cu(II)-Chi-Gra were compared in detail to characterize the signal amplification ability of this platform. The results showed that this immunosensor exhibited excellent analytical performance in the detection of NDV in the concentration range of 100.13 to 105.13 EID50/0.1 mL, and it had a detection limit of 100.68 EID50/0.1 mL, which was calculated based on a signal-to-noise (S/N) ratio of 3. The resulting immunosensor also exhibited high sensitivity, good reproducibility and acceptable stability.

The H3 subtype of avian influenza virus (AIV) is widespread in avian species and is frequently isolated in surveillance projects; thus, we have developed a more effective diagnostic approach of a monoclonal antibody (mAb)-based sandwich ELISA for the H3 AIV detection. First, we have produced the essential reagent of mAb against AIV H3 strains with the development of an mAb-Mouse immunization with a purified H3-subtype AIV strain and cell fusion to generate hybridoma cells. These cells were screened with hemagglutination inhibition (HI) tests, and optimal cells were subcloned. We chose a hybridoma cell line that steadily secreted a specific H3-subtype AIV mAb, designated 9F12, that belongs to the IgG1 subclass and has a K-type light chain. 9F12 was shown to bind specifically to the H3-subtype AIV antigen by both immunofluorescence assay and Western blot analysis. Finally, a 9F12-based sandwich ELISA was successfully developed and used to specifically test for this antigen. The sandwich ELISA conditions were optimized, and the specificity and sensitivity were validated. The results for clinical sample detection were consistent with viral isolation. Consequently, the 9F12-based sandwich ELISA is a specific, sensitive, robust, rapid and versatile diagnostic tool for H3-subtype AIV and provides a promising strategy for effective influenza virus prevention and control.

Hepatitis-hydropericardium syndrome (HHS) is a severe disease that causes 20 to 80% mortality in chickens aged 3 to 6 wk. Fowl aviadenovirus serotype 4 (FAdV-4) plays an important role in the etiology of HHS. Since 2015, outbreaks of HHS have been reported in several provinces of China; however, details regarding the FAdV-4 genome properties are lacking. In the present study, the complete genomes of 9 isolates responsible for these outbreaks in Guangxi Province, China, were sequenced. To investigate the molecular characteristics of these FAdV-4 isolates, we compared their genomes with those of other reported pathogenic and nonpathogenic FAdV-4 isolates. A variable number of GA repeats were observed in the isolates of this study. Each of the isolates GX2017-01, GX2017-02, GX2018-08, and GX2019-09 had 11 GA repeats; GX2017-03, GX2017-04, and GX2017-05 each had 10 GA repeats, while GX2017-06 and GX2018-07 each had 8 GA repeats. We observed several deletions and distinct amino acid mutations in the major structural genes of these isolates when compared with non-Chinese isolates. We found 2 novel putative genetic markers in the hexon protein, one present in GX2017-02, in which aspartic acid (D) was changed to tyrosine (Y), and another present in each of isolates GX2018-08 and GX2019-09, in which serine (S) was changed to arginine (R), when compared with selected Chinese and some non-Chinese isolates. Moreover, the phylogenetic analysis revealed that all the isolates of this study were clustered within FAdV-C. We found that these isolates were closely related to other recently isolated Chinese strains. The data presented in this study will not only increase the understanding of the molecular epidemiology and genetic diversity of FAdV-4 isolates in China but also has an important reference value of the major factors that determine the virulence of FAdV-4 strains.

The aim of the current study was to determine the genomic sequence of parvovirus strain GX-Tu-PV-1, which was isolated from a turkey in Guangxi Province, South China. The analysis showed that the genome sequence of GX-Tu-PV-1 was 81.3% to ∼99.3% similar to those of other turkey parvoviruses (TuPVs) and 79.8% to ∼92.1% related to chicken parvovirus (ChPV). This study will help in understanding the epidemiology and molecular characteristics of parvovirus in turkeys.

Immunochromatography test strips that use metal particles constructed from Au, Fe3O4, and Au/Fe3O4 nanoparticles were developed for the rapid detection of avian influenza virus subtype H7 (AIV H7). The principle of this immunochromatography test strip was based on a sandwich immunoreaction in which AIV H7 antigens bind specifically to their corresponding antibodies on a nitrocellulose membrane. An antibody–metal (Au, Fe3O4 or Au/Fe3O4) nanoparticle conjugate was used as a label and coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and a control zone, an anti-H7 polyclonal antibody and an anti-IgG antibody were immobilized onto the nitrocellulose membrane, respectively. Positive samples displayed brown/red lines in the test and control zones of the nitrocellulose membrane, whereas negative samples resulted in a brown/red line only in the control zone. The limit of detection (LOD) of the Au/Fe3O4 nanoparticle-based immunochromatography test strips was found to be 103.5 EID50 (EID50: 50% Egg Infective Dose), which could be visually detected by the naked eye within 15 min. In addition, 200 clinical samples were tested using the Au/Fe3O4 nanoparticle-based immunochromatography test strip to estimate its performance, and seven were positive for AIV H7. In summary, the Au/Fe3O4 nanoparticle-based immunochromatography test strip offers a simple and cost-effective tool for the rapid detection of AIV H7.

Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.

Mycoplasma bovis (MB) and bovine herpes virus 1 (BHV-1) are two important pathogens that cause bovine respiratory disease in the beef feedlot and dairy industries. The aim of this study was to develop and validate a duplex fluorescence-based loop-mediated isothermal amplification (DLAMP) assay for simultaneous detection of MB and BHV-1. Two sets of specific primers for each pathogen were designed to target the unique sequences of the MB uvrC gene and the BHV-1 gB gene. The inner primer for BHV-1 was synthesized with the fluorophore FAM at the 5′ end to detect the BHV-1 gB gene, and the inner primer for MB was synthesized with the fluorophore CY5 at the 5′ end to detect the MB uvrC gene. The DLAMP reaction conditions were optimized for rapid and specific detection of MB and BHV-1. The DLAMP assay developed here could specifically detect MB and BHV-1 without cross-reaction with other known non-target bovine pathogens. The sensitivity of this DLAMP assay was as low as 2 × 102 copies for recombinant plasmids containing the MB and BHV-1 target genes. In a detection test of 125 clinical samples, the positive rates for MB, BHV-1 and co-infection were 44.8%, 13.6% and 1.6%, respectively. Furthermore, the sensitivity and specificity of DLAMP were determined as 95%–96.6% and 100%, respectively, of those of field sample detection by the real-time polymerase chain reaction (PCR) assay recommended by the World Organisation for Animal Health. Overall, DLAMP provides a rapid, sensitive and specific assay for the identification of MB and BHV-1 in clinical specimens and for epidemiological surveillance.