Molecular characterization of emerging chicken and turkey parvovirus variants and novel strains in Guangxi, China.

Avian parvoviruses cause several enteric poultry diseases that have been increasingly diagnosed in Guangxi, China, since 2014. In this study, the whole-genome sequences of 32 strains of chicken parvovirus (ChPV) and 3 strains of turkey parvovirus (TuPV) were obtained by traditional PCR techniques. Phylogenetic analyses of 3 genes and full genome sequences were carried out, and 35 of the Guangxi ChPV/TuPV field strains were genetically different from 17 classic ChPV/TuPV reference strains. The nucleotide sequence alignment between ChPVs/TuPVs from Guangxi and other countries revealed 85.2-99.9% similarity, and the amino acid sequences showed 87.8-100% identity. The phylogenetic tree of these sequences could be divided into 6 distinct ChPV/TuPV groups. More importantly, 3 novel ChPV/TuPV groups were identified for the first time. Recombination analysis with RDP 5.0 revealed 15 recombinants in 35 ChPV/TuPV isolates. These recombination events were further confirmed by Simplot 3.5.1 analysis. Phylogenetic analysis based on full genomes showed that Guangxi ChPV/TuPV strains did not cluster according to their geographic origin, and the identified Guangxi ChPV/TuPV strains differed from the reference strains. Overall, whole-genome characterizations of emerging Guangxi ChPV and TuPV field strains will provide more detailed insights into ChPV/TuPV mutations and recombination and their relationships with molecular epidemiological features.

Pathogenicity and molecular characteristics of fowl adenovirus serotype 4 with moderate virulence in Guangxi Province, China.

The GX2020-019 strain of fowl adenovirus serotype 4 (FAdV-4) was isolated from the liver of chickens with hydropericardium hepatitis syndrome in Guangxi Province, China, and was purified by plaque assay three times. Pathogenicity studies showed that GX2020-019 can cause typical FAdV-4 pathology, such as hydropericardium syndrome and liver yellowing and swelling. Four-week-old specific pathogen-free (SPF) chickens inoculated with the virus at doses of 103 median tissue culture infectious dose (TCID50), 104 TCID50, 105 TCID50, 106 TCID50, and 107 TCID50 had mortality rates of 0, 20, 60, 100, and 100%, respectively, which were lower than those of chickens inoculated with other highly pathogenic Chinese isolates, indicating that GX2020-019 is a moderately virulent strain. Persistent shedding occurred through the oral and cloacal routes for up to 35 days postinfection. The viral infection caused severe pathological damage to the liver, kidney, lung, bursa of Fabricius, thymus, and spleen. The damage to the liver and immune organs could not be fully restored 21 days after infection, which continued to affect the immune function of chickens. Whole genome analysis indicated that the strain belonged to the FAdV-C group, serotype 4, and had 99.7-100% homology with recent FAdV-4 strains isolated from China. However, the amino acid sequences encoded by ORF30 and ORF49 are identical to the sequences found in nonpathogenic strains, and none of the 32 amino acid mutation sites that appeared in other Chinese isolates were found. Our research expands understanding of the pathogenicity of FAdV-4 and provides a reference for further studies.

Explore how immobilization strategies affected immunosensor performance by comparing four methods for antibody immobilization on electrode surfaces.

Among the common methods used for antibody immobilization on electrode surfaces, which is the best available option for immunosensor fabrication? To answer this question, we first used graphene-chitosan-Au/Pt nanoparticle (G-Chi-Au/PtNP) nanocomposites to modify a gold electrode (GE). Second, avian reovirus monoclonal antibody (ARV/MAb) was immobilized on the GE surface by using four common methods, which included glutaraldehyde (Glu), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS), direct incubation or cysteamine hydrochloride (CH). Third, the electrodes were incubated with bovine serum albumin, four different avian reovirus (ARV) immunosensors were obtained. Last, the four ARV immunosensors were used to detect ARV. The results showed that the ARV immunosensors immobilized via Glu, EDC/NHS, direct incubation or CH showed detection limits of 100.63 EID50 mL-1, 100.48 EID50 mL-1, 100.37 EID50 mL-1 and 100.46 EID50 mL-1 ARV (S/N = 3) and quantification limits of 101.15 EID50 mL-1, and 101.00 EID50 mL-1, 100.89 EID50 mL-1 and 100.98 EID50 mL-1 ARV (S/N = 10), respectively, while the linear range of the immunosensor immobilized via CH (0-105.82 EID50 mL-1 ARV) was 10 times broader than that of the immunosensor immobilized via direct incubation (0-104.82 EID50 mL-1 ARV) and 100 times broader than those of the immunosensors immobilized via Glu (0-103.82 EID50 mL-1 ARV) or EDC/NHS (0-103.82 EID50 mL-1 ARV). And the four immunosensors showed excellent selectivity, reproducibility and stability.

An Enzyme-Free Sandwich Amperometry-Type Immunosensor Based on Au/Pt Nanoparticle-Functionalized Graphene for the Rapid Detection of Avian Influenza Virus H9 Subtype.

Avian influenza virus H9 subtype (AIV H9) has contributed to enormous economic losses. Effective diagnosis is key to controlling the spread of AIV H9. In this study, a nonenzymatic highly electrocatalytic material was prepared using chitosan (Chi)-modified graphene sheet (GS)-functionalized Au/Pt nanoparticles (GS-Chi-Au/Pt), followed by the construction of a novel enzyme-free sandwich electrochemical immunosensor for the detection of AIV H9 using GS-Chi-Au/Pt and graphene-chitosan (GS-Chi) nanocomposites as a nonenzymatic highly electrocatalytic material and a substrate material to immobilize capture antibodies (avian influenza virus H9-monoclonal antibody, AIV H9/MAb), respectively. GS, which has a large specific surface area and many accessible active sites, permitted multiple Au/Pt nanoparticles to be attached to its surface, resulting in substantially improved conductivity and catalytic ability. Au/Pt nanoparticles can provide modified active sites for avian influenza virus H9-polyclonal antibody (AIV H9/PAb) immobilization as signal labels. Upon establishing the electrocatalytic activity of Au/Pt nanoparticles on graphene towards hydrogen peroxide (H2O2) reduction for signal amplification and optimizing the experimental parameters, we developed an AIV H9 electrochemical immunosensor, which showed a wide linear range from 101.37 EID50 mL-1 to 106.37 EID50 mL-1 and a detection limit of 100.82 EID50 mL-1. This sandwich electrochemical immunosensor also exhibited high selectivity, reproducibility and stability.

Screening of interferon-stimulated genes against avian reovirus infection and mechanistic exploration of the antiviral activity of IFIT5.

Avian reovirus (ARV) infection can lead to severe immunosuppression, complications, and secondary diseases, causing immense economic losses to the poultry industry. In-depth study of the mechanism by which the innate immune system combats ARV infection, especially the antiviral effect mediated by interferon, is needed to prevent and contain ARV infection. In this study, ARV strain S1133 was used to artificially infect 7-day-old specific pathogen-free chickens. The results indicated that ARV rapidly proliferated in the immune organs, including the spleen, bursa of Fabricius, and thymus. The viral load peaked early in the infection and led to varying degrees of pathological damage to tissues and organs. Real-time quantitative PCR revealed that the mRNA levels of interferon and multiple interferon-stimulated genes (ISGs) in the spleen, bursa of Fabricius, and thymus were upregulated to varying degrees in the early stage of infection. Among the ISGs, IFIT5, and Mx were the most upregulated in various tissues and organs, suggesting that they are important ISGs for host resistance to ARV infection. Further investigation of the role of IFIT5 in ARV infection showed that overexpression of the IFIT5 gene inhibited ARV replication, whereas inhibition of the endogenously expressed IFIT5 gene by siRNA promoted ARV replication. IFIT5 may be a positive feedback regulator of the innate immune signaling pathways during ARV infection and may induce IFN-α production by promoting the expression of MAD5 and MAVS to exert its antiviral effect. The results of this study help explain the innate immune regulatory mechanism of ARV infection and reveal the important role of IFIT5 in inhibiting ARV replication, which has important theoretical significance and practical application value for the prevention and control of ARV infection.

Molecular characterization of chicken anemia virus in Guangxi Province, southern China, from 2018 to 2020.

BACKGROUND: Chicken anemia virus (CAV) causes chicken infectious anemia, which results in immunosuppression; the virus has spread widely in chicken flocks in China. OBJECTIVES: The aim of this study was to understand recent CAV genetic evolution in chicken flocks in Guangxi Province, southern China. METHODS: In total, 350 liver samples were collected from eight commercial broiler chicken farms in Guangxi Province in southern China from 2018 to 2020. CAV was detected by conventional PCR, and twenty CAV complete genomes were amplified and used for the phylogenetic analysis and recombination analysis. RESULTS: The overall CAV-positive rate was 17.1%. The genetic analysis revealed that 84 CAVs were distributed in groups A, B, C (subgroups C1-C3) and D. In total, 30 of 47 Chinese CAV sequences from 2005-2020 belong to subgroup C3, including 15 CAVs from this study. There were some specific mutation sites among the intergenotypes in the VP1 protein. The amino acids at position 394Q in the VP1 protein of 20 CAV strains were consistent with the characteristics of a highly pathogenic strain. GX1904B was a putative recombinant. CONCLUSIONS: Subgroup C3 was the dominant genotype in Guangxi Province from 2018-2020. The 20 CAV strains in this study might be virulent according to the amino acid residue analysis. These data help improve our understanding of the epidemiological trends of CAV in southern China.

Survey of low pathogenic avian influenza viruses in live poultry markets in Guangxi Province, Southern China, 2016-2019.

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.

Dynamic Changes in the Expression of Interferon-Stimulated Genes in Joints of SPF Chickens Infected With Avian Reovirus.

Avian reovirus (ARV) can induce many diseases as well as immunosuppression in chickens, severely endangering the poultry industry. Interferons (IFNs) play an antiviral role by inducing the expression of interferon-stimulated genes (ISGs). The effect of ARV infection on the expression of host ISGs is unclear. Specific-pathogen-free (SPF) chickens were infected with ARV strain S1133 in this study, and real time quantitative PCR was used to detect changes in the dynamic expression of IFNs and common ISGs in joints of SPF chickens. The results showed that the transcription levels of IFNA, IFNB, and several ISGs, including myxovirus resistance (MX), interferon-induced transmembrane protein 3 (IFITM3), protein kinase R (PKR), oligoadenylate synthase (OAS), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), interferon-stimulated gene 12 (ISG12), virus inhibitory protein (VIPERIN), interferon-alpha-inducible protein 6 (IFI6), and integrin-associated protein (CD47), were upregulated in joints on days 1-7 of infection (the levels of increase of MX, IFIT5, OAS, VIPERIN, ISG12, and IFI6 were the most significant, at hundreds-fold). In addition, the expression levels of the ISGs encoding zinc finger protein 313 (ZFP313), and DNA damage-inducible transcript 4 (DDIT4) increased suddenly on the 1st or 2nd day, then decreased to control levels. The ARV viral load in chicken joints rapidly increased after 1 day of viral challenge, and the viral load remained high within 6 days of viral challenge. The ARV viral load sharply decreased starting on day 7. These results indicate that in SPF chicken joints, many ISGs have mRNA expression patterns that are basically consistent with the viral load in joints. IFNA, IFNB, and the ISGs MX, IFITM3, PKR, OAS, IFIT5, ISG12, VIPERIN, IFI6, and CD47 play important roles in defending against ARV invasion, inhibiting ARV replication and proliferation, and promoting virus clearance. These results enrich our understanding of the innate immune response mechanisms of hosts against ARV infection and provide a theoretical basis for prevention and control of ARV infection.

Gallus NME/NM23 nucleoside diphosphate kinase 2 interacts with viral σA and affects the replication of avian reovirus.

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.

Epidemiological Surveillance of Parvoviruses in Commercial Chicken and Turkey Farms in Guangxi, Southern China, During 2014-2019.

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.

Genetic characterization of fowl aviadenovirus 4 isolates from Guangxi, China, during 2017-2019.

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.

Electrochemical immunosensor with Cu(I)/Cu(II)-chitosan-graphene nanocomposite-based signal amplification for the detection of newcastle disease virus.

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.

[Preparation of monoclonal antibody against hemagglutinin of H4 subtype avian influenza and establishment of sandwich ELISA].

Objective To prepare the monoclonal antibodies (mAb) against hemagglutinin of H4 subtype avian influenza virus (AIV), and develop a sandwich ELISA for the detection of H4 subtype AIV. Methods The BALB/c mice were immunized with inactive H4 subtype AIV. A mAb against H4 subtype AIV, designated as 6G4, was obtained by cell fusion, hemagglutination inhibition (HI) screening and subcloing. Immuofluorescence cytochemistry and Western blotting were used to detect the reactivity of 6G4 with H4 subtype AIV, and the specificity, broad spectrum and stability of 6G4 were characterized by HI assay. Subclass of 6G4 was determined by kit. With chicken polyclonal antibody against H4 subtype AIV as coated antibody, 6G4 mAb as capture antibody and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody, a sandwich ELISA for the detection of H4 subtype AIV was established by optimization of the reaction conditions and serial verification. Results 6G4 belonged to IgG1 subclass, and the light chain belonged to κ. It could secrete antibody stably and had good reactivity, specificity, broad spectrum and stability. ELISA based on 6G4 was specific, sensitive, accurate and suitable for the detection of a large number of samples. Conclusion We successfully achieved the anti-H4 subtype AIV mAb, and developed the sandwich ELISA for the detection of H4 subtype AIV.

Production and identification of monoclonal antibodies and development of a sandwich ELISA for detection of the H3-subtype avian influenza virus antigen.

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.

Study of the activation of the PI3K/Akt pathway by the motif of σA and σNS proteins of avian reovirus.

The present study was conducted to determine whether avian reovirus (ARV) activates the phosphatidylinositol 3-kinase-dependent Akt (PI3K/Akt) pathway according to the PXXP or YXXXM motifs of σA and σNS proteins. Gene splicing by overlap extension PCR was used to change the PXXP or YXXXM motifs of the σA and σNS genes. Plasmid constructs that contain mutant σA and σNS genes were generated and transfected into Vero cells, and the expression levels of the corresponding genes were quantified according to immunofluorescence and Western blot analyses. The Akt phosphorylation (P-Akt) profile of the transfected Vero cells was examined by flow cytometry and Western blot. The results showed that the σA and σNS genes were expressed in the Vero cells, and P-Akt expression in the σA mutant groups (amino acids 110-114 and 114-117) was markedly decreased. The results indicated that the σA protein of ARV activates the PI3K/Akt pathway via the PXXP motif. The results of this study reveal the mechanisms by which ARV manipulates the cellular signal transduction pathways, which may provide new ideas for novel drug targets.

Molecular Characterization of Parvovirus Strain GX-Tu-PV-1, Isolated from a Guangxi Turkey.

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.

Altered gene expression profiles of the MDA5 signaling pathway in peripheral blood lymphocytes of chickens infected with avian reovirus.

Avian reovirus (ARV) is a member of the genus Orthoreovirus in the family Reoviridae and causes a severe syndrome including viral arthritis that leads to considerable losses in the poultry industry. Innate immunity plays a significant role in host defense against ARV. Here, we explored the interaction between ARV and the host innate immune system by measuring mRNA expression levels of several genes associated with the MDA5 signaling pathway. The results showed that expression peaks for MDA5, MAVS, TRAF3, TRAF6, IRF7, IKKɛ, TBK1 and NF-κB occurred at 3 days postinfection (dpi). Moreover, type I IFN (IFN-α, IFN-ß) and IL-12 expression levels peaked at 3 dpi, while type II IFN (IFN-γ), IL-6, IL-17 and IL-18 expression reached a maximum level at 1 dpi. IL-8 changed at 5 dpi, and IL-1ß and TNF-α changed at 7 dpi. Interestingly, several key IFN-stimulated genes (ISGs), including IFITM1, IFITM2, IFITM5, Mx1 and OASL, were simultaneously upregulated and reached maximum values at 3 dpi. These data indicate that the MDA5 signaling pathway and innate immune cytokines were induced after ARV infection, which would contribute to the ARV-host interaction, especially at the early infection stage.

Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay.

To date, nine neuraminidase (NA) subtypes of avian influenza virus (AIV) have been identified in poultry and wild birds. Rapid and effective methods for differentiating these nine NA subtypes are needed. We developed and validated a rapid, sensitive, and robust method utilizing a GeXP analyzer-based multiplex RT-PCR assay and capillary electrophoresis for the simultaneous differentiation of the N1 to N9 subtypes in a single-tube reaction. Ten pairs of primers-nine subtype-specific pairs and one pan-AIV pair-were screened and used to establish the GeXP multiplex RT-PCR assay. A single subtype was detected using the developed GeXP assay; the N1 to N9 AIV subtypes individually generated two target peaks: the NA subtype-specific peak and the general AIV peak. Different concentrations of multiplexed subtypes were tested with this GeXP assay and the peaks of the corresponding NA subtypes were generated, suggesting that this GeXP assay is useful for identifying NA subtypes in mixed samples. Moreover, no peaks were generated for other important avian viruses, indicating negative results and validating the lack of cross-reactions between AIV subtypes and other avian pathogens. RNA templates synthesized through in vitro transcription were used to analyze the sensitivity of the assay; the limit of detection was 100 copies per reaction mixture. The results obtained from clinical samples using this GeXP method were consistent with the results of the neuraminidase inhibition (NI) test, and the ability of the GeXP assay to identify mixed infections was superior to amplicon sequencing of isolated viruses. In conclusion, this GeXP assay is proposed as a specific, sensitive, rapid, high-throughput, and versatile diagnostic tool for nine NA subtypes of AIV.

Development of duplex fluorescence-based loop-mediated isothermal amplification assay for detection of Mycoplasma bovis and bovine herpes virus 1.

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.

Characterization of an Avian Influenza Virus H9N2 Strain Isolated from Dove in Southern China.

We report here the complete genome sequence of strain H9N2, an avian influenza virus (AIV) isolated from dove in Guangxi, China. Phylogenetic analysis showed that it was a novel reassortant AIV derived from chicken, duck, and wild bird. This finding provides useful information for understanding the H9N2 subtype of AIV circulating in southern China.