Hypervirulent fowl adenovirus serotype 4 elicits early innate immune response and promotes virus-induced cellular autophagy in the spleen.

The recent emergence of hepatitis-hydropericardium syndrome caused by highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in significant economic losses to the poultry industry. However, the early innate immune response of immune organs within 24 hpi and the induction of autophagy in vivo after FAdV-4 infection have not been fully elucidated. In this study, 35-day-old specific pathogen-free (SPF) chickens were artificially infected with hypervirulent FAdV-4, which resulted in a mortality rate of up to 90%. The results showed that FAdV-4 infection rapidly triggered the innate immune response in vivo of chickens, with the spleen eliciting a stronger innate immune response than the thymus and bursa. During the early stage of viral infection within 24 hpi, the main receptors TLR3/7/21, MDA5, and cGAS were activated via the NF-κB and TBK1/IRF7-dependent signaling pathways, which up-regulated production of inflammatory cytokines and type I interferons. Additionally, the expression levels of the autophagy-related molecules LC3B, Beclin1, and ATG5 were significantly up-regulated at 24 hpi, while degradation of SQSTM1/p62 was observed, suggesting that FAdV-4 infection elicits a complete autophagy response in the spleen. Besides, the colocalization of Fiber2 and LC3B suggested that FAdV-4 infection induced autophagy which benefits FAdV-4 replication in vivo. This study provides new insights into the immunoregulation signal pathways of the early innate immunity in response to hypervirulent FAdV-4 infection in vivo within 24 hpi and the close relationship between viral replication and autophagy.

Rapid detection of pigeon adenovirus 2 using a TaqMan real-time PCR assay.

Pigeons infected with aviadenoviruses have been found worldwide. Recently, pigeon adenovirus 2 (PiAdV-2) has been widely distributed in racing pigeons in Germany. However, the epidemiology of this virus remains unclear due to the lack of a specific detection platform for PiAdV-2. In this study, we first detected PiAdV-2 positivity in racing pigeons (designated FJ21125 and FJ21128, which share 100% nucleotide identity with each other based on the fiber 2 gene) in Fujian, Southeast China. These genes shared 99.8% nucleotide identity with PiAdV-2 (GenBank No. NC_031501) but only 54.1% nucleotide identity with PiAdV-1 (GenBank No. NC024474). Then, the TaqMan-qPCR assay for the detection of PiAdV-2 was established based on fiber 2 gene characterization. The established assay had a correlation coefficient of 1.00, with an amplification efficiency of 99.0%. The minimum detection limit was 34.6 copies/µL. Only PiAdV-2 exhibited a positive fluorescent signal, and no signal was detected for other pathogens (including PiCV, FAdV-4, FAdV-8a, EDSV, PPMV-1, RVA and PiHV). The assay has good reproducibility, with a coefficient of variation less than 2.42% both intragroup and intergroup. The distributions of PiAdV-2 in fecal samples from YPDS (35 samples) and healthy (43 samples) racing pigeons from different geographical areas were investigated and were 37.14% (YPDS) and 20.93% (healthy), respectively. In summary, we developed a TaqMan-qPCR platform for the detection of PiAdV-2 infection with high sensitivity, specificity, and reproducibility. We confirmed the presence of PiAdV-2 in China, and our data suggested that there is no indication of a correlation between YPDS and PiAdV-2. This study provides more information on the pathogenesis mechanism and epidemiological surveillance of PiAdV-2.

Codon usage bias of goose circovirus and its adaptation to host.

Goose circovirus (GoCV), a potential immunosuppressive virus possessing a circular single-stranded DNA genome, is widely distributed in both domesticated and wild geese. This virus infection causes significant economic losses in the waterfowl industry. The codon usage patterns of viruses reflect the evolutionary history and genetic architecture, allowing them to adapt quickly to changes in the external environment, particularly to their hosts. In this study, we retrieved the coding sequences (Rep and Cap) and the genome of GoCV from GenBank, conducting comprehensive research to explore the codon usage patterns in 144 GoCV strains. The overall codon usage of the GoCV strains was relatively similar and exhibited a slight bias. The effective number of codons (ENC) indicated a low overall extent of codon usage bias (CUB) in GoCV. Combined with the base composition and relative synonymous codon usage (RSCU) analysis, the results revealed a bias toward A- and G-ending codons in the overall codon usage. Analysis of the ENC-GC3s plot and neutrality plot suggested that natural selection plays an important role in shaping the codon usage pattern of GoCV, with mutation pressure having a minor influence. Furthermore, the correlations between ENC and relative indices, as well as correspondence analysis (COA), showed that hydrophobicity and geographical distribution also contribute to codon usage variation in GoCV, suggesting the possible involvement of natural selection. In conclusion, GoCV exhibits comparatively slight CUB, with natural selection being the major factor shaping the codon usage pattern of GoCV. Our research contributes to a deeper understanding of GoCV evolution and its host adaptation, providing valuable insights for future basic studies and vaccine design related to GoCV.

Rapid detection of goose megrivirus using TaqMan real-time PCR technology.

The aim of this study was to develop an efficient and accurate platform for the detection of the newly identified goose megrivirus (GoMV). To achieve this goal, we developed a TaqMan real-time PCR technology for the rapid detection and identification of GoMV. Our data showed that the established TaqMan real-time PCR assay had high sensitivity, with the lowest detection limit of 67.3 copies/µL. No positive signal can be observed from other goose origin viruses (including AIV, GPV, GoCV, GHPyV, and GoAstV), with strong specificity. The coefficients of variation of repeated intragroup and intergroup tests were all less than 1.5%, with excellent repeatability. Clinical sample investigation data from domestic Minbei White geese firstly provided evidence that GoMV can be transmitted both horizontally and vertically. In conclusion, since the TaqMan real-time PCR method has high sensitivity, specificity, and reproducibility, it can be a useful candidate tool for GoMV epidemiological investigation.

Simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting analysis.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/µL (for DuCV-1) and 60.6 copies/µL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.

Genomic analysis reveals the population structure and antimicrobial resistance of avian Pasteurella multocida in China.

OBJECTIVES: To investigate the population structure and antimicrobial resistance (AMR) of avian Pasteurella multocida in China. METHODS: Utilizing WGS analysis, we explored the phylogeny using a dataset of 546 genomes, comprising avian P. multocida isolates from China (n = 121), the USA (n = 165), Australia(n = 153), Bangladesh (n = 3) and isolates of other hosts from China (n = 104). We examined the integrative and conjugative element (ICE) structures and the distribution of their components carrying resistance genes, and reconstructed the evolutionary history of A:L1:ST129 (n = 110). RESULTS: The population structure of avian P. multocida in China was dominated by the A:L1:ST129 clone with limited genetic diversity. A:L1:ST129 isolates possessed a broader spectrum of resistance genes at comparatively higher frequencies than those from other hosts and countries. The novel putative ICEs harboured complex resistant clusters that were prevalent in A:L1:ST129. Bayesian analysis predicted that the A:L1:ST129 clone emerged around 1923, and evolved slowly. CONCLUSIONS: A:L1:ST129 appears to possess a host predilection towards avian species in China, posing a potential health threat to other animals. The complex AMR determinants coupled with high frequencies may strengthen the population dominance of A:L1:ST129. The extensive antimicrobial utilization in poultry farming and the mixed rearing practices could have accelerated AMR accumulation in A:L1:ST129. ICEs, together with their resistant clusters, significantly contribute to resistance gene transfer and facilitate the adaptation of A:L1:ST129 to ecological niches. Despite the genetic stability and slow evolution rate, A:L1:ST129 deserves continued monitoring due to its propensity to retain resistance genes, warranting global attention to preclude substantial economic losses.

Rapid detection of goose astrovirus genotypes 2 using real-time reverse transcription recombinase polymerase amplification.

BACKGROUND: Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. RESULTS: The real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/µL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. CONCLUSIONS: The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.

Rapid detection of pigeon Megrivirus using TaqMan real-time PCR technology.

Megriviruses have been identified from fecal samples in wild pigeons in Hong Kong (China) and Hungary. In this study, the genomic sequences of pigeon Megriviruses (PiMeVs) were downloaded from GenBank and compared. Based on the genetic comparison results, a pair of primers and TaqMan probe were designed based on the conserved sequences of the 3C gene (located in the P3 gene coding region), and a TaqMan real-time PCR method (TaqMan-qPCR) was established. The standard curve of the TaqMan-qPCR had an axial intercept of 39.74 and a slope of -3.2475 with a linear correlation (R2) of 1.00 and an efficiency of 103.2%. No cross-amplification signal was found from other pigeon viruses (such as avian influenza virus, pigeon paramyxovirus type I, pigeon torque teno virus, pigeon adenovirus, and pigeon circovirus). The limit of detection concentration was 53.6 copies/µL. The intra- and interassay results were less than 1.0% based on the reproducibility test. Furthermore, field samples investigation by the established TaqMan-qPCR method showed that positive signals can be found from racing pigeon fecal samples and embryos. Thus, our data suggested that this visible TaqMan-qPCR method is sensitive, specific, and reproducible. Moreover, we first confirmed the presence of pigeon Megrivirus infection in racing pigeon embryos, indicating that the virus may be vertically transmitted. This study provides a reference basis for further understanding the epidemiology of PiMeVs.

Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Rapid diagnosis of different goose astrovirus genotypes with Taqman-based duplex real-time quantitative PCR.

The epidemic of goose astrovirus (GoAstV) caused huge losses to the poultry industry. Epidemiological studies in China revealed 2 circulating genotypes of GoAstV, but there is a lack of differential diagnosis tools. By analyzing all published genomes of GoAstV, this study designed specific PCR primers and Taqman probes to recognize different genotypes of GoAstV. After optimization and verification, this study developed a Taqman-based real-time quantitative PCR method that is capable of differential diagnosis. The established qPCR exhibited detection limitations of 100 copies/µL or 10 copies/µL, respectively, for GoAstV genotype 1 and genotype 2, and showed no false positive for other common avian viruses. This method was then used to analyze 72 samples collected from different regions in Jiangxi, and the results were verified by genome sequencing and phylogenetic analysis. These results revealed a complex coinfection of GoAstV different genotypes in China, highlighting the importance of long-term focus on the prevalence and genome evolution of GoAstV.

Rapid diagnosis of duck Tembusu virus and goose astrovirus with TaqMan-based duplex real-time PCR.

The mixed infection of duck Tembusu virus (DTMUV) and goose astrovirus (GoAstV) is an important problem that endangers the goose industry. Although quantitative PCR has been widely used in monitoring these two viruses, there is no reliable method to detect them at the same time. In this study, by analyzing the published genomes of DTMUV and goose astrovirus genotype 2 (GoAstV-2) isolated in China, we found that both viruses have high conservation, showing 96.5 to 99.5% identities within different strains of DTMUV and GoAstV, respectively. Subsequently, PCR primers and TaqMan probes were designed to identify DTMUV and GoAstV-2, and different fluorescent reporters were given to two probes for differential diagnosis. Through the optimization and verification, this study finally developed a duplex TaqMan qPCR method that can simultaneously detect the above two viruses. The lower limits of detection were 100 copies/µL and 10 copies/µL for DTMUV and GoAstV-2 under optimal condition. The assay was also highly specific in detecting one or two viruses in various combinations in specimens, and provide tool for clinical diagnosis of mixed infections of viruses in goose.

Characterization and evaluation of an oral vaccine via nano-carrier for surface immunogenic protein (Sip) delivery against Streptococcus agalactiae infection.

Streptococcus agalactiae causes systemic disease in a variety of wild and farmed fish, resulting in high levels of morbidity and mortality, as well as serious economic losses to the Nile tilapia aquaculture industry. The development of economic and applicable oral vaccines is therefore urgently needed for the sustainable development of Nile tilapia aquaculture. In this study, mesoporous silica nanoparticles (MSNs) were fabricated using sol-gel synthesis technology, and the antigens of surface immunogenic protein (Sip) was loaded into MSNs to develop a nanovaccine MSNs-Sip@HP55. The results showed that the prepared nanovaccine exhibited pH-controlled release, which could survive in the simulated gastric environment (pH 1.5), and release antigens in the simulated intestinal environment at pH 7.4. The nanovaccine could induce innate and adaptive immune responses in Nile tilapia. When the challenge doses were 1.5 × 106, 1.18 × 106, and 0.88 × 106 CFU/mL, the relative protection rates in immunized Nile tilapia were 63.33 %, 64.23 %, and 76.31 %, respectively. Taken together, the nanovaccine exhibited a high antigen utilization rate and was easily administered orally via feeding, which could protect Nile tilapia against challenge with S. agalactiae in large-scale farms. Oral vaccine based on MSNs carriers is a potentially promising strategy for the development of fish vaccines.

WITHDRAWN: Molecular Evolution and Amino Acid Characteristics of Main Antigen Genes of Clinical Duck-Derived H5N6 Subtype Avian Influenza Virus in East China from 2015 to 2019.

This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause.

Differential metabolism-associated gene expression of duck pancreatic cells in response to two strains of duck hepatitis A virus type 1.

Several outbreaks of duck hepatitis A virus type 1 (DHAV-1), which were characterized by yellow coloration and hemorrhage in pancreatic tissues, have occurred in China. The causative agent is called pancreatitis-associated DHAV-1. The mechanisms involved in pancreatitis-associated DHAV-1 infection are still unclear. Transcriptome analysis of duck pancreas infected with classical-type DHAV-1 and pancreatitis-associated DHAV-1 was carried out. Deep sequencing with Illumina-Solexa resulted in a total of 53.9 Gb of clean data from the cDNA library of the pancreas, and a total of 29,597 unigenes with an average length of 993.43 bp were generated by de novo sequence assembly. The expression levels of D-3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which are involved in glycine, serine, and threonine metabolism pathways, were significantly downregulated in ducks infected with pancreatitis-associated DHAV-1 compared with those infected with classical-type DHAV-1. These findings provide information regarding differences in expression levels of metabolism-associated genes between ducks infected with pancreatitis-associated DHAV-1 and those infected with classical-type DHAV-1, indicating that intensive metabolism disorders may contribute to the different phenotypes of DHAV-1-infection.

First report of the multiresistance gene cfr in Pasteurella multocida strains of avian origin from China.

OBJECTIVES: The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr gene in Pasteurella multocida of avian origin from China. METHODS: A total of 113 P. multocida isolates were collected from sick poultries (ducks, chickens and geese) from 2003 to 2016 in Southern China and were screened for the presence of the cfr gene by PCR. The cfr-carrying P. multocida strains were subjected to antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blot hybridisation, conjugative transfer and analysis of genetic environment of the cfr gene. RESULTS: Among 113 P. multocida isolates, strains FJ6671 and FJ6683 from Muscovy duck harboured the cfr gene and presented a multiresistant phenotype. The cfr gene in the two strains was located on an ∼40-kb conjugative plasmid in different genetic environments, including ISApl12-cfr-IS26 and IS26-cfr-IS256. CONCLUSIONS: These results demonstrate plasmid-carried cfr in P. multocida and suggest that transposition and homologous recombination mediated by IS26, ISApl1 and IS256 might have played an important role in transfer of the cfr gene in P. multocida. To the best of our knowledge, this is the first report of the cfr gene in P. multocida. Active and ongoing surveillance of cfr in P. multocida is urgently warranted.

Specific detection and differentiation of classic goose parvovirus and novel goose parvovirus by TaqMan real-time PCR assay, coupled with host specificity.

BACKGROUND: Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. RESULTS: After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/µl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. CONCLUSIONS: We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

Specific detection of the novel goose astrovirus using a TaqMan real-time RT-PCR technology.

Recently, a novel goose astrovirus (N-GoAstV) was discovered in China, with the transmission route of N-GoAstV unclear. In this study, we developed a TaqMan-based real-time RT-PCR (qRT-PCR) assay for the detection of N-GoAstV infection. After the optimization of the qRT-PCR assay conditions, the results demonstrated that the lower limit of detection for N-GoAstV was 33.4 copies/µL. No cross-reactivity was observed with other goose-origin viruses. Intra-assay and inter-assay variability were ≤1.36% and 2.34%, respectively. N-GoAstV was detected in both field samples, embryos and newly hatched goslings by qRT-PCR assay, provided the view that N-GoAstV may be both horizontally and vertically transmitted. The established qRT-PCR method showed high specificity, sensitivity, and reproducibility, which can be used in future investigations on the pathogenesis and epidemiology of N-GoAstV.