Epidemiological investigation of fowl adenovirus (FAdV) infections in ducks and geese in Shandong Province, China.

RESEARCH HIGHLIGHTS: Samples of suspected FAdV-infected waterfowl from farms in Shandong Province were collected from 2019 to 2022.Single infections with FAdV were less frequent than mixed infections.477 out of 792 samples (60.23%) tested positive for FAdV nucleic acids.Detection rate of FAdV was 65.47% in fattening duck farms, 55.73% in breeder duck farms and 54.55% in fattening geese farms.

Pathogenicity of duck circovirus and novel goose parvovirus co-infection in SPF ducks.

Duck circovirus (DuCV) is one of the most prevalent infectious viruses in the duck industry in China. Although the clinical signs vary, it often causes immunosuppression in the host and leads to secondary infection with other pathogens. Novel goose parvovirus (NGPV) mainly infects ducks and causes short beak and dwarfism syndrome in ducks. However, the incidence of infection in ducks has increased in recent years, and the phenomenon of mixed infection with DuCV is common, resulting in more severe clinical morbidity. However, there are no systematic studies evaluating the presence of mixed infections. In order to investigate the synergistic pathogenicity of DuCV and NGPV co-infection in SPF ducks, a comparative experiment using DuCV and NGPV co-infection and mono-infection bird models was established. The results showed that the clinical signs of short beak, dwarfism and immunosuppression were more obvious in DuCV and NGPV co-infected ducks; the tissue damage of target organs was more serious, and the viral titre in organs and cloacal swabs were more significant compared with those of SPF ducks infected with only one virus. The results indicated that co-infection with DuCV and NGPV could promote viral replication and cause more severe tissue damage and immunosuppression than single virus infection. The present study reveals that the co-infection of NGPV and DuCV has a synergistic pathogenic effect from the aspect of pathogenicity, and the conclusions drawn not only clarify the direction of the subsequent research on the mechanism of co-infection of NGPV and DuCV, but also provide a scientific basis for the research on the co-infection of immunosuppressive pathogens and other pathogens.

Genetic characteristics and pathogenicity of avian pox virus for a new host, Cherry Valley breeder ducks in China.

Cherry Valley breeder ducks in Shandong province in northern China have experienced swollen eyes, lachrymation, pox scabs on glabrous or glabrous skin, depression and dysentery since 2021. The spread of this infectious disease has seriously affected the breeder ducks in major Cherry Valley duck farms in China. The virus causing clinical signs in Cherry Valley breeder ducks was isolated by chicken embryo inoculation. We successfully isolated a strain of duck pox virus from diseased breeder ducks by virus replication. We have also successfully conducted experiments for duck pox disease using the isolated strains to infect ducklings. By comparison with 22 pox viruses already published in GenBank, the virus strain obtained in this study was most homologous (about 99.7%) to the strain isolated from infected domestic ducks in Guangxi, China in 2014 (KJ192189), and belonged to the same A5 subtype. Since there were no previous cases of avian pox virus infecting white or Cherry Valley duck breeds, this study identified a new host for avian pox virus infection and provided theoretical support and data for the development of avian pox virus research.RESEARCH HIGHLIGHTS Avian pox virus can infect a new host type - Cherry Valley breeder ducks.The avian pox virus isolated from Cherry Valley breeder ducks showed highest identity with Guangxi hemp duck-derived avian pox virus.Cherry Valley breeder ducks were infected with avian pox virus of subtype A5.

Co-circulation of multiple genotypes of ARV in poultry in Anhui, China.

ABSTRACTPoultry production in China has been experiencing a high incidence of broiler arthritis /tenosynovitis caused by avian orthoreovirus (ARV) since 2013. In the spring of 2020 severe arthritis cases from broiler flocks were identified in a large-scale commercial poultry company in Anhui Province, China. Diseased organs from dead birds were sent for diagnosis to our laboratory. ARVs, including seven broiler-isolates and two breeder-isolates, were successfully harvested and sequenced. Interestingly, the genotypes of ARVs isolated from infected chickens were inconsistent between different flocks, or even between different houses on the same flocks. Pathogenicity testing in chicks confirmed that the seven broiler-isolates were pathogenic strains, which could cause arthritis in infected chickens. Subsequently, a total of 89.66% serum samples collected from apparently healthy adult broiler flocks not vaccinated against ARV tested positive for ARV antibodies, suggesting that low and high virulence reovirus strains may be co-circulating in the farm. To this end, we collected dead embryos of unhatched chicken eggs for pathogen tracing, and the two ARV breeder-isolates isolated indicated that vertical transmission from breeders to progeny should not be underestimated for the prevalence of ARV within broiler flocks. The findings have implications for the evidenced-based formulation of prevention and control strategies.

Localization and distribution of goose astrovirus 2 antigens in different tissues at different times.

Goose astrovirus 2 (GAstV-2) causes visceral gout in goslings and has resulted in significant economic losses in the goose industry of China since its outbreak in 2017. To further investigate the distribution and localization of GAstV-2 in different tissues at different times, a monoclonal antibody (mAb)-based immunohistochemical (IHC) assay was developed to detect GAstV-2. A total of 80 1-day-old healthy goslings were inoculated with GAstV-2 via the oral (n = 40) and intramuscular routes (n = 40). GAstV-2 in the tissues of interest was detected using the established IHC assay. The results showed that positive signals were detected in most tissues at 1 day post-infection (dpi). Viral antigens were mainly distributed in the cytoplasm, and the staining intensity was higher in the renal tubular epithelial cells than in other cells. Taken together, our data demonstrated that GAstV-2 has a broad tissue tropism and primarily targets the kidneys. These results are likely to provide a scientific basis for further elucidation of the pathogenesis of GAstV-2.

Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics.

In China, drastic losses in the economy have been caused by the Tembusu virus (TMUV), the causative agent of the egg-drop syndrome, to the duck-raising industry. To succeed in preventing and controlling infections, extant techniques must be upgraded to achieve fast detection of viruses. This work is the first attempt to present the development of a recombinase polymerase amplification (RPA)-based clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas13a approach for the TMUV infection diagnosis, where the CRISPR-Cas13a system is exploited, i.e., the programmability of CRISPR RNA (crRNA) and the promiscuous RNase collateral cleavage of Cas13a upon recognition of target RNAs. A prokaryotic expression system was utilized for the expression of LwCas13a soluble protein, while its purification was accomplished by nickel-nitrilotriacetic acid (Ni-NTA) agarose. In the design of a particular crRNA, the target used was the TMUV NS3 RNA transcribed in vitro. The signals used for the Cas13a activity validation were an RNA-bound fluorescent group (single-stranded) and a quenching fluorophore. In the present work, a specific high-sensitivity enzymatic molecular detection system termed RPA-based CRISPR-Cas13a was established by combining Cas13a with T7 transcription and RPA for sensitive detection of TMUV at room temperature. This system can detect 102 copies of the target TMUV DNA standard/µL within 50 min. A comparison revealed that the specificity was superior to that for other avian viruses. Furthermore, the RPA-based CRISPR-Cas13a detection system was successfully applied for clinical samples, and its performance is comparable to the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR). Being satisfyingly reliable, simple, specific, and sensitive, our RPA-based CRISPR-Cas13a detection system could be expanded and universalized for identifying other viruses, enabling quick detection in the field with a portable lateral flow dipstick.

Pathogenicity of novel goose-origin astrovirus causing gout in goslings.

BACKGROUND: A novel goose-origin astrovirus (GoAstV) has broken out across China in recent years, causing gout in goslings with a mortality rate of around 50%. However, our understanding of the dynamic distribution, tissue tropism and pathogenesis of GoAstV is incomplete. In order to assess its pathogenicity, one-day-old goslings were inoculated separately with GoAstV via oral and subcutaneous injection routes. RESULTS: Clinical symptoms, gross and microscopic lesions, blood biochemical parameters and viral loads were detected and recorded for 20 days after infection. Typical gout was observed in experimental goslings. GoAstV can be replicated in tissues and cause pathological damage, especially in the kidney, liver, heart and spleen. Virus-specific genomic RNA was detected in blood, cloacal swabs and all representative tissues, and virus shedding was detected up to 20 days after inoculation, suggesting that GoAstV has a wide tissue tropism and spread systematically after inoculation. The viral copy numbers examined in kidney were the highest, followed by spleen and liver. CONCLUSION: This experiment determined the accurate value of viral loads and biochemical indicators of GoAstV-induced goslings. These findings increase our understanding of the pathogenicity of GoAstV in goslings and provide more reference for future research.

A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China.

BACKGROUND: In China, Newly emerging duck reovirus (NDRV) variants have been causing major disease problems in cherry valley ducks. NDRV has the potential to cause high morbidity and 5-50% mortality rates. Severe hemorrhagic-necrosis in the liver and spleen were commonly seen in NDRV affected ducks. The availability of upgraded methods for rapid diagnosis of newly emerging DRV variants is crucial for successful DRV infection control and prevention. RESULTS: In this study, we present a TaqMan-based real-time PCR assay (RT-qPCR) for the detection of NDRV infection. Using the conserved regions within the NDRV genome, we designed the specific primers and probe. The lower limit of detection for NDRV infection was 10 copies/µL (Ct values: 38.3) after the optimization of the RT-qPCR conditions. By cross-checking with other duck viral pathogens, no cross-reactivity was observed confirming the assay was highly specific for the detection of NDRV. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assay variability was less than 2.91%(Intra-assay variability of Ct values: 0.07-1.48%; Interassay variability of Ct values: 0.49-2.91%). This RT-qPCR and conventional PCR (cPCR) detected one hundred and twenty samples of NDRV infection from different regions. The result shows that the positive rates were 94.17 and 84.17% respectively. The detection rate of RT-qPCR rapid detection assay was 10% higher than that of the cPCR method. CONCLUSION: This research developed a highly sensitive, specific, reproducible and versatile of RT-qPCR for quantitatively detecting NDRV. It can be used to study the pathogenesis and epidemiology investigation of NDRV.

Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China.

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5 dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China. RESEARCH HIGHLIGHTS A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China. The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains. The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection. These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

Pathogenicity of fowl adenovirus (FAdV) serotype 4 strain SDJN in Taizhou geese.

Hydropericardium hepatitis syndrome (HHS) is a fatal disease in chickens, mainly caused by fowl adenovirus serotype 4 (FAdV-4). Since June 2015, HHS has appeared in many provinces in China. The disease has spread from broilers to laying hens, breeders and Cherry Valley ducks, seriously endangering the health of the poultry industry in China. In July 2016, an infectious disease was noticed in a goose farm in Jinan, Shandong Province, China, and hydropericardium was the main finding in post mortem investigations. In the actual study, we isolated a FAdV-4 strain from the livers of naturally-infected goslings and designated it as SDJN. We first evaluated its pathogenicity by inoculating Taizhou geese at 10, 20, and 30 days of age with 10-7.15EID50/0.2 ml doses of the SDJN strain in 1 ml allantoic fluid via subcutaneous injection or oral infection. Clinical signs and pericardial effusion appeared in geese infected subcutaneously at 10 days of age, whereas 20- and 30-day-old geese were not susceptible to FAdV-4. The results of real-time PCR showed that the replication ability of FAdV-4 in geese correlated with the age. Furthermore, results from clinical chemistry showed that FAdV-4 damaged the liver and kidney in geese and the results paralleled viral load and gross lesions. Consequently, FAdV-4 was pathogenic in geese, and the pathogenicity was related to age and mode of infection. This study is the first experimental infection of FAdV-4 in geese, which will provide a basis for further understanding of the disease. RESEARCH HIGHLIGHTS Pathogenicity tests with a FAdV-4 were conducted in geese, which included data on clinical signs, gross pathology, histopathology, clinical chemistry and viral load. FAdV-4 could replicate in geese and HHS was successfully induced. Pathogenicity of FAdV-4 in geese was related to the age and routes of infection.

Integrated Analysis of miRNA and mRNA Expression Profiles in Spleen of Specific Pathogen-Free Chicken Infected with Avian Reticuloendotheliosis Virus Strain SNV.

The Reticuloendotheliosis virus (REV) primarily causes avian severe immunosuppression, in addition to other symptoms, which include avian dwarfing syndrome and chronic tumors in lymphoid and other tissue. To date, REV's molecular mechanisms leading to immunosuppression have not been fully elucidated. In the current study, we aimed to elucidate the role of microRNAs (miRNA) in regulating gene expression during REV infections. Therefore, we used a high-dose spleen necrosis virus (SNV) model of REV to inoculate one-day-old specific pathogen-free (SPF) chickens, thereby inducing congenital infections. We analyzed miRNA and mRNA expression profiles using Next Generation Sequencing (NGS) in a total of 19 spleen samples that were collected at 7, 14, and 21 days post infection (dpi). The results showed that 63 differentially expressed miRNAs (DEmiRNAs) (30 known miRNAs and 33 novel miRNAs) and 482 differentially expressed target genes (DETGs) were identified. Integration analysis identified 886 known miRNA⁻mRNA and 580 novel miRNA⁻mRNA interaction pairs, which involved miRNAs that were inversely correlated with the above DETGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DETGs were considerably enriched in the immune-relevant pathways category, such as immune system, cell growth and death, signaling molecules and interaction, signal transduction, etc. We further verified selected immune-relevant miRNA and their DETGs while using quantitative RT-PCR (qRT-PCR). Overall, our data revealed valuable immune-related miRNA⁻mRNA interaction information that occurred during REV infections, thereby broadening our understanding of the REV-induced immunosuppression.

Complete genome sequence of a novel avian orthoreovirus isolated from gosling, China.

Avian orthoreovirus (ARV) has been considered as a significant pathogen causing great infectious diseases to the avian, like broiler and waterfowl. The genome of this novel ARV(Reo/SDPY/Goose) was completely sequenced by next-generation sequencing. The complete genome was found to be 23517 bp in length with 10 segments. Although the Reo/SDPY/Goose was isolated from the gosling, it shares great similarity, no matter which segment within the genome, with those published as avian-origin reovirus. Genomic analysis revealed that this virus was distinct from published ARV strains and met criteria to become a novel ARV strain.

Comparative Transcriptomic Analysis of Immune-Related Gene Expression in Duck Embryo Fibroblasts Following Duck Tembusu Virus Infection.

Duck is a major waterfowl species in China, providing high-economic benefit with a population of up to 20⁻30 billion per year. Ducks are commonly affected by severe diseases, including egg-drop syndrome caused by duck Tembusu virus (DTMUV). The immune mechanisms against DTMUV invasion and infection remain poorly understood. In this study, duck embryo fibroblasts (DEFs) were infected with DTMUV and harvested at 12 and 24 h post-infection (hpi), and their genomes were sequenced. In total, 911 (764 upregulated and 147 downregulated genes) and 3008 (1791 upregulated and 1217 downregulated) differentially expressed genes (DEGs) were identified at 12 and 24 hpi, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that DEGs were considerably enriched in immune-relevant pathways, including Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, Chemokine signaling pathway, NOD-like receptor signaling pathway, and Hematopoietic cell lineage at both time points. The key DEGs in immune system included those of the cytokines (IFN α2, IL-6, IL-8L, IL-12B, CCR7, CCL19, and CCL20), transcription factors or signaling molecules (IRF7, NF-κB, STAT1, TMEM173, and TNFAIP3), pattern recognition receptors (RIG-I and MDA5), and antigen-presenting proteins (CD44 and CD70). This suggests DTMUV infection induces strong proinflammatory/antiviral effects with enormous production of cytokines. However, these cytokines could not protect DEFs against viral attack. Our data revealed valuable transcriptional information regarding DTMUV-infected DEFs, thereby broadening our understanding of the immune response against DTMUV infection; this information might contribute in developing strategies for controlling the prevalence of DTMUV infection.

Preparation and evaluation of goose reovirus inactivated vaccine.

BACKGROUND: Infection with Goose Reovirus (GRV) can cause serious economic losses in the goose breeding industry. In this study, the GRV allantoic fluid was concentrated and used as an antigen in a formalin-inactivated oil-emulsion vaccine. RESULTS: When 6 day-old geese were inoculated, antibodies against GRV became detectable at 6 days post-vaccination, their concentration peaked at 3 weeks. These antibodies were maintained for longer than 2 weeks. As the most susceptible age for GRV infection is birds under 2 weeks of age this vaccine should provide adequate cover for the most at risk birds. When geese were exposed to reovirus at different time intervals after immunization, the data revealed that the vaccine can provide a protection rate of 80%. The developed vaccine has good stability and could be stored at 4 °C for at least 12 months. CONCLUSION: These results indicate that the developed GRV vaccine is safe, effectively absorbed, efficacious in inducing a rapid immune response, and effective in controlling GRV infection. Our results should be useful for the application of vaccines for controlling GRV in different goose flocks.

Comparison of four molecular assays for the detection of Tembusu virus.

Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/µL, 20 copies/µL, 2 copies/µL, and 20 copies/µL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.

Evaluation of histopathological changes, viral load and immune function of domestic geese infected with Newcastle disease virus.

Outbreaks of Newcastle disease in flocks of geese with high morbidity and mortality in southern and eastern China have been reported frequently since the late 1990s, which broke the traditional view that geese are considered to be the natural reservoir of Newcastle disease virus (NDV) but show few or no clinical signs after infection. In this present study, geese were infected intranasally with a local strain of NDV. Clinical disease and gross pathology were observed. Serum and immune organs were collected from geese sequentially euthanized or after disease-associated death. We studied the histopathology of immune organs by haematoxylin and eosin staining and NDV fusion protein was detected in tissues by immunohistochemistry. At the same time, the SYBR Green I real-time polymerase chain reaction assay was used to detect the viral load from the collected samples. Serum samples were tested for NDV-specific antibodies and avian influenza virus (AIV)-specific antibodies by haemagglutination inhibition (HI) test. The results showed that severe lesions and numerous positive reactions of NDV antigen were detected in the immune organs. High viral loads developed in immune organs of infected geese, correlating with the severity of clinical signs and lesions in the tissues. Furthermore, the infected geese developed low HI antibody titres to both AIV and NDV. The present study showed that the replication and dissemination of the NDV isolate was widespread in immune organs of geese. The study revealed that waterfowl may not only be a natural reservoir of NDV but also become susceptible to disease and may play a major role in the epidemiology of Newcastle disease.

Isolation, Identification, and Pathogenicity of a Goose Astrovirus Genotype 1 Strain in Goslings in China.

Goose astrovirus genotype 1 (GAstV-1) has emerged in goose farms in some provinces of China in recent years and is considered to be one of the pathogens of gout in goslings in China. However, few studies have been conducted on the dynamic distribution, tissue tropism, and pathogenesis of GAstV-1 in goslings. In 2022, an epidemiological investigation of goose astrovirus (GAstV) in goslings was conducted in seven provinces of China. During the investigation, a GAstV-1 designated as GAstV-JSXZ was identified in the kidney of an 8-day-old gosling and was successfully isolated from a goose embryo. The full genome sequence of GAstV-JSXZ was determined using the next-generation sequencing technique. The complete genome of GAstV-JSXZ was 7299-nt-long. Interestingly, the phylogenetic analysis revealed that Chinese GAstV-1 has formed two distinct subgroups based on the ORF 2 genomes, designated GAstV-1 1a and GAstV-1 1b. The GAstV-JSXZ shared the highest identity with GAstV-1 1a strain FLX and TZ03 in nucleotides (ORF1a: 98.3-98.4%; ORF1b: 92.3-99.1%; ORF2: 95.8-98.8%) and amino acid sequences (ORF1a: 99.4-99.5%; ORF1b: 98.2-98.8%; ORF2: 97.0-99.4%). To evaluate the pathogenicity of GAstV-1, 1-day-old goslings were inoculated with the virus by oral and subcutaneous injection routes, respectively. The results revealed that the virus causes extensive pathological organ damage, especially in the kidney, liver, and thymus. Virus-specific genomic RNA could be detected in the cloacal swabs and tissues of infected goslings throughout the experiment. The viral copy numbers examined in the kidney and intestine were the highest, followed by the liver and spleen. These results are likely to provide a new understanding of the pathogenicity of GAstV-1 in geese.

Role of long non-coding RNA DLY6E in regulating TMUV infection.

Long non-coding RNA (lncRNA) is a type of RNA with a length greater than 200 nt and lacking coding ability. In recent years, a considerable number of lncRNAs have been found to have important functions. The lncRNA plays an important role in growth and development, body metabolism, immune function, and regulation of viral replication. A lncRNA, MSTRG8505.2, was screened and named lncRNA DLY6E, which was a new duck-derived lncRNA. The lncRNADLY6E in this study has a complex secondary structure, specifically distributed in the heart, liver and other organs. The expression of lncRNA DLY6E was significantly up-regulated after TMUV infection, which was time-dependent and non-dose-dependent. Overexpression of three structural proteins and seven non-structural proteins of TMUV in DEF cells showed no significant difference in the expression of lncRNADLY6E. Meanwhile, using lipopolysaccharides (LPS) and poly (I:C) to stimulate DEF cells, the results showed that the induced expression of lncRNA DLY6E was associated with the dsRNA-related TLR3/RIG-I/MDA5 pathway rather than the LPS activated signaling pathway. To further explore the function of lncRNA DLY6E, an eukaryotic expression vector was constructed. Overexpression of lncRNA DLY6E in DEF cells can increase the replication of TMUV. After overexpression of lncRNADLY6E, the transcriptional level of its target gene LY6E was detected, and the results showed that lncRNADLY6E did not act through its target gene. Overexpression of lncRNA DLY6E significantly inhibited the mRNA levels of OAS, Mx and PKR, suggesting that lncRNA DLY6E may promote the virus by inhibiting the transcription of antiviral proteins in innate immunity. This phenomenon provides new ideas for the prevention and control of TMUV, which is worth further thinking and exploration.

Pathogenicity studies and molecular characterization of DPV infection in ducklings.

In the spring of 2023, 10 to 21-day-old chicks in a broiler duck farm in Shandong Province, China, developed swelling of the head and neck, moist eyes with mucous discharge, difficulty in walking, shrinking of the neck, and loose and disorganized coat. Anatomical observation revealed hemorrhages in the esophageal mucosa, myocardium, and liver, and severe hemorrhages in the trachea with copious inflammatory secretions. Soon after, similar symptoms appeared in a large number of ducks in the flock, which eventually led to the elimination of all the 20,000-odd newly introduced ducklings on the farm, resulting in huge economic losses. We detected duck plague virus in the tissues of liver, spleen and lungs of diseased and dead ducks, and successfully isolated the pathogenic strain, named SD423, by inoculating duck embryos and inoculating duck embryo fibroblasts. We successfully conducted animal regression experiments with the isolated strain, and the experimental animals in the 1 d of age group showed symptoms of swollen eyes and tearing, shrinking of the neck, crouching, and hemorrhage in organs such as the liver and intestines successively from the 3rd d. We sequenced the whole genome of the isolated duck plague strain, and by comparing the homology with the published duck plague virus whole sequences in Genbank, the virus strain obtained in this study had the highest homology with the Chinese virulent strain SD (MN518864.1), with nucleotide (nt) homology of about 99.90% and amino acid (aa) homology of about 99.75%, which indicated that the isolate is a virulent strain. Previously, it was reported that the natural infection of duck plague virus mainly occurs above 30 d of age, but the duck plague virus found in this study can naturally infect ducklings up to 20 d of age, and the mortality rate is as high as 100%. In this study, the pathogenicity test and whole genome sequence analysis of this isolate provided data support and theoretical basis for further research on pathogenicity and virulence-related gene analysis of duck plague virus.

Pathogenicity of duck circovirus 1 in experimentally infected specific pathogen-free ducks.

Ducks infected with duck circovirus (DuCV) show symptoms such as feather loss, growth retardation and low body weight in the flock. The virus induces immunosuppression and increases the prevalence of infection with other pathogens. However, most studies on duck circovirus were focused on coinfection, and fewer studies had been conducted on the pathogenicity of duck circovirus alone. The aim of the present study was to investigate the pathogenesis of DuCV-1 in experimentally infected specific pathogen-free ducks. In this study, we sequenced the whole genome of a strain of duck circovirus and identified the virus genotype as DuCV-1b. This strain of duck circovirus was named SDLH(OR567883). Animal pathogenicity experiments were then conducted, wherein specific pathogen-free ducks were infected by mucosal injection and abdominal injection. Infected ducks were sampled for 4 consecutive weeks after infection and showed symptoms of dwarfism. We further examined the replication of DuCV-1 in the ducks. The highest virus titers in the 2 infection groups were found in the liver and spleen, with different results for the different routes of infection. Pathological sections of duck organs were made and it was found that organs such as the liver and spleen were damaged by DuCV-1. In conclusion, our experimental results indicate that DuCV-1 can infect ducks individually and cause widespread organ damage in infected ducks.