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Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is associated with high mortality in Pekin ducklings. σC is an outer capsid protein encoded by the S1 genome segment of NDRV which mediates attachment to host cells. Our previous studies using immunoprecipitation and mass spectrometry found that σC coprecipitated with some host proteins including Translocation-associated membrane protein 1 (TRAM1). However, the interaction between σC and TRAM1 has not been further confirmed experimentally. In this study, we utilized coimmunoprecipitation assays, glutathione S-transferase pull-down, and confocal microscopy to confirm the interaction between σC and TRAM1. In addition, knockdown of TRAM1 using siRNA and overexpression of TRAM1 gene were conducted to explore its effect on virus replication. The result showed that TRAM1 silencing benefits while overexpression inhibits viral replication. This study confirms the important role TRAM1 during NDRV infection which can help develop new approaches for NDRV disease prevention and control.

Duck adenoviruses (DAdVs) include serotype 1 (DAdV-1) in the genus Atadenovirus and serotypes 2-4 (DAdV-2, 3, and 4) in the genus Aviadenovirus. DAdV-3 was initially isolated from Chinese Muscovy ducks in 2014, whereby the infected ducks exhibited yellowing and hemorrhaging in the liver, along with slight pericardial effusion, swelling, and hemorrhaging in the kidneys. In recent years, duck adenovirus infections have appeared in Muscovy duck farms in Fujian, Zhejiang, Anhui, Guangdong, and other places in China. They have an incidence rate of 40 to 55% and a mortality rate of 35 to 43%, resulting in great losses to the duck breeding industry. In this study, 7 DAdV-3 strains, designated as TZ193, FJPT20161124, GX20170519, FJZZ, GDMM, AHAQ, and GDHS were isolated from Muscovy ducks in different provinces of China during 2016–2019, and their complete genomics were sequenced. Their genomes all exhibited significant deletions in ORF67, which also had G to A transitions at the 41st and 977th nt positions, resulting in a stop codon. The pathogenicity of TZ193, a novel isolate of DAdV-3, was investigated in Muscovy ducks. TZ193 caused characteristic lesions of swelling as well as hemorrhagic liver and kidney in the infected ducklings. Moreover, the mortality rate of TZ193 in 5-day-old domestic ducks was 100%. Our data provide concrete evidence for the identification of the DAdV-3 novel variant mutant in China, which effects increased mortality in ducks. This highlights the necessity for monitoring the specific molecular epidemiology of novel DAdV-3 mutants and the development of new vaccines in the future.

Duck short beak and dwarfism syndrome (SBDS) is a viral infectious disease caused by novel goose parvovirus (NGPV), which has been responsible for serious economic losses to the Chinese duck industry in recent years. Currently, there is no effective vaccine against this disease. In this study, we developed an inactivated virus vaccine candidate for SBDS based on NGPV strain DS15 isolated from a duck in China. Immune efficacy was evaluated in 112 ducks, which were randomly divided into vaccination, challenge-control, vaccination-challenge, and blank control groups (28 per group). Clinical characteristics, antibodies, virus excretion, viremia, and pathological changes were monitored. No morbidity or death was observed in the immunized ducks, which showed normal weight and a good mental state. High levels of serum antibodies (optical density at 450 nm of ~ 0.63) were detected in ducks immunized with the inactivated vaccine at 7 days post-vaccination (dpv), and the titer of virus-neutralizing antibodies increased from 1:2

A novel duck picornavirus, designated as duck/AH15/CHN/2015, was isolated and identified from Cherry Valley ducks with short beak and dwarfism syndrome in 2015 in Anhui province of China. Duck/AH15/CHN/2015 has the highest degree of amino acid sequence identity (approximately 43 %) with duck hepatitis A viruses (DHAV) Complete genome analysis revealed that duck/AH15/CHN/2015 possesses a typical picornavirus-like genomic organization, 5' UTR-L-P1 (VP0-VP3-VP1)-P2 (2A1-2A2- 2B-2C)-P3 (3A-3B-3C-3D)-3'UTR-poly (A). The 5'UTR contains a potential type IV internal ribosome entry site, while a conserved "barbell"-like structure is found at the 3'UTR, which is similar to DHAV. Compared to the closest related DHAVs, two unrelated 2A proteins were predicted in duck/AH15/CHN/2015, while three unrelated 2A proteins were presented in DHAVs. Based on the amino acid identity comparison and phylogenetic analysis of P1, 2C, and 3CD (3C and 3D), duck/AH15/CHN/2015 was closely related to but distinct from DHAVs, and it was proposed to be a member of a novel species in the genus Avihepatovirus of the family Picornaviridae.

Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is currently an infectious agent for ducks. Studies on NDRV replication and pathogenesis have been hampered by the lack of an available reverse-genetics system. In this study, a plasmid-based reverse-genetics system that is free of helper viruses has been developed. In this system, 10 full-length gene segments of wild-type NDRV TH11 strain are transfected into BSR-T7/5 cells that express bacteriophage T7 RNA polymerase. Production of infectious virus was shown by the inoculation of cell lysate derived from transfected cells into 10-day-old duck embryos. The in vivo growth kinetics and infectivity of the recombinant strains were identical to those of the wild-type strain. These viruses grew well and were genetically stable both in vitro and in vivo. Altogether, these results show the successful production of an infectious clone for NDRV. The infectious clone reported will be further used to elucidate the mechanisms of host tropism, viral replication and pathogenesis, as well as immunological changes induced by NDRV.

Novel duck reovirus (NDRV) disease emerged in China in 2011 and continues to cause high morbidity and about 5.0 to 50% mortality in ducklings. Currently there are no approved vaccines for the virus. This study aimed to assess the efficacy of a new vaccine created from the baculovirus and sigma C gene against NDRV. In this study, a recombinant baculovirus containing the sigma C gene was constructed, and the purified protein was used as a vaccine candidate in ducklings. The efficacy of sigma C vaccine was estimated according to humoral immune responses, cellular immune response and protection against NDRV challenge. The results showed that sigma C was highly expressed in Sf9 cells. Robust humoral and cellular immune responses were induced in all ducklings immunized with the recombinant sigma C protein. Moreover, 100% protection against lethal challenge with NDRV TH11 strain was observed. Summary, the recombinant sigma C protein could be utilized as a good candidate against NDRV infection.

An unknown infectious disease in Cherry Valley ducks (Anas platyrhynchos domesticus) characterized by short beak and strong growth retardation occurred in China during 2015. The causative agent of this disease, tentatively named duck short beak and dwarfism syndrome (DSBDS), as well as the evolutionary relationships between this causative agent and all currently known avian-origin parvoviruses were clarified by virus isolation, transmission electron microscope (TEM) observation, analysis of nuclear acid type, (RT-)PCR identification, whole genome sequencing, and NS1 protein sequences-based phylogenetic analyses. The results indicated that the causative agent of DSBDS is closely related with the goose parvovirus-like virus, which is divergent from all currently known avian-origin parvoviruses and should be a novel duck parvovirus (NDPV).

Background Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR3 is involved in the recognition of double-stranded RNA. This study was designed to explore the relationship between duTLR3 and duck reovirus (DRV) infection. Methods In this study, we cloned and performed a molecular characterization of the complete sequence of Peking duck TLR3 (duTLR3). The expression level of duTLR3 was also determined, along with the relative levels of Mx and IFN-α mRNA after DRV infection. Results The duTLR3 gene is 2776-bp long and encodes an 895-amino-acid-long protein. Sequence analysis of the product revealed the complete transcript of Peking duck TLR3, including the 88-bp 5′UTR, the 2688-bp coding sequence (ORF), and the 76-bp 3′UTR and poly(A) tail. DuTLR3 was found to share a high amino acid sequence similarity with TLR3 from Jing ding duck (99.6 %), Muscovy duck (97.1 %) and chicken (86.3 %). Additionally, the tissue distribution of duTLR3 suggested that it was abundantly expressed in various tissues, especially in the trachea, esophagus and pancreatic gland. Duck reovirus (DRV) infection resulted in high mRNA expression levels of duTLR3 in the spleen, liver, lung and brain. Conclusion These results suggest that duTLR3 may play an important role in anti-viral defense mechanisms. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0434-x) contains supplementary material, which is available to authorized users.

A Neisseria strain, Neisseria sp. AH-N10, was isolated from liver of a Chinese Peking duck and characterized using a number of phenotypic and genotypic approaches. Based on scanning electron microscopy examination, the isolated strain has the typical structure of Neisseria species. Sequence comparison of 16S rRNA gene and phylogenetic analysis suggest that Neisseria sp. AH-N10 is closely related to Neisseria canis, which was previously isolated from a human dog bite wound. Animal infection experiments demonstrated that the isolated Neisseria sp. AH-N10 is pathogenic in ducks and mice. The pathogenicity to humans and evolutional origin of this Neisseria strain should be further investigated.

It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens. IMPORTANCE An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.

The complete genome sequence of a novel duck orthoreovirus, designated DRV strain TH11(DRV-TH11), was determined and characterized. The DRV-TH11 genome is comprised of 23,417 bp and its genome organization is more similar to that of avian orthoreoviruses (ARVs) of chicken origin than other reoviruses. The results of comparative sequence analysis and dendrograms based on the µB- and σC-encoding genes indicated that TH11 may be derived from the reassortment of ARVs and classic Muscovy duck reovirus (MDRV). A possible recombinant event was identified using the SimPlot program, and it occurred in the M2 segment. The results indicated that reassortment and mutation play a role in the evolution of duck reovirus.

To the Editor: In 2011, an unidentified disease in Pekin ducks (Anas platyrhynchos) was reported in People’s Republic of China. The infection caused death in 40% of ducks of various age and 35%–40% mortality in different flocks. Clinical signs included unstable gait, weakness in legs, and diarrhea. At necropsy, large necrotic foci were observed in the spleens. All classical endemic and emerging viruses, such as duck enteritis virus, duck hepatitis virus, duck flavivirus, duck parvovirus, and avian influenza virus, could be excluded as the causative agent by PCR and serologic methods. To identify the cause of the disease, we tested tissue from affected ducks and subsequently isolated a novel duck-pathogenic orthoreovirus from the livers of affected ducks. Avian orthoreoviruses (ARVs) belong to the family Reoviridae, genus Orthoreovirus (1). The virions are nonenveloped, with icosahedral symmetry and a double capsid containing 10 double-stranded RNA segments that can be separated by polyacrylamide gel electrophoresis into 3 size classes: large (L1–L3), medium (M1–M3), and small (S1–S4) (2,3). ARVs cause a range of diseases in chicken, including viral arthritis/tenosynovitis, and are associated with respiratory disease, enteric disease, inclusion body hepatitis, hydropericardium, runting stunting syndrome, malabsorption syndrome, and sudden death. ARVs also have been isolated from the Muscovy duck (Cairina moschata). Muscovy duck reovirus infection caused illness in 30% and death in 20% of ducks on poultry farms in Israel (4). In China, reovirus infection has been reported in Muscovy ducklings, with a resulting death rate of 10%–30% since 1997 (5). The isolated reovirus was highly pathogenic to 1-day-old Muscovy ducklings by experimental infection. However, the Muscovy duck reovirus isolate was nonpathogenic for Pekin ducks when inoculated subcutaneously (4). Since 2007, three isolates of orthoreovirus were confirmed in Pekin ducks from several duck farms in China. However, experiment infection with the isolates did not cause death (6). In 2011, farmers and veterinarians in China reported to the Animal Health Services and National Research Institutes an unidentified disease in ducks that spread rapidly around the county. We conducted further investigation to identify the causative agent of this disease. The diseased ducks showed depression and leg weakness. Large necrotic foci were observed in the spleens of the dead ducks. Histopathologic examination showed necrotic foci and granulomas in the spleen. Focal hepatic necrosis and proliferation of bile ducts were seen in the liver. Virus isolation from liver homogenate was conducted in duck embryo fibroblast cultures. At 48 hours after infection, a strong cytopathic effect was observed, including syncytium formation. All duck embryos experimentally infected with the isolate died within 48–72 hours after infection. The dead embryos showed swollen livers with petechial hemorrhages. Spherical, spiked virus particles, consistent with those of members of the family Reoviridae, were observed by electron microscopy. As reported (7), the diameter of the particles was ≈85 nm (Figure A1). The RNA extracted from DRV-infected duck embryo fibroblast cultures showed 10 dsRNA segments in 3 size classes (L1–3, M1–3, and S1–4) on polyacrylamide gel electrophoresis. The isolate was designated as novel duck reovirus, DRV-TH11. The pathogenicity of DRV-TH11 was tested by infecting 10-day-old Pekin ducks subcutaneously at a dose of 4 × 104.5 50% tissue culture infective dose. Experimental infection caused death on day 3 after infection. The clinical signs and histopathologic examination show the same features as the naturally infected ducks. For phylogenetic analyses, the S2 gene was amplified by reverse transcription PCR with avian reovirus–specific primers. The complete sequence of the S2 gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JQ664689","term_id":"387862751","term_text":"JQ664689"}}JQ664689) was aligned with 30 published orthoreovirus sequences, including data on all 3 newly obtained sequences from Pekin duck reovirus in China in 2008 and 2011. Phylogenetic relationship was assessed by using the neighbor-joining method based on a Tamura 3-parameter model and bootstrap analysis (1,000 replicates) as implemented in MEGA5 (8). The phylogenetic tree shows that the complete sequence of S2 gene is distinct but clusters closely with sequences from all 3 Pekin duck isolates within the ARVs serogroup, which suggests that the novel virus is an ARV-like virus within the genus Orthoreovirus (Figure). Figure Phylogenetic relationship between DRV-TH11 isolate and orthoreovirus of the avian orthoreovirus (ARV) and mammalian orthoreovirus (MRV). ARV includes chicken reovirus, Muscovy duck reovirus, and Pekin duck reovirus. GenBank accession numbers of the sequences ... In summary, we isolated a novel duck-pathogenic orthoreovirus from the liver of affected Pekin ducks. The regression test in its natural host animal showed that the newly isolated virus caused their deaths. This finding highlights the need to prevent and control this highly transmissible infectious agent. Further study is needed to determine what role the newly isolated DRV played in the 2011 outbreaks on many of the duck farms in China.

Until recently, there was no cell line that could produce continuously high-titer duck hepatitis virus type 1 (DHV-1). In this study, a duck embryo fibroblast (DEF) cell line was established, and the susceptibility of this cell line to DHV-1 was determined. The primary culture of DEF cells was from a duck embryo that was partially digested with trypsin. Digested tissue pieces were cultured at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. The cultured DEF cells, which had the morphology of fibroblast, proliferated to 100% confluence four days later. An immortalized DEF cell line, named DEF-TA, was established and subcultured to passage 33, and the susceptibility of that cell line to DHV-1 was determined. In the DHV-1 susceptibility tests, cytopathic effects and the propagation of virus were observed in DEF-TA cells after DHV-1 infection. This continuous DHV-1-susceptible DEF cell line may serve as a valuable cell line for studies of cell-virus interactions and the pathogenesis of DHV-1 and may be useful for the development of an inactivated vaccine.

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against duck hepatitis virus type 1 (DHV-1). The VP1 gene of DHV-1 was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/VP1, was transfected into BHK-21 cells and the antigenicity of the expressed protein was confirmed using an indirect immunofluorescence and western blot assay. Immunogenicity was studied in ducklings. Ducklings were injected intramuscularly two times with pSCA/VP1 at 14 days intervals. Anti-DHV-1 antibodies were detected by ELISA, the lymphocyte proliferation response was also tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method and neutralizing antibodies were measured by microneutralization tests. Our results showed that DHV-1-specific antibodies, neutralizing antibodies and lymphocyte proliferation were well induced in ducklings. Furthermore, all the ducklings were protected against challenge with wild DHV-1. In conclusion, we demonstrate that the suicidal DNA vaccine is a promising vaccine candidate facilitating the prevention of duck hepatitis caused by DHV-1.