Complete genomic sequence of goose-origin reovirus from China.

We report the full-genome sequence of a goose-origin reovirus (GRV) strain 03G from Zhejiang Province, China. This is the first report of the complete genomic sequence (segments 1 to 10) of GRV. Phylogenetic analyses of the sequence suggest that GRV 03G represents a new species distinct from other established species within the avian reovirus (ARV) group of orthoreoviruses.

Complete genome sequence of a novel flavivirus, duck tembusu virus, isolated from ducks and geese in china.

Duck tembusu virus (DTMUV) is an emerging agent that causes a severe disease in ducks. We report herein the first complete genome sequences of duck tembusu virus strains YY5, ZJ-407, and GH-2, isolated from Shaoxing ducks, breeder ducks, and geese, respectively, in China. The genomes of YY5, ZJ-407, and GH-2 are all 10,990 nucleotides (nt) in length and encode a putative polyprotein of 3,426 amino acids. It is flanked by a 5' and a 3' noncoding region (NCR) of 94 and 618 nt, respectively. Knowledge of the whole sequence of DTMUV will be useful for further studies of the mechanisms of virus replication and pathogenesis.

Construction of a full-length infectious bacterial artificial chromosome clone of duck enteritis virus vaccine strain.

BACKGROUND: Duck enteritis virus (DEV) is the causative agent of duck viral enteritis, which causes an acute, contagious and lethal disease of many species of waterfowl within the order Anseriformes. In recent years, two laboratories have reported on the successful construction of DEV infectious clones in viral vectors to express exogenous genes. The clones obtained were either created with deletion of viral genes and based on highly virulent strains or were constructed using a traditional overlapping fosmid DNA system. Here, we report the construction of a full-length infectious clone of DEV vaccine strain that was cloned into a bacterial artificial chromosome (BAC). METHODS: A mini-F vector as a BAC that allows the maintenance of large circular DNA in E. coli was introduced into the intergenic region between UL15B and UL18 of a DEV vaccine strain by homologous recombination in chicken embryoblasts (CEFs). Then, the full-length DEV clone pDEV-vac was obtained by electroporating circular viral replication intermediates containing the mini-F sequence into E. coli DH10B and identified by enzyme digestion and sequencing. The infectivity of the pDEV-vac was validated by DEV reconstitution from CEFs transfected with pDEV-vac. The reconstructed virus without mini-F vector sequence was also rescued by co-transfecting the Cre recombinase expression plasmid pCAGGS-NLS/Cre and pDEV-vac into CEF cultures. Finally, the in vitro growth properties and immunoprotection capacity in ducks of the reconstructed viruses were also determined and compared with the parental virus. RESULTS: The full genome of the DEV vaccine strain was successfully cloned into the BAC, and this BAC clone was infectious. The in vitro growth properties of these reconstructions were very similar to parental DEV, and ducks immunized with these viruses acquired protection against virulent DEV challenge. CONCLUSIONS: DEV vaccine virus was cloned as an infectious bacterial artificial chromosome maintaining full-length genome without any deletions or destruction of the viral coding sequence, and the viruses rescued from the DEV-BAC clone exhibited wild-type phenotypes both in vitro and in vivo. The generated infectious clone will greatly facilitate studies on the individual genes of DEV and applications in gene deletion or live vector vaccines.

Study on the Protective Immunity Induced by Pseudotyped Baculovirus Expressing the E Protein of Tembusu Virus in Ducklings.

The Duck Tembusu virus (DTMUV), a pathogenic flavivirus, has been causing significant economic losses in the Chinese poultry industry since 2010. This virus can severely decrease egg production and inhibit the growth of laying ducks and ducklings. While many vaccines have been developed to prevent DTMUV infection, fresh outbreaks continue to occur, as few effective vaccines are available. The E glycoprotein of DTMUV is the primary target for inducing protective immunity in the natural host. Therefore, we conducted an investigation and successfully developed a recombinant baculovirus containing the DTMUV E gene. Ducklings were then vaccinated with the purified protein derived from this virus as a potential vaccine candidate. Our findings demonstrated that the E glycoprotein of DTMUV was highly expressed in Sf9 cells. The vaccination of ducklings with the recombinant baculovirus Bac-E resulted in the induction of strong humoral and cellular immune responses. Most significantly, we observed that the vaccine provided 100% protective immunity against lethal challenges with the DTMUV YY5 strain.

Inactivation Performance of Pseudorabies Virus as African Swine Fever Virus Surrogate by Four Commercialized Disinfectants.

This study was based on similar physicochemical characteristics of pseudorabies virus (PRV) and African swine fever virus (ASFV). A cellular model for evaluation of disinfectants was established with PRV as an alternative marker strain. In the present study, we evaluated the disinfection performance of commonly used commercialized disinfectants on PRV to provide a reference for the selection of good ASFV disinfectants. In addition, the disinfection (anti-virus) performances for four disinfectants were investigated based on the minimum effective concentration, onset time, action time, and operating temperature. Our results demonstrated that glutaraldehyde decamethylammonium bromide solution, peracetic acid solution, sodium dichloroisocyanurate, and povidone-iodine solution effectively inactivated PRV at concentrations 0.1, 0.5, 0.5, and 2.5 g/L on different time points 30, 5, 10, and 10 min, respectively. Specifically, peracetic acid exhibits optimized overall performance. Glutaraldehyde decamethylammonium bromide is cost effective but requires a long action time and the disinfectant activity is severely affected by low temperatures. Furthermore, povidone-iodine rapidly inactivates the virus and is not affected by environmental temperature, but its application is limited by a poor dilution ratio such as for local disinfection of the skin. This study provides a reference for the selection of disinfectants for ASFV.

Recombinant pseudorabies virus (PRV) expressing stabilized E2 of classical swine fever virus (CSFV) protects against both PRV and CSFV.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

Intergrated Transcriptomic and Proteomic Analysis Revealed the Differential Responses to Novel Duck Reovirus Infection in the Bursa of Fabricius of Cairna moschata.

The bursa of Fabricius is an immunologically organ against the invasion of duck reovirus (DRV), which is a fatal bird virus belonging to the Reoviridae family. However, responses of the bursa of Fabricius of Cairna moschata to novel DRV (NDRV) infection are largely unknown. Transcriptomes and proteomes of the samples from control and two NDRV strain (HN10 and JDm10) with different virulence were analyzed. Differentially expressed genes and differential accumulated proteins were enriched in the serine protease system and innate immune response clusters. Most of the immune-related genes were up-regulated under both JDm10/HN10 infections. However, the immune-related proteins were only accumulated under HN10 infection. For the serine protease system, coagulation factor IX, three chains of fibrinogen, and complements C8, C5, and C2s were significantly up-regulated by the HN10 infection, suggesting that the serine protease-mediated immune system might be involved in the resistance to NDRV infection. For the innate and adaptive immune system, RIG-I, MDA5, MAPK20, and IRF3 were significantly up-regulated, indicating their important roles against invaded virus. TLR-3 and IKBKB were only up-regulated in the liver cells, MAPK20 was only up-regulated in the bursa of Fabricius cells, and IRAK2 was only up-regulated in the spleen samples. Coagulation factor IX was increased in the bursa of Fabricius, not in the liver and spleen samples. The data provides a detailed resource for studying the proteins participating in the resistances of the bursa of Fabricius of duck to NDRV infections.

Development and Evaluation of a Monoclonal Antibody-Based Blocking Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies against Novel Duck Reovirus in Waterfowl Species.

The novel duck reovirus (NDRV) is an emerging pathogen that causes disease in various waterfowl species. Since the outbreak, it has caused huge economic losses to the duck industry in China. A rapid, reliable, and high-throughput method is required for epidemiological investigation and evaluation of vaccine immunogenicity. A good first step would be establishing an enzyme-linked immunosorbent assay (ELISA) that could detect NDRV antibodies in different breeds of ducks and geese from the serum and egg yolk. This study used a recombinant NDRV σB protein and a corresponding horseradish peroxidase (HRP)-labeled monoclonal antibody to develop a blocking ELISA (B-ELISA). The cutoff value of the B-ELISA was 37.01%. A total of 212 serum samples were tested by the B-ELISA, and the virus neutralization test (VNT) was the gold standard test. The sensitivity and specificity of the B-ELISA were 92.17% (106/115) and 97.94% (95/97), respectively. The agreement rates between the B-ELISA and VNT were 94.81% (kappa value, 0.896). The B-ELISA could specifically recognize anti-NDRV sera without cross-reacting with other positive serums for other major diseases in ducks and geese. The inter- and intra-assay coefficients of variation (CVs) of the B-ELISA and VNT assays were acceptable. In conclusion, the novel B-ELISA could be a rapid, simple, safe, and economically attractive alternative to the VNT in assessing duck flocks' immunity status and in epidemiological surveillance in multiple waterfowl species. IMPORTANCE NDRV disease is a new epidemic disease in waterfowl that first appeared in China. Compared with the classical DRV (CDRV), NDRV is associated with more severe symptoms, a higher mortality rate, and a broader host range. NDRV has become the prevalent genotype in China. At present, there are no commercially available diagnostic products for the NDRV disease. VNT, as the gold standard serologic test, is not only time-consuming and laborious, but also has high requirements for facilities and equipment, which is not suitable for clinical application. Conventional ELISA requires specific antispecies conjugates that are not currently available. B-ELISA not only has the advantage of higher analysis specificity, but also can be used to test specific antibodies against different waterfowl species, because no species-specific conjugates are required in such detection. Therefore, it is necessary to establish a B-ELISA for the detection of antibodies against NDRV in waterfowl species.

Distinct Whole Transcriptomic Profiles of the Bursa of Fabricius in Muscovy Ducklings Infected by Novel Duck Reovirus with Different Virulence.

Novel duck reovirus (NDRV) is a newly identified reovirus that brings about more severe damage on multiple organs and mortality in various species of waterfowl. We previously characterized the transcriptomic profiles responding to NDRV in the bursa of Fabricius of Muscovy ducklings, which is a major immunological organ against virus infection. However, the molecular mechanisms of variant cell responses in the bursa of Fabricius to NDRV with different virulence is unclear. Here, we conducted a whole transcriptomic analysis to study the effects of two strains, HN10 (virulent NDRV) and JDm10 (artificially attenuated NDRV), on the bursa of Fabricius of Muscovy ducklings. We harvested a large number of differentially expressed genes (DEGs) of the bursa of Fabricius specially induced by HN10 and JDm10, and we found that HN10 induced DEGs enriched in differentiation and development in multiple organs beyond JDm10. Moreover, the ceRNA regulatory network also indicated the different connections among mRNA, lncRNA and miRNA. Interestingly, we further noticed that a population of differential expressed miRNA could particularly target to transcripts of HN10 and JDm10. We took miR-24 as an example and observed that miR-24 could reduce the transcription of GLI family zinc finger 3 (Gli3) and membrane-associated guanylate kinase, WW and PDZ domain containing 1 (Magi1) via recognition 3' UTR of these two genes by a dual luciferase reporter gene assay in vitro. However, this effect could be compromised by HN10 infection or the ectopic over-expression of the putative miR-24 targeting regions in L1 and L3 fragments of HN10. Taken together, we examined and proposed a novel regulatory competitive mechanism between transcripts of NDRV and Muscovy ducklings for miRNA. These findings may advance the understanding of the molecular pathogenesis of NDRV in Muscovy ducklings, and help provide the potential targets for vaccine and drug development against NDRV.

Genomic and epidemiological characteristics provide insights into the phylogeographic spread of goose astrovirus in China.

Goose astrovirus (GAstV) is an emerging pathogen with a wide distribution in China that causes visceral gout and leads to significant economic losses in the goose industry. Here, 10 GAstV strains were isolated from different farms in southeast China. We performed an integrated analysis of the full-genome sequences of these new strains alongside comprehensive epidemiological surveillance information from the database. Interestingly, the results showed two distinct genotypes of GAstV, which were evolutionarily distant from each other. Group I GAstVs were closely related to DAstV IV, and group II strains were classified with duck astrovirus (DAstV) II and turkey astrovirus (TAstV) II. Further investigation showed that among the GAstV I strains, ZJC14 and AHDY differed from FLX. Comparative analysis of 58 available genomes clustered the GAstV II strains into two subgroups. We identified two major mutation sites, 456 (E/D) and 540 (L/Q), in the capsid protein, which were related to distinct subgroups according to evolution. GAstV II subgroup 1a strains are the predominant strains in the current prevalent epidemiology. Phylogeographic analysis based on 90 reported cases from 13 provinces revealed the complexity and severity of GAstV epidemics in China, within which Henan, Anhui and Jiangsu provinces have suffered great impacts. According to these phylogeographic investigations, following the initial introduction of GAstV from Hunan Province, the dispersal of GAstV with different subgenotypes on a nationwide scale may be explained by the live gosling trade. Our findings have important implications for the evolution and dispersal of GAstV and will contribute to understanding the potential risk of GAstV.

Isolation and Pathogenicity of a Novel Goose Astrovirus from Overfed Adult Landaise Geese in China.

Goose astrovirus (GAstV) is an important pathogen causing visceral gout and high mortality in goslings, which has broken out and spread across China. In 2021, a disease characterized by urate deposition on the visceral surface and 30% mortality occurred in commercial adult Landaise geese in Zhejiang Province, China. A systematic study identified an infecting astrovirus, designated ZJCX, that was efficiently isolated from a diseased goose with a chicken hepatocellular carcinoma cell line (LMH). In contrast to other GAstVs originating from goslings, ZJCX caused cytopathogenic effects in LMH cells, and the crystalline arrangement of viral particles was observed through transmission electron microscopy. Indeed, phylogenetic analysis and nucleotide homology comparison revealed that ZJCX isolate belongs to the genotype II cluster of GAstVs and displays 97.8-98.4% identity with other GAstV II strains. However, several specific mutations occurred in the polyprotein and capsid protein regions. Moreover, a pathogenicity assessment of ZJCX with a gosling model was conducted, and typical visceral gout was reproduced and led to 18% mortality. The viral loads of ZJCX in the blood, kidney, and liver were detected with specific primers after inoculation, which demonstrated that the kidney and liver presented viral loads peaking at seven days post-inoculation (dpi). Biochemical parameter examination showed that AST, ALT, γ-GT, UA, and BUN levels were significantly increased by GAstV, whereas body weight was reduced. Overall, this study indicated that the GAstV isolate could infect adult geese, and the results regarding the viral loads and biochemical parameters induced by ZJCX provide insight into GAstV pathogenicity.

Proteomic analysis of host cellular proteins co-immunoprecipitated with duck enteritis virus gC.

Duck enteritis virus (DEV), the causative agent of duck viral enteritis, causes a contagious, lethal viral disease in Anseriformes (waterfowls). In virus infection, host-virus interaction plays a crucial role in virus replication and pathogenesis. In our previous study, mRFP was fused with the C-terminus of DEV glycoprotein C (gC) to construct a fluorescent-tag DEV virus rgCRFP. In the current study, fluorescent fusion protein (gC-mRFP) was used as the proteomic probe. Co-immunoprecipitation and mass spectrometric analysis of proteins from rgCRFP-infected chicken embryo fibroblasts using commercial anti-RFP antibody led to the identification of a total of 21 gC interacting host proteins. Out of these 21 proteins, the interaction of seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) with DEV gC protein was validated using membrane-bound split-ubiquitin yeast two-hybrid system (MbYTH) and bimolecular fluorescence complementation (BiFC) analyses. It indicated direct interaction between these proteins with DEV gC protein. This study has furthered the current understanding of DEV virus infection and pathogenesis. SIGNIFICANCE: gC is an crucial glycoprotein of duck enteritis virus that plays an important role in the viral life cycle. Uncovering the interaction between virus-host is very important to elucidate the pathogenic mechanism of the virus. In this study, host factors interacting with DEV gC have been discerned. And seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) have been further validated to interact with DEV gC using MbYTH and BiFC analyses. These outcomes could shed light on how DEV manipulates the cellular machinery, which could further our understanding of DEV pathogenesis.

Simultaneous tracking of capsid VP26, envelope protein gC localization in living cells infected with double fluorescent duck enteritis virus.

Duck enteritis virus (DEV) can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of DEV vaccine strain pE1 (pDEV-EF1) has been constructed in our previous study. Based on pE1, a recombinant mutated clone pDL (pVP26CFP-gCRFP), which carries a red fluorescent protein (mRFP) gene fused to the viral envelope protein gC in combination with a cyan fluorescent protein (CFP) gene fused to the viral capsid VP26, was constructed by two-step Red/ET recombination and the recombinant virus rDL (rVP26CFP-gCRFP) was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate transfection. Western blot analysis revealed that VP26-CFP and gC-mRFP were both expressed in fusion forms in rDL-infected CEFs, and subcellular localization study showed that gC-mRFP was mainly localized in whole cell at 36, 48 h post infection (p.i.); and then mostly migrated to the cytoplasm after 60 h.p.i., ; whereas VP26-CFP was localized in the nucleus in all stages of virus infection. Additionally, viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells by transmission electron microscopy, indicating that exogenous gene insertion has no effect on virus assembly. This study has laid a foundation for visually studying localization, transportation of DEV capsid proteins and envelope glycoproteins as well as virus assembly, virion movement and virus-cell interaction.

The phosphoproteomic responses of duck (Cairna moschata) to classical/novel duck reovirus infections in the spleen tissue.

Duck reovirus (DRV) is a fatal member of the genus Orthoreovirus in the family Reoviridae. The disease caused by DRV leads to huge economic losses to the duck industry. Post-translational modification is an efficient strategy to enhance the immune responses to virus infection. However, the roles of protein phosphorylation in the responses of ducklings to Classic/Novel DRV (C/NDRV) infections are largely unknown. Using a high-resolution LC-MS/MS integrated to highly sensitive immune-affinity antibody method, phosphoproteomes of Cairna moschata spleen tissues under the C/NDRV infections were analyzed, producing a total of 8,504 phosphorylation sites on 2,853 proteins. After normalization with proteomic data, 392 sites on 288 proteins and 484 sites on 342 proteins were significantly changed under the C/NDRV infections, respectively. To characterize the differentially phosphorylated proteins (DPPs), a systematic bioinformatics analyses including Gene Ontology annotation, domain annotation, subcellular localization, and Kyoto Encyclopedia of Genes and Genomes pathway annotation were performed. Two important serine protease system-related proteins, coagulation factor X and fibrinogen α-chain, were identified as phosphorylated proteins, suggesting an involvement of blood coagulation under the C/NDRV infections. Furthermore, 16 proteins involving the intracellular signaling pathways of pattern-recognition receptors were identified as phosphorylated proteins. Changes in the phosphorylation levels of MyD88, NF-κB, RIP1, MDA5 and IRF7 suggested a crucial role of protein phosphorylation in host immune responses of C. moschata. Our study provides new insights into the responses of ducklings to the C/NDRV infections at PTM level.

Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60.

Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus-insect cell system, mammalian-luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.

Comparative proteomic analysis revealed complex responses to classical/novel duck reovirus infections in the spleen tissue of Cairna moschata.

Duck reovirus (DRV), a member of the genus Orthoreovirus in the family Reoviridae, was first isolated from Muscovy ducks. The disease associated with DRV causes great economic losses to the duck industry. However, the responses of duck (Cairna moschata) to the classical/novel DRV (C/NDRV) infections are largely unknown. To reveal the relationship of pathogenesis and immune response, the proteomes of duck spleen cells under the control and C/NDRV infections were compared. In total, 5986 proteins were identified, of which 5389 proteins were quantified. The different accumulated proteins (DAPs) under the C/NDRV infections showed displayed various biological functions and diverse subcellular localizations. The proteins related to the serine protease system were siginificantly changed, suggesting that the activated serine protease system may play an important role under the C/NDRV infections. Furthermore, the differences in the responses to the C/NRDV infections between the duck liver and spleen tissues were compared. Only a small number of common DAPs were identified in both liver and spleen tissues, suggesting diversified pattern involved in the responses to the C/NRDV infections. However, the changes in the proteins involved in the serine protease systems were similar in both liver and spleen cells. Our data may give a comprehensive resource for investigating the responses to C/NDRV infections in ducks. SIGNIFICANCE: A newly developed MS/MS-based method involving isotopomer labels and 'tandem mass' has been applied to protein accurate quantification in current years. However, no studies on the responses of duck (Cairna moschata) spleen tissue to the classical/novel DRV (C/NDRV) infections have been performed. As a continued study of our previous report on the responses of duck liver tissue to the C/NDRV infections, the current study further compared the differences in the responses to the C/NRDV infections between the duck liver and spleen tissues. Our results will provide an opportunity to reveal the relationship of pathogenesis and immune response and basic information on the pathogenicity of C/NDRV in ducks.

Comparative proteomic analysis revealed complex responses to classical/novel duck reovirus infections in Cairna moschata.

Duck reovirus (DRV) is an typical aquatic bird pathogen belonging to the Orthoreovirus genus of the Reoviridae family. Reovirus causes huge economic losses to the duck industry. Although DRV has been identified and isolated long ago, the responses of Cairna moschata to classical/novel duck reovirus (CDRV/NDRV) infections are largely unknown. To investigate the relationship of pathogenesis and immune response, proteomes of C. moschata liver cells under the C/NDRV infections were analyzed, respectively. In total, 5571 proteins were identified, among which 5015 proteins were quantified. The differential expressed proteins (DEPs) between the control and infected liver cells displayed diverse biological functions and subcellular localizations. Among the DEPs, most of the metabolism-related proteins were down-regulated, suggesting a decrease in the basal metabolisms under C/NDRV infections. Several important factors in the complement, coagulation and fibrinolytic systems were significantly up-regulated by the C/NDRV infections, indicating that the serine protease-mediated innate immune system might play roles in the responses to the C/NDRV infections. Moreover, a number of molecular chaperones were identified, and no significantly changes in their abundances were observed in the liver cells. Our data may give a comprehensive resource for investigating the regulation mechanism involved in the responses of C. moschata to the C/NDRV infections.

Development and application of an indirect ELISA for the detection of antibodies to novel duck reovirus.

A novel duck reovirus (N-DRV) disease emerged in China in 2000 and it has become an epidemic genotype. A test for detection of virus-specific antibodies in serum samples would be useful for epidemiological investigations. Currently, Currently, serological assays for N-DRV diagnosis are not available. A test for detection of virus-specific antibodies in serum samples would be useful for epidemiological investigations. In this study, a highly sensitive and specific indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to N-DRV was developed. The outer capsid (σC) of N-DRV was cloned and expressed in Escherichia coli as a coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. Furthermore, the specificity of σC-ELISA assay was confirmed by cross checking with other duck viral pathogens. In comparison with the western blot, the sensitivity and specificity of the σC-ELISA was 92.6% and 88.9%, respectively, and agreement of two tests was excellent with κ value of 0.786 (p < 0.05). A serological survey was performed using the assay on serum samples from different age and species of duck flocks in the Zhejiang and Jiangsu Province, China. The seropositive rate of the 1209 serum samples was 57.7%. In conclusion, the developed σC-ELISA assay is a very specific and sensitive test that will be useful for large-scale serological survey in N-DRV infection and monitoring antibodies titers against N-DRV.

Genomic characteristics of a novel reovirus from Muscovy duckling in China.

A new reovirus was isolated from a sick Muscovy duckling with hemorrhagic-necrotic lesions in the liver in Zhejiang, China in 2000 and was tentatively denoted a new type of Muscovy duck reovirus (N-MDRV ZJ00M). This reovirus was propagated in a chicken fibroblast cell line (DF-1) with obvious cytopathic effects. The reovirus's genome was 23,419 bp in length with an approximately 50% G+C content and 10 dsRNA segments encoding 12 proteins. The length of the genomic segments was similar to those of avian reoviruses (ARVs), which range from 3959 nt (L1) to 1191 nt (S4) in size. All of the segments have the conserved terminal sequences 5'-GCUUUUU…UUCAUC-3', and all of the genome segments, with the exception of S1, apparently encoded one single primary translation product. The genome analysis revealed that the S1 segment of N-MDRV is a tricistronic gene that encodes the overlapping ORFs for p10, p18, and σC. This finding is similar to that found for ARVs but distinct from that found for classical MDRV and GRV, which have a bicistronic S4 segment that encodes p10 and σC and do not encode p18. The amino acid (aa) alignments of the putative proteins encoded by the main ORF in each segment revealed a high similarity (14.1-100%) to the counterpart proteins encoded by other ARV species from the avian orthoreoviruses (e.g., ARV, classical MDRV and N-MDRV) in the Orthoreovirus genus, particularly with N-MDRV (94.6-100%). The phylogenetic analysis of the nucleotide sequences of all 10 genome segments revealed that N-MDRV ZJ00M is distinct from all other described reovirus species groups but is a separated from the ARV (including MDRV and GRV) species within orthoreovirus species group II and grouped into the classical MDRV and GRV genogroup with the N-MDRV isolates. The MDRV genogroup can be further divided into two genotype clusters. The morphological and pathological analyses and the genetic characterization of N-MDRV ZJ00M suggest that it belongs to genotype 2 (N-MDRV). In addition, the RT-PCR assays of DRV diseased duckling and gosling samples collected from different regions of China during 2000-2013 indicate that N-MDRV is currently the prevalent genotype in China.