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Duck spleen marble-like necrosis disease (DSMND) has been prevalent in Chinese duck farms since 2016. The etiological study was carried out in this study using etiological detection, pathogen isolation, whole genome sequencing, and artificial infection test. A highly pathogenic Riemerella anatipestifer (RA) strain was determined as the etiologic agent. Phylogenomic analysis showed that this RA strain was closely related to duck origin RA strain RCAD0421 and chicken origin RA strain S63. The clinical symptoms and pathological changes of artificial infection ducks were similar to those of clinical cases. This is the first identification of RA as the pathogen of DSMND, which provides a theoretical basis for this disease' s prevention and control. • An unidentified disease designated duck spleen marble-like necrosis disease (DSMND). • The viral pathogens were excluded as the causative agent for DSMND. • The causative agent of DSMND was identified as a kind of high pathogenicity RA strain. [ABSTRACT FROM AUTHOR]

Abstract To obtain a deletion mutant of Muscovy duck-origin goose parvovirus (MDGPV) and to analyze its biological characteristics, the pMDGPVPT plasmid, which contains a full-length DNA infectious clone of the MDGPV PT strain, was used in this study as the template. The E-box at nt 315 of the left inverted terminal repeat sequence (L-ITR) was deleted by overlap extension PCR to obtain the infectious recombinant plasmid p-PTΔE315. The p-PTΔE315 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac and the rescued deletion mutant virus r-PTΔE315 was generated. Experiments to demonstrate the novel deletion mutant virus' biological characteristics showed that r-PTΔE315 can cause typical lesions after infection of Muscovy duck embryos. Compared with its parent strain PT, the virulence of r-PTΔE315 and its proliferation ability in Muscovy duck embryos were attenuated, but its ability to replicate in MDEF cells was enhanced. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence. Highlights • In this study, we constructed a full-length infectious clone (pMDGPVPT). • Deletion of E-box (E315) may improve the adaptability of MDGPV to MDEF cells. • Deletion of E-box (E315) decreased MDGPV replication in Muscovy duck embryos. [ABSTRACT FROM AUTHOR]

The complete sequence of a reovirus, strain NP03 associated with necrotic focus formation in the liver and spleen of Muscovy ducklings in Fujian Province, China in 2009, was determined and compared with sequences of other waterfowl and chicken-origin avian reoviruses (ARVs). Sequencing of the complete genomes of strain NP03 showed that they consisted of 23,418 bp and were divided into 10 segments, ranging from 1191 bp (S4) to 3959 bp (L1) in length, and all segments contained conserved sequences in the 5′ non-coding region (GCUUUU) and 3' non-coding region (UCAUC). Pairwise sequence comparisons demonstrated that NP03 strain showed the highest similarity with novel waterfowl origin reoviruses (WRVs). The genome analysis revealed that the S1 segment of novel WRV is a tricistronic gene, encoding the overlapping open reading frames (ORFs) for p10, p18, and σC, similar to the ARV S1 gene, but distinct from classical WRV S4 genome segment, which contained two overlapping ORFs encoding p10 and σC. Phylogenetic analyses of the nucleotide sequences of all 10 segments revealed that NP03 strain was clustered together with other novel WRVs and were distinct from classical WRVs and chicken-origin ARVs. The analyses also showed possible intra-segmental reassortment events in the segments encoding λA, λB, μB, μNS, σA, and σNS between novel and classical WRVs. Potential recombination events detection in segment L1 suggests that NP03 strain may be recombinants of novel WRVs. Based on our genetic analyses, multiple reassortment events, intra-segmental recombination, and accumulation of point mutations have possibly contributed to the emergence of this novel genotype of WRV, identified in China. • There were two genotypes of waterfowl reovirus (WRV), which mainly causes the outbreaks in China. • The novel WRV strain NP03 likely derived from multiple reassortment events. • Possible intra-segmental recombination event was identified. [ABSTRACT FROM AUTHOR]

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on Taq Man-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 101 copies/μL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay. The use of β-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established Taq Man-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of Taq Man fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity Taq Man-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection. • A Taq Man-MGB real-time RT-PCR was developed for detection MDRV. • The assay includes an internal control of the β-actin gene. • The Taq Man-MGB real-time RT-PCR offered rapid, effective and reliable for the detection of MDRV in Muscovy ducks. [ABSTRACT FROM AUTHOR]

An unclassified calicivirus (CV) detected in geese was recently reported and proposed as a new member of the family Caliciviridae. There is limited information about the epidemiology, etiology and detection method of goose-origin CV (GCV) to date. In this study, an EvaGreen based fluorescence quantitative real-time RT-PCR assay was developed and optimized for the detection of GCVs. The assay sensitively detected GCV RNA template with a good linear standard curve. We also demonstrated the specificity and reproducibility of the detection method for GCVs. Thus, the method developed in this study will benefit the investigation of possible sporadic outbreaks of CV infections in geese, as well as epidemiological and etiological studies of GCVs. • An EvaGreen based real-time RT-PCR assay was developed for detection and quantitation of newly emerging goose caliciviruses. • The assay provides quantitative data that may be used to inform epidemiological and etiological studies. [ABSTRACT FROM AUTHOR]

Duckling short beak and dwarfism syndrome virus (SBDSV), a newly identified goose parvovirus, causes devastating disease in domestic waterfowl and considerable economic losses to Chinese waterfowl industry. The molecular pathogenesis of SBDSV infection, nature and dynamics of host immune responses against SBDSV infection remained elusive. In this study, we systematically explored the relative mRNA expression profiles of major innate immune-related genes in SBDSV infected duck embryo fibroblasts. We found that SBDSV infection effectively activated host innate immune responses and resulted in significant up-regulation of IFN-β and several vital IFN-stimulated genes (ISGs). These up-regulation responses were mainly attributed to viral genomic DNA and dsRNA replication intermediates. Importantly, the expression of cGAS was significantly induced, whereas the expression of other DNA receptors including DDX41, STING, ZBP1, LSM14A and LRRFIP1 have no significant change. Furthermore, SBDSV infection also activates the up-regulation of TLR3 and inhibited the expression of TLR2 and TLR4; however, no effect was observed on the expression of TLR1, TLR5, TLR7, TLR15 and TLR21. Intriguingly, SBDSV infection significantly up-regulated the expression of RNA sensors such as MDA5 and LGP2, and resulted in a delayed but significant up-regulation of RIG-I gene. Taken together, these data indicate that host multiple sensors including DNA sensor (cGAS) and RNA sensors (TLR3, MDA5 and LGP2) are involved in recognizing a variety of different pathogen associated molecular patterns (PAMPs) including viral genomic ssDNA and dsRNA replication intermediates, which trigger an effective antiviral innate immune response. • SBDSV, a new number of goose parvovirus, causes devastating disease in domestic waterfowl. • The relative mRNA expression profiles of innate immune-related genes in SBDSV infected DEFs were systematically explored. • SBDSV infection activates host innate immune responses and results in significant up-regulation of IFN-β and vital ISGs. • Host multiple sensors are involved in recognizing pathogen associated molecular patterns (PAMPs). [ABSTRACT FROM AUTHOR]

Novel duck reovirus (NDRV) is pathogenic to young ducks, which is characterized by hemorrhagic spots and necrotic foci of the livers and necrotic foci of spleens. However, the effect of NDRV infection on the composition of the host's intestinal microbiota remains poorly understood. In this study, three-day-old Muscovy ducklings were inoculated with either the virulent NDRV strain NP03 or sterile Hank's solution. Through Illumina MiSeq sequencing, the whole cecal microbiota of healthy and NDRV infected ducklings was examined. The results showed that the gut microbiota was mainly dominated by Firmicutes, Proteobacteria and Bacteroidestes in both healthy and NDRV infected ducks. NDRV infection altered the relative abundance of bacteria. Specifically, families Ruminococcaceae and Lachnospiraceae were remarkably reduced, whereas Escherichia_Shigella belonging to family Enterobacteriaceae was significantly increased. Collectively, NDRV infection in Muscovy ducks resulted in a shift of the gut microbiota, including a net loss of probiotic bacteria with a compensatory expansion of pathogenic bacteria. These results provide new insights into the potential pathogenic mechanisms of NDRV. • The gut microbiota of Muscovy ducklings was mainly dominated by Firmicutes, Proteobacteria and Bacteroidestes. • NDRV infection remarkably reduced the abundance of families Ruminococcaceae and Lachnospiraceae. • The relative abundance of Escherichia_Shigella was significantly increased after NDRV infection. [ABSTRACT FROM AUTHOR]

Muscovy duck-origin goose parvovirus (MDGPV) is a causative agent of MDGPV-associated Derzsy's disease. To evalute the role of the cis -acting element E-box (CACATG) deletion on MDGPV eplication, an infectious plasmid clone p-PTΔE287, having one E-box deletion at nucleotide (nt) 287 of the left inverted terminal repeat sequence (L-ITR), was constructed by overlap extension PCR deleting the 287CACATG292 motif from the plasmid pMDGPVPT containing the full-length genome of the virulent MDGPV strain PT. The p-PTΔE287 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac, resulting in successful rescue of the deletion mutant virus r-PTΔE287. Compared with its parental virus PT, the virulence and the replication ability of r-PTΔE287 were reduced. In addition, we examined the ability of r-PTΔE287 to manipulate cell cycle progression. The results showed that r-PTΔE287 replication results in G0/G1 phase accumulation of infected duck embryo liver mesenchymal stem cells (BMSCs) and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Taken together, introducing 287CACATG292 element deletion into MDGPV PT genomic DNA that induced rescued mutant virus (r-PTΔE287) cell cycle arrest function at the G0/G1 phase, which might inhibit MDGPV replication and virus progeny production. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence. • We constructed an E-box motif (CACATG)-deleted MDGPV. • Deletion of E-box (E287) decreased MDGPV replication in Muscovy duck embryos. • The mutant virus r-PTΔE287 may induce cell cycle arrest at G0/G1 phase. [ABSTRACT FROM AUTHOR]