Shaohua Shi

Tembusu Virus in Ducks, China

In China in 2010, a disease outbreak in egg-laying ducks was associated with a flavivirus. The virus was isolated and partially sequenced. The isolate exhibited 87%–91% identity with strains of Tembusu virus, a mosquito-borne flavivirus of the Ntaya virus group. These findings demonstrate emergence of Tembusu virus in ducks.

Complete Genome Sequence of Avian Tembusu-Related Virus Strain WR Isolated from White Kaiya Ducks in Fujian, China

ABSTRACT Avian tembusu-related virus, which was first identified in China, is an emerging virus causing serious economic loss to the Chinese poultry industry. We report here the complete genome sequences of avian tembusu-related virus strain WR, isolated from a White Kaiya duck with disease characterized by an abrupt decrease in egg laying with ovarian hemorrhage, which will help in further understanding the molecular and evolutionary characteristics and pathogenesis of avian tembusu-related virus, the new flavivirus affecting ducks in Southern China.

The development of a rapid SYBR Green I-based quantitative PCR for detection of Duck circovirus

This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.

Comparative analysis of transcriptional profiles of retinoic-acid-induced gene I-like receptors and interferons in seven tissues from ducks infected with avian Tembusu virus.

Avian Tembusu virus (ATV), an emerging virus that mainly infects laying and breeding ducks in China, has caused severe economic loss in duck industry. However, there have been no reports about host innate immune responses during ATV infection and its correlation with clinical signs or pathology. To identify the roles of these immune factors in the innate host response to ATV infection, quantitative real-time PCR (qPCR) was used to analyze the transcriptional profiles on the genes encoding two retinoic-acid-induced gene I (RIG-I)-like receptors (RLRs) and two interferons (INF-α and INF-γ) in seven tissues of an ATV-infected shelduck. After infection with ATV, both RLR genes were significantly upregulated (P < 0.05) in all seven tissues. The peak expression levels of the two RLR genes were observed at 24 hours postinfection (hpi) and were higher in non-lymphoid tissues (liver, lung, kidney, and ovary) than in lymphoid tissues (thymus, spleen and bursa). Although the transcription levels of both IFN genes were also upregulated, they showed different time-dependent expression patterns compared with those of the RLR genes. In addition, the highest mRNA expression of the two IFN genes was observed in the ovary at 6 hpi. This observation suggests that the ovary is the primary target tissue in ATV infection and explains the clinical characteristics of the primary pathological changes in the ovaries of ATV-infected ducks. Our results, for the first time, elucidate the differential and coordinated expression profiles of two RLRs and two IFNs in an ATV-infected shelduck.

Genetic Diversity and Genotype Analysis of Duck Circovirus

Abstract To investigate the genetic diversity and genotype of duck circovirus (DuCV), nine full-length DuCV genomes were determined from clinical samples. Multiple sequence alignment and phylogenetic analyses were performed on the nine viral genome sequences as well as on 27 genome sequences retrieved from the GenBank database. Pairwise analysis showed that the determined genome sequences have a genome organization identical to the 27 sequences and share 83.3%–99.8% identity among themselves and 82.6%–99.9% with the other 27 sequences. Phylogenetic analysis revealed that all 36 viral genome sequences are divided into two lineages, DuCV1 and DuCV2, in which the nucleotide diversity between genome sequences in these two lineages ranged from 13.2%–17.4%; these may be regarded as two types of viruses. Viruses under DuCV1 and DuCV2 are further clustered into different sublineages. When analyzed using the method for genotype definition proposed by Grau-Roma et al., these different sublineages can be defined as genotypes DuCV1a, DuCV1b, DuCV2a, DuCV2b, and DuCV2c. In addition, the viral sequences obtained from mainland China are different in genomic size and share a diversity of no less than 13.2%, including the sequences that came from all genotypes. This suggests that the DuCVs prevalent in domestic duck flocks in China are ecologically divergent.

Epidemiological investigation and genome analysis of duck circovirus in Southern China

Duck circovirus (DuCV), a potential immunosuppressive virus, was investigated in Southern China from March 2006 to December 2009 by using a polymerase chain reaction (PCR) based method. In this study, a total of 138 sick or dead duck samples from 18 different farms were examined with an average DuCV infection rate of ∼35%. It was found that ducks between the ages of 40∼60 days were more susceptible to DuCV. There was no evidence showing that the DuCV virus was capable of vertical transmission. Farms with positive PCR results exhibited no regularly apparent clinical abnormalities such as feathering disorders, growth retardation or lower-than-average weight. The complete genomes of 9 strains from Fujian Province and 1 from Zhejiang Province were sequenced and analyzed. The 10 DuCV genomes, compared with others genomes downloaded from GenBank, ranged in size from 1988 to 1996 base pairs, with sequence identities ranging from 83.2% to 99.8%. Phylogenetic analysis based on genome sequences demonstrated that DuCVs can be divided into two distinct genetic genotypes, Group I (the Euro-USA lineage) and Group II (the Taiwan lineage), with approximately 10.0% genetic difference between the two types. Molecular epidemiological data suggest there is no obvious difference among DuCV strains isolated from different geographic locations or different species, including Duck, Muscovy duck, Mule duck, Cheery duck, Mulard duck and Pekin duck.

Complete Genome Sequence of a Duck Hepatitis A Virus 1 Isolated from a Pigeon in China

ABSTRACT We report here the complete genome sequence of a duck hepatitis A virus 1 (DHAV-1), strain FJ1220, isolated from a dead pigeon in eastern China. DHAV-1 FJ1220 has high homology of up to 99.6% to the DHAV-1 strain Du/CH/LGD/111238 but relatively low homology to strains FFZ05 and FZ05. An amino acid hypervariable region in the VP1 protein of FJ1220 has the motif 180TPSGR184 replaced by 180ALSRG184 compared to strains FFZ05 and FZ05.

Development of a restriction length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses

A restriction fragment length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses (GPV and MDPV) was developed based on comparison of the NS gene of GPV and MDPV. Both GPV and MDPV genomic DNA can be amplified with 641 bp using the specific PCR primers. The PCR fragments can be cut into 463 bp and 178 bp only in the case of MDPV-derived PCR products, whereas the GPV-derived PCR products cannot. The method established in this study can be used to differentiate GPV and MDPV with high specificity and precision, by using a direct PCR kit and QuickCut enzyme, as quickly as conventional PCR.

Full Genome Sequence of Egg Drop Syndrome Virus Strain FJ12025 Isolated from Muscovy Duckling

ABSTRACT Egg drop syndrome virus (EDSV) strain FJ12025 was isolated from a 9-day-old Muscovy duckling. The results of the sequence showed that the genome of strain FJ12025 is 33,213 bp in length, with a G+C content of 43.03%. When comparing the genome sequence of strain FJ12025 to that of laying duck original strain AV-127, we found 50 single-nucleotide polymorphisms (SNPs) between the two viral genome sequences. A genomic sequence comparison of FJ12025 and AV-127 will help to understand the phenotypic differences between the two viruses.

Complete Genomic Sequence of the Virulent Bacteriophage RAP44 of Riemerella anatipestifer

SUMMARY. A virulent Riemerella anatipestifer bacteriophage, RAP44, belonging to the Siphoviridae family of tailed phages, was previously isolated from feces of healthy Muscovy ducks in China. A complete genomic sequence analysis indicates that the phages genome consists of a linear, double-stranded DNA molecule of 49,329 nucleotides. Eighty open reading frames (ORF) were identified. Putative functions could be assigned to 24 of the ORFs. The location of these genes was consistent with organization of the genome in a modular format which includes modules for host cell lysis, tail morphogenesis, head morphogenesis, and DNA replication and modification modules. Until now, no R. anatipestifer phage genome sequence has been reported in the literature. Therefore, this study represents the first complete genomic and molecular description of the R. anatipestifer phage. RESUMEN. Secuencia genómica completa del bacteriófago virulento RAP44 de Riemerella anatipestifer. Un bacteriófago virulento de Riemerella anatipestifer, RAP44, que pertenece a la familia Siphoviridae que incluye fagos con cola, fue aislado anteriormente de las heces de patos reales sanos en China. El análisis completo de la secuencia genómica indica que el genoma del fago consiste de una molécula de ADN lineal, de doble cadena de 49,329 nucleótidos. Ochenta marcos de lectura continuos (ORF) fueron identificados. Se pudieron asignar funciones supuestas a 24 de los ORFs. La ubicación de estos genes fue compatible con la organización del genoma en un formato modular que incluye módulos para la lisis de la célula huésped, para morfogénesis de la cola, y de la cabeza, y para la replicación y modificación del ADN. Hasta ahora, ninguna secuencia de un fago de R. anatipestifer se había reportado en la literatura. Por lo tanto, este estudio representa la primera descripción genómica y molecular completa de un fago de R. anatipestifer.