Youxiang Diao

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.

Characterization of a Tembusu virus isolated from naturally infected house sparrows (Passer domesticus) in Northern China.

The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV-SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species.

Tembusu Virus in Human, China

SUMMARY Tembusu virus (TMUV) infection in ducks, geese and house sparrows was reported in China. To confirm the emergence of TMUV in humans, we investigated TMUV as a possible infection in duck industry workers in Shandong, China. Of 132 serum samples tested, 95 (71.9%) had TMUV antibodies. In oral swabs detection, 63 (47.7%) samples were positive for TMUV RNA. Nucleotide sequences of 277 bp coding the partial NS3 protein showed more than 99.5% identity with other duck TMUV strains, which can cause severe egg drop in ducks. These findings contribute to the realization that TMUV may be overlooked as a zoonotic transmission in China.

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China.

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%–94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%–81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160–176 and 306–322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.

Hydropericardium Hepatitis Syndrome Emerged in Cherry Valley Ducks in China.

Since June 2015, a highly pathogenic disease occurred in duck flocks in China, causing pericardial effusion, enlarged discoloured liver, renal enlargement and haemorrhagic lung with a mortality ranging from 5% to 20%. Previous study confirmed that Fowl adenovirus group C (FAdV-C) and some field FAdVs isolates had been identified as causative agents of hydropericardium hepatitis syndrome (HHS) in chickens and geese world widely. In this study, we firstly report the isolation of FAdV-C from ducks with HHS. The two isolates, designated as SDSX and SDJX, were separated from liver samples using 9-day-old SPF chicken embryos and could cause severe cytopathic effects in duck and chicken embryonic kidney cells. The entire ORF sequences of hexon gene of the two isolates were amplified, sequenced and analysed by restriction fragment length polymorphism. Phylogenetic analysis of loop 1 sequences of hexon gene of FAdVs revealed that the two isolates were closely related to FAdV-C isolates, which could cause HHS in chickens. Experimental infection indicated that the isolate was high pathogenicity to 20-day-old ducks. Our study shows that the recently emerged HHS in ducks was caused by FAdV-C and may possess a potential risk to other poultry flocks.

Rapid detection of Tembusu virus by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP).

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of Tembusu virus (TMUV) infection. The reaction was performed in one step in a single tube at 64°C for 45 min, with SYBR Green I dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10 copies/μl, and no cross-reaction with other avian viruses was observed. The assay was evaluated further for the diagnosis of TMUV in field samples and compared with conventional RT-PCR, demonstrating that results of the RT-LAMP assay corresponded to those of conventional RT-PCR. In conclusion, RT-LAMP with SYBR Green I dye was shown to be a sensitive, simple assay for the rapid diagnosis of TMUV infection in ducks.

Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein.

Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.

Experimental reproduction of beak atrophy and dwarfism syndrome by infection in cherry valley ducklings with a novel goose parvovirus-related parvovirus.

Infection of clinically susceptible ducks, including cherry valley and Muscovy ducks, with a novel goose parvovirus (GPV)-related virus (N-GPV) can result in beak atrophy and dwarfism syndrome (BADS). To obtain new insights into the host range and pathogenic potential of this novel waterfowl parvovirus, cherry valley ducklings (n=20) were experimentally infected with N-GPV strain SDLC01. An equal number of ducklings served as uninfected controls. The appearance of clinical signs, histopathological changes, viral shedding, and seroconversion was monitored for 20 days post-infection. Infection status of all ducks was monitored using indirect ELISA, virus neutralization test, nested PCR, clinical indicators, and microscopic examination. Three ducks developed the typical clinical, gross, and histological changes of BADS. By study day 6, the infected ducks had seroconverted to N-GPV. The antibodies raised were neutralizing against the SDLC01 strain in vitro. Here we successfully developed an experimental infection model for studying the pathogenicity and role of N-GPV in BADS.

Evidence for Vertical Transmission of Novel Duck-Origin Goose Parvovirus-Related Parvovirus.

In 2015, novel duck-origin goose parvovirus-related parvovirus (N-GPV) infection progressively appeared in commercial Cherry Valley duck flocks in North China. Diseased ducks were observed to have beak atrophy and dwarfism syndrome (BADS). A previous study showed that a high seropositive rate for N-GPV indicated a latent infection in most breeder duck flocks. To investigate this possibility in hatching eggs collected from N-GPV-infected breeder ducks, 120 eggs were collected at various stages of embryonic development for viral DNA detection and an N-GPV-specific antibody test. N-GPV DNA was present in nine hatching eggs, eleven duck embryo and eight newly hatched ducklings. Of the newly hatched ducklings, 58.33% (21/36) were seropositive. Further, two isolates were obtained from a 12-day-old duck embryo and a newly hatched duckling. N-GPV infection did not reduce the fertilization rate and hatchability. These results indicate possible vertical transmission of N-GPV and suggest that it may be transmitted from breeder ducks to ducklings in ovo.

Development of a TaqMan-based real-time PCR assay for the detection of Novel GPV

The newly emerged disease, duck beak atrophy and dwarfism syndrome (BADS), is caused by novel goose parovirus (N-GPV). Although N-GPV infection has severe consequences, few methods for detecting this virus have been developed. Therefore, the availability of rapid and reliable molecular diagnostic methods would aid future studies of this novel virus. Clinical specimens from 138 suspected cases of N-GPV infection and 120 cloacal swabs from breeding ducks were used in this study. The targeted sequence of N-GPV cloned into the pMD18-T vector was used to generate the N-GPV DNA standard curve. The specificity of the assay was validated using duck plague virus, GPV, duck hepatitis virus, avian influenza virus, duck reovirus, tembusu virus, and fowl adenovirus. The lowest limit of detection was 8.8×101copies/μL with a good linear standard curve (Y=-3.3682X+37.220, R2=0.9953) over a wide range of input DNA, of which the concentration was between 8.8×101 to 8.8×108copies/μL. The results show that the real-time PCR assay is a highly sensitive, specific, reproducible, and versatile method for quantitatively detecting N-GPV DNA, and thus can be used to detect this virus, thereby facilitating epidemiological investigations of animals with BADS.