Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay

Wen, Xingjian; Cheng, Anchun; Wang, M. S.; Jia, Renyong; Zhu, Dekang; Chen, S.; Liu, M. F.; Liu, F.; Chen, X. Y.

doi:10.3382/ps.2014-04024

Cited by 26 publications

(16 citation statements)

References 44 publications

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“…Mean pairwise nucleotide sequence identities within groups 1, 2 and 3 were calculated to be 98.2%, 97.4% and 99.1%, respectively; the average genetic divergences between these groups are 7.2% (G1/G2), 9.8% (G1/G3) and 8.0% (G2/G3), showing high levels of genetic diversity. DHAV‐3 strains isolated from China from 2010 to 2015 are monophyletic and belong to group 1, which is consistent with the findings of previous studies (Li et al., ; Wen et al., ). Virulent and low‐pathogenicity strains from South Korea clustered into group 2, revealing a clear geographic pattern, and virulent strains from Vietnam clustered separately into groups 1 and 3.…”

Section: Resultssupporting

confidence: 91%

“…The collected samples were homogenized, diluted 1:10 in phosphate‐buffered saline (PBS) and centrifuged at 1,500 g for 10 min to obtain supernatant for RNA extraction and virus isolation. Total RNA was extracted from the supernatant using RNAiso Plus (TaKaRa, China) according to the manufacturer's instructions and detected by a reverse‐transcription PCR assay, as previously described (Wen et al., ). Positive liver samples from different farms were selected and used for virus isolation in 9‐day‐old DHAV antibody‐free duck embryos according to the previously described procedures (Swayne et al., ).…”

Section: Methodsmentioning

confidence: 99%

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Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015

Wen

Zhu

Cheng

et al. 2017

Transbound Emerg Dis

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Duck hepatitis A virus (DHAV) is the most common aetiologic agent of duck virus hepatitis (DVH), causing substantial economic losses in the duck industry worldwide. In China, officially approved DHAV-1 live-attenuated vaccines have been used widely to vaccinate breeder ducks since 2013. However, following the reports of DVH outbreaks, it has become necessary to assess the epidemiological situation of this virus in China. We conducted molecular epidemiological analyses of 32 DHAV field isolates while analysing the samples from ducks suspected of having hepatitis collected from commercial duck farms in China between May 2010 and December 2015. Considerable changes were observed in the epidemiology of DHAV-1 and DHAV-3 in China over time. A higher number of DHAV-1 strains were isolated during 2010-2012, coinciding with the widespread use of officially approved DHAV-1 live vaccine strains beginning in 2013. In contrast, a higher rate of DHAV-3 causing DHAV infections was observed between 2013 and 2015. Phylogenetic analyses based on the full-length VP1 gene were performed on these field isolates and using reference strains available in GenBank. DHAV-1 field isolates were evaluated in two groups: one group closely related to prototype strains and circulating in China between 2010 and 2012 and another group exhibiting genetic and serological differences from prototype strains. All DHAV-3 strains isolated in this study were grouped as monophyletic, which has become the predominant viral type, particularly in Shandong and Sichuan provinces, since 2013. In conclusion, these data provide updated information on the genetic and serological diversity of DHAV-1 and DHAV-3, and our findings may serve as a foundation for the prevention of, and vaccine development for, DHAV in China.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015

Wen

Zhu

Cheng

et al. 2017

Transbound Emerg Dis

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“…Cytokine expression levels were measured by the 2 −ΔΔCt method with relative quantification. Differences in expression levels of the various genes between strains CH and CH60 at each time point (24,36,48, 60 and 72 hpi) were analyzed using Student's t test and were considered significant as follows: *P < 0.05; **P < 0.01; ****P < 0.0001.…”

Section: Discussionmentioning

confidence: 99%

“…One-day-old Cherry Valley ducks were purchased from the poultry farm of Sichuan Agricultural University and were raised in isolators. The ducks were confirmed to be free of DHAV-1 or IgG against DHAV-1 by one step reverse-transcription PCR 48 and indirect ELISA 49 detection in serum samples.…”

Section: Methodsmentioning

confidence: 96%

Cytokine storms are primarily responsible for the rapid death of ducklings infected with duck hepatitis A virus type 1

Xie

Wang

Cheng

et al. 2018

Sci Rep

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Duck hepatitis A virus type 1 (DHAV-1) is one of the most harmful pathogens in the duck industry. The infection of adult ducks with DHAV-1 was previously shown to result in transient cytokine storms in their kidneys. To understand how DHAV-1 infection impacts the host liver, we conducted animal experiments with the virulent CH DHAV-1 strain and the attenuated CH60 commercial vaccine strain. Visual observation and standard hematoxylin and eosin staining were performed to detect pathological damage in the liver, and viral copy numbers and cytokine expression in the liver were evaluated by quantitative PCR. The CH strain (108.4 copies/mg) had higher viral titers than the CH60 strain (104.9 copies/mg) in the liver and caused ecchymotic hemorrhaging on the liver surface. Additionally, livers from ducklings inoculated with the CH strain were significantly infiltrated by numerous red blood cells, accompanied by severe cytokine storms, but similar signs were not observed in the livers of ducklings inoculated with the CH60 strain. In conclusion, the severe cytokine storm caused by the CH strain apparently induces hemorrhagic lesions in the liver, which might be a key factor in the rapid death of ducklings.

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“…Based on phylogenetic and neutralization assays, DHAV has been divided into three distinct genotypes: DHAV-1, DHAV-2, and DHAV-3 [1,9]. DHAV-1 is the most common type and has spread worldwide, and the development of accurate detection methods is essential [10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

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Background The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. Methods In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. Results Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and V max and K m values were determined to be 16.52 nmol/min and 50.78 μM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. Conclusions This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the V max and K m values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.

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Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay

Cited by 26 publications

References 44 publications

Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015

Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015

Cytokine storms are primarily responsible for the rapid death of ducklings infected with duck hepatitis A virus type 1

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

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