Differential metabolism-associated gene expression of duck pancreatic cells in response to two strains of duck hepatitis A virus type 1.

Several outbreaks of duck hepatitis A virus type 1 (DHAV-1), which were characterized by yellow coloration and hemorrhage in pancreatic tissues, have occurred in China. The causative agent is called pancreatitis-associated DHAV-1. The mechanisms involved in pancreatitis-associated DHAV-1 infection are still unclear. Transcriptome analysis of duck pancreas infected with classical-type DHAV-1 and pancreatitis-associated DHAV-1 was carried out. Deep sequencing with Illumina-Solexa resulted in a total of 53.9 Gb of clean data from the cDNA library of the pancreas, and a total of 29,597 unigenes with an average length of 993.43 bp were generated by de novo sequence assembly. The expression levels of D-3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which are involved in glycine, serine, and threonine metabolism pathways, were significantly downregulated in ducks infected with pancreatitis-associated DHAV-1 compared with those infected with classical-type DHAV-1. These findings provide information regarding differences in expression levels of metabolism-associated genes between ducks infected with pancreatitis-associated DHAV-1 and those infected with classical-type DHAV-1, indicating that intensive metabolism disorders may contribute to the different phenotypes of DHAV-1-infection.

Proteomics reveals the effect of type I interferon on the pathogenicity of duck hepatitis A virus genotype 3 in Pekin ducks.

Duck hepatitis A virus genotypes 3 (DHAV-3) has become the most prevalent pathogen of duck viral hepatitis (DVH) in Asian duck industry in recent years. Previous studies on the pathogenic mechanism of DHAV-3 mainly focused on examine host gene expression levels. However, the study about host protein expression levels has not been reported. For this, proteomics analysis on livers of infected 7-day-old Pekin ducks with DHAV-3 112803 strain was performed to screen differentially expressed proteins. A total of 3,385 proteins were identified, and we found 39 proteins in the challenged group (CH) were significantly up-regulated and 15 proteins were significantly down-regulated in comparison with control group (CON). GO results showed that 9 of the top 20 GO terms were involved in type I interferon regulation, and the KEGG pathway enrichment results showed that innate immune responses were significantly enriched, such as RIG-1-like, Toll-like and NOD-like receptor signaling pathways. Notably, interaction between 11 up-regulation proteins promoted interferon-induced protein synthesis and supported viral genome replication, which could aggravate inflammatory response and liver damage. These findings, together with RT-qPCR verification of related genes, support the view that the type I interferon may play an extremely important role in the pathogenic mechanism of DHAV-3.

Quantitative Proteomic Analysis Uncovers the Mediation of Endoplasmic Reticulum Stress-Induced Autophagy in DHAV-1-Infected DEF Cells.

Autophagy is a tightly regulated catabolic process and is activated in cells in response to stress signals. Despite extensive study, the interplay between duck hepatitis A virus type 1 (DHAV-1) and the autophagy of host cells is not clear. In this study, we applied proteomics analysis to investigate the interaction mechanism between DHAV-1 and duck embryo fibroblast (DEF) cells. In total, 507 differentially expressed proteins (DEPs) were identified, with 171 upregulated proteins and 336 downregulated proteins. The protein expression level of heat shock proteins (Hsps) and their response to stimulus proteins and zinc finger proteins (ZFPs) were significantly increased while the same aspects of ribosome proteins declined. Bioinformatics analysis indicated that DEPs were mainly involved in the "response to stimulus", the "defense response to virus", and the "phagosome pathway". Furthermore, Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to the lipidation form of LC3-II increased, and the conversion rate decreased when DEF cells were processed with 4-phenylbutyrate (4-PBA). These findings indicated that DHAV-1 infection could cause endoplasmic reticulum (ER) stress-induced autophagy in DEF cells, and that ER stress was an important regulatory factor in the activation of autophagy. Our data provide a new clue regarding the host cell response to DHAV-1 and identify proteins involved in the DHAV-1 infection process or the ER stress-induced autophagy process.

The VP3 protein of duck hepatitis A virus mediates host cell adsorption and apoptosis.

Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, -8 and -9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.

The pathogenicity of duck hepatitis A virus types 1 and 3 on ducklings.

Duck hepatitis A virus (DHAV) is one of the pathogens that cause fatal duck viral hepatitis (DVH) in ducklings, which is an acute and contagious disease with a high mortality rate. Despite a continuing official duck vaccination program, DHAV infection remains a major threat to the duck industry. Considerable changes were observed in the epidemiology of DHAV-1/-3 in China over time. Therefore, comparing the pathogenicity of different DHAV serotypes can provide a theoretical basis for the diagnosis and prevention of DVH. In this study, we systematically investigated the effects of infection with DHAV-1/-3 field strains on clinical signs, gross lesions, histopathological changes, viral RNA detection, enzymatic systems, and metabolite concentrations. The results demonstrated that the major macroscopic and microscopic lesions in ducks infected with DHAV-1/-3 in the liver, brain, spleen, pancreas, and kidneys exhibited no significant differences. After 24 h of infection, DHAV quickly appeared in blood and major organs. Significant changes in clinical chemical markers together with histopathological lesions and viral RNA detection indicated that the liver is the major target organ for both viruses, resulting in impaired of liver integrity and function. In addition, we found that both viruses were able to invade both central and peripheral immune organs. Also lipase plasma activity was substantially affected by DHAV-1/-3, indicating that the integrity and function of the pancreas was compromised. However, there was no significant difference in pathogenicity between DHAV-1 and -3. The results of this study provide new insights into the pathogenesis of DHAV-1/3, two viruses that cause serious depression, metabolic disorders, and immunosuppression.

Comparative pathogenicity of two subtypes (hepatitis-type and pancreatitis-type) of duck hepatitis A virus type 1 in experimentally infected Muscovy ducklings.

Duck hepatitis A virus type 1 (DHAV-1) causes acute hepatitis with high morbidity and mortality in ducklings of the genera Cairina and Anas and is characterized by ecchymotic haemorrhage and necrosis of the liver surface. Since September 2011, a new subtype of DHAV-1 (named pancreatitis-type DHAV-1) has been isolated. This new subtype is characterized by yellowish or haemorrhagic pancreatitis, but with no significant pathological changes in the liver. To further investigate the difference in pathogenicity between hepatitis-type DHAV-1 and pancreatitis-type DHAV-1, we infected Muscovy ducklings with a hepatitis-type DHAV-1 strain, FZ86, or a pancreatitis-type DHAV-1 strain, MPZJ1206, and then compared the resulting gross lesions, histopathological changes, viral distribution and cellular apoptosis in the liver and pancreas of Muscovy ducklings. The results suggested that FZ86 induced a more efficient viral propagation in the liver than MPZJ1206, and the gross and histopathological lesions were also limited to the liver. However, MPZJ1206 induced more effective viral replication in the pancreas than FZ86. The MPZJ1206-infected Muscovy ducklings showed an obviously yellowed and haemorrhagic pancreas, but with no significant pathological changes in the liver. Furthermore, FZ86 induced notable hepatocyte apoptosis and increased the expression of caspase-3 in the liver, whereas MPZJ1206 caused apoptosis in a large number of acinar epithelial cells and elevated the expression of caspase-3 in the pancreas. Taken together, these results demonstrated that pancreatitis-type DHAV-1 has many new pathogenic features which distinguish it from the hepatitis-type DHAV-1. RESEARCH HIGHLIGHTS Pancreatitis-type DHAV-1 (MPZJ1206) was characterized by pancreatic haemorrhage and yellow discolouration, but with no obvious haemorrhage and necrosis in the liver. Pancreatitis-type DHAV-1 (MPZJ1206) exhibits many new pathogenic features which distinguish it from the hepatitis-type DHAV-1 (FZ86).

Isolation, identification and attenuation of a pathogenic duck hepatitis virus type 1 in China, and complete genomic sequence comparison between the embryo-passaged, attenuated derivatives and their parent.

Duck viral hepatitis (DVH) is an acute and fatal disease of young ducklings characterized by rapid transmission and damages. The most important agent of DVH is duck hepatitis virus 1 (DHV-1). The effective control of DVH was achieved by active immunization of 1-day-old duck- lings with an attenuated DHV-1 virus vaccine. However, the attenuated virus might reverse to virulence. In this study, a DHV-1 strain, Du/CH/LBJ/090809, was identified and its genomic se- quences were determined. The genome of Du/CH/LBJ/090809 is composed of 7,692 nt excluding poly A and the virus was clustered into genotype A by comparing with other referenced DHV-1 strains. Du/CH/LBJ/090809 could lead to 30% mortality of 10-day-old specific pathogen free (SPF) ducklings. The virus was passaged serially in SPF chicken embryonated eggs and three vi- ruses, passage 16 (P16), P29 and P40, were selected for genomic analysis. P29 and P40 were used to evaluate the attenuation in duckling by inoculating the virus to 10-day-old SPF ducklings. Re- sults of vaccination-challenge assay showed that the inactivated virus P40 could evoke protection against the pathogenic parent virus. Nucleotide and amino acid sequences of the genomes of Du/ CH/LBJ/090809, P16, P29 and P40 were compared. Changes both in nucleotides and amino acids, which might be contributed to the decreasing in virulence by chicken embryo-passaging of DHV- 1, were observed. We speculated that these changes might be important in the adaption and at- tenuation of the virulent virus. Additionally, strains obtained in this study will provide potential candidate in the development of vaccines against DHV-1.

PI3KC3-dependent autophagosomes formation pathway is of crucial importance to anti-DHAV activity of Chrysanthemum indicum polysaccharide.

We previously reported that Chrysanthemum indicum polysaccharide (CIPS) effectively inhibited the replication of duck hepatitis A virus (DHAV). However, the inhibition mechanisms are still unclear. Autophagy plays important role in virus genomic replication. Therefore, in present study, the effect of autophagy on DHAV genome replication as well as the influence of CIPS on autophagy were studied. qPCR, western blot, and ELISA methods were applied to observe the autophagy and analyze the inhibition mechanisms of CIPS on DHAV. Results showed that DHAV infection increased the expression level of LC3-II and interdicted the degradation of p62. Treating with rapamycin benefited DHAV gene expression level. What's more, DHAV infection and rapamycin treatment also promoted the expression of PI3KC3 and increased the concentration of PI3P. However, CIPS treatment significantly downregulated the expressions of LC3-II and PI3KC3 induced by DHAV and rapamycin, and consequently inhibited autophagosomes formation. As a result, DHAV replication was inhibited.

Comparative pathogenicity of different subtypes of duck hepatitis A virus in Pekin ducklings.

Duck hepatitis A virus (DHAV) is a major pathogen of viral hepatitis in ducks, which is a fatal and contagious disease of young ducklings. Despite the identification of numerous DHAV strains (e.g. DHAV-3, DHAV-2, DHAV-1 and DHAV-1a), the pathogenic differences among the different subtypes have not been evaluated. The objective of this study was to compare the pathogenic properties of three epidemic strains DHAV-3, DHAV-1, and DHAV-1a in mainland China, in a Pekin duckling infection model. We evaluated the pathogenicity of these different subtypes by investigating clinical signs, macroscopic and microscopic lesions, immunohistochemical examination, and viral RNA detection after experimental inoculation of Pekin ducklings with the three different DHAV strains. There was no significant difference in pathogenicity between DHAV-3 and DHAV-1. Pathogenicity of DHAV-1a differed significantly from that of classical duck hepatitis A (DHAV-3 or DHAV-1), in that there were no clinical signs of opisthotonos. More importantly, pancreatic bleeding or yellowing, and spleen swelling and bleeding were the predominant lesions in the DHAV-1a group, while liver and spleen lesions were the main signs in classical hepatitis (DHAV-1/3). Our findings indicate that there are differences in the pathogenicity of different subtypes of DHAV in ducklings, which may be useful for understanding the biological characteristics of the different subtypes of DHAV in ducks.

Incompatible Translation Drives a Convergent Evolution and Viral Attenuation During the Development of Live Attenuated Vaccine.

Live attenuated vaccines are widely used to protect humans or animals from pathogen infections. We have previously developed a chicken embryo-attenuated Duck Hepatitis A Virus genotype 1 (DHAV-1) vaccine (CH60 strain). This study aims to understand the mechanisms that drive a virulent strain to an attenuated virus. Here, we systematically compared five DHAV-1 chicken embryo attenuated strains and 68 virulent strains. Phylogenetic analysis indicated that duck virulent strains isolated from different geographic regions of China undergo a convergent evolution in the chicken embryos. Comparative analysis indicated that the codon usage bias of the attenuated strains were shaped by chicken codons usage bias, which essentially contributed to viral adaption in the unsuitable host driven by incompatible translation. Of note, the missense mutations in coding region and mutations in untranslated regions may also contribute to viral attenuation of DHAV-1 to some extent. Importantly, we have experimentally confirmed that the expression levels of four viral proteins (2A3pro, 2A3pro, 3Cpro, and 3Dpro) in the liver and kidney of ducks infected with an attenuated strain are significantly lower than that infected with a virulent strain, despite with similar virus load. Thus, the key mechanisms of viral attenuation revealed by this study may lead to innovative and easy approaches in designing live attenuated vaccines.

Establishment and evaluation of the goose embryo epithelial (GEE) cell line as a new model for propagation of avian viruses.

In this study, we report the establishment and characterization of a new epithelial cell line, goose embryonated epithelial cell line (GEE), derived from embryonic goose tissue. The purified GEE cell line can efficiently grow over 65 passages in the M199 medium supplemented with 10% fetal bovine serum at 37°C. Immunofluorescence assay was used to identify purified GEE cells as epithelial cell line by detecting expression of the Keratin-18 and -19. Further characterizations demonstrated that the GEE cell line can be continuously subcultured with (i) a high capacity to replicate for over 65 passages, (ii) a spontaneous epithelial-like morphology, (iii) constant chromosomal features and (iv) without an evidence of converting to tumorigenic cells either in vitro or in vivo study. Moreover, the GEE cell line can be effectively transfected with plasmids expressing reporter genes of different avian viruses, such as VP3, VP1 and F of goose parvo virus (GPV), duck hepatitis virus (DHV), and Newcastle disease virus (NDV), respectively. Finally, the established GEE cell line was evaluated for avian viruses infection susceptibility. Our results showed that the tested GPV, DHAV and NDV were capable to replicate in the new cell line with titers a comparatively higher to the ones detected in the traditional culture system. Accordingly, our established GEE cell line is apparently a suitable in vitro model for transgenic, and infection manipulation studies.

Pathogenicity of duck hepatitis A virus type 3 and innate immune responses of the ducklings to virulent DHAV-3.

Duck virus hepatitis caused by duck hepatitis A virus (DHAV) is an acute and contagious disease. To better understand the pathogenic mechanism of DHAV-3 in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DHAV-3 could infect many tissues, leading to pathological lesions, especially on the livers and spleen, and the host immune responses are activated in infection. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the livers and spleen, and antiviral innate immune response was established, but not sufficient to restrict the virus replication of lethal dose. Many major pattern recognition receptors (PRRs) (RIG-1, MDA5, and TLR7) are involved in the host immune response to DHAV-3, and the expression of interferon (IFNα, IFNß and IFNγ) and antiviral proteins (MX, OAS and PKR) are also up-regulated in the liver and spleen. The expression of most cytokines (IL-1ß, IL-2 and IL-6) was also up-regulated to different degrees and was various; the expression of IL-2 increased most significantly in liver. Our data provide a foundation for further study of the pathogenicity of duck virus hepatitis and extend our understanding of the immune responses of ducklings to DHAV-3 infection.

Treatment effect of a flavonoid prescription on duck virus hepatitis by its hepatoprotective and antioxidative ability.

CONTEXT: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and lethal disease of young ducklings. However, there is still no effective drug to treat DVH. OBJECTIVE: This study assessed the curative effect on DVH of a flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) as well as the hepatoprotective and antioxidative effects of BLIN. MATERIALS AND METHODS: MTT method was used to test the anti-DHAV-1 ability of BLIN in vitro. We then treated ducklings by BLIN (3 mg per duckling, once a day for 5 days) to evaluate the in vivo efficacy. To study the hepatoprotective and antioxidative roles of BLIN in its curative effect on DVH, we investigated the hepatic injury evaluation biomarkers and the oxidative stress evaluation indices of the ducklings. RESULTS: On duck embryonic hepatocytes, DHAV-1 inhibitory rate of BLIN at 20 µg/mL was 69.3%. The survival rate of ducklings treated by BLIN was about 35.5%, which was significantly higher than that of virus control (0.0%). After the treatment of BLIN, both the hepatic injury and the oxidative stress of infected ducklings alleviated. At the same time, a significant positive correlation (p < 0.05) existed between the hepatic injury indices and the oxidative stress indices. CONCLUSIONS: BLIN showed a significant curative effect on DVH. The antioxidative and hepatoprotective effects of BLIN made great contributions to the treatment of DVH. Furthermore, BLIN is expected to be exploited as a new drug for the clinical treatment of DVH.

Establishment and characterization of duck embryo epithelial (DEE) cell line and its use as a new approach toward DHAV-1 propagation and vaccine development.

The primary cell culture was derived from duck embryonic tissue, digested with collagenase type I. The existence of cell colonies with epithelial-like morphology, named duck embryo epithelial (DEE), were purified and optimally maintained at 37°C in M199 medium supplemented with 5% fetal bovine serum. The purified cells were identified as epithelial cell line by detecting Keratin-18 expression using immunofluorescence assay. Our findings demonstrated that DEE cell line can be propagated in culture with (i) a great capacity to adhere, (ii) a great proliferation activity, and (iii) a population doubling time of approximately 18h. Chromosomal features of the DEE cell line were remained constant after the 50th passage. Further characterizations of DEE cell line showed that cell line can normally be grown even after several passages and never converted to tumorigenic cells either in vitro or in vivo study. Susceptibility of DEE cell line was determined for transfection and duck hepatitis A type 1 virus (DHAV-1)-infection. Interestingly, the 50% egg lethal dose (ELD50) of the propagated virus in DEE cell line was higher than ELD50 of the propagated virus in embryonated eggs. Finally, DEE cell line was evaluated to be used as a candidate for DHAV-1 vaccine development. Our results showed that the propagated DHAV-1 vaccine strain SDE in DEE cell line was able to protect ducklings against DHAV-1 challenge. Taken together, our findings suggest that the DEE cell line can serve as a valuable tool for DHAV-1 propagation and vaccine production.

Identification and expression analysis of the interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) gene in duck (Anas platyrhynchos domesticus).

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

Virulent and attenuated strains of duck hepatitis A virus elicit discordant innate immune responses in vivo.

Previous studies of duck hepatitis A virus infection have focused only on the pathogenicity and host response of one strain. Here, we show that the virulent SH strain and the attenuated FC64 strain induced varied pathogenicity, apoptosis and immune responses in the livers of 1-day-old ducklings. SH infection caused apoptosis and visible lesions in the liver; serum aspartate aminotransferase, alanine transaminase, alkaline phosphatase, γ-glutamyltransferase and total bilirubin activities were markedly upregulated; and ducklings died at 36 h post-infection (p.i.). However, FC64 infection did not induce significant symptoms or impair liver function, and all of the infected ducklings remained healthy. In addition, both virus strains replicated well in the liver, spleen and intestine, whilst the SH strain replicated more efficiently than FC64. IFN-γ, IL-2, inducible nitric oxide synthase and nitric oxide were strongly induced by SH infection, and may be associated with the pathogenicity of the SH strain. IFN-α, IFN-ß, IFN-stimulated transmembrane protein 1, IFN-stimulated gene 12, 2',5'-oligoadenylate synthetase-like and IL-6 were moderately induced by SH infection at 24 h p.i., and dramatically induced by FC64 infection at 36 h p.i. The intensive induction of cytokines by FC64 may be involved in restriction of virus replication and stimulation of adaptive immune responses. Ducklings inoculated with FC64 produced high levels of antiviral antibodies within 45 days p.i. The low virulence and strong immune response of FC64 rendered this strain a good vaccine candidate, as confirmed by a protective assay in this study.

Effect of age on the pathogenesis of DHV-1 in Pekin ducks and on the innate immune responses of ducks to infection.

Duck hepatitis virus (DHV) affects 1-week-old but not 3-week-old ducks, and it causes a more severe disease in the younger ducks. These differences may be partially due to the host response to DHV infection. In order to understand this difference, we characterized the pathobiology of and innate immune response to DHV infection in 1-day-old (1D) and 3-week-old (3 W) ducks. Viral RNA was detected in duck livers at 24, 36 and 72 h after inoculation with DHV at a dose of 10(3) LD50. Virus-induced pathology ranged from no clinical signs to severe disease and death, and it was more severe in the 1D ducks. Infection with DHV induced up-regulation of gene expression of Toll-like receptor (TLR)-7, TLR3, retinoic-acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), interleukin (IL)-6, interferon (IFN)-α, interferon-induced transmembrane protein 1 (IFITM1), interferon-stimulated gene 12 (ISG12), and 2'-5' oligoadenylate synthetase-like gene (OASL) in the livers of 3 W ducks. Of these, IL-6, OASL and ISG12 mRNA levels were more than 100-fold higher in infected 3 W ducks than in mock-infected ducks of the same age. These genes were induced much less in infected 1D ducklings. We present evidence that a lower level of viral replication in the hepatocytes of 3 W ducks, whose basal level of cytokines is higher than that in 1D ducklings, may be related to the strong innate immunity induced. From our data, we conclude that duck age plays an important role in the pathogenicity of and innate immune responses to DHV.

An experimental study of the pathogenicity of a duck hepatitis A virus genotype C isolate in specific pathogen free ducklings.

Duck hepatitis A virus genotype C (DHAV-C), recognized recently, is one of the pathogens causing fatal duck viral hepatitis in ducklings, especially in Asia. To demonstrate the pathogenesis of the DHAV-C isolate, 3-day-old specific pathogen free ducklings were inoculated subcutaneously with a DHAV-C isolate and the clinical signs were observed. Virus distribution, histological and apoptotic morphological changes of various tissues were examined at different times post inoculation. The serial, characteristic changes included haemorrhage and swelling of the liver. Apoptotic cells and virus antigen staining were found in all of the tissues examined. Where more virus antigen staining was detected, there were more severe histopathological and apoptotic changes. The amount of virus antigen and the histological and apoptotic morphological changes agreed with each other and became increasingly severe with length of time after infection. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages and monocytes in immune organs such as the bursa of Fabricius, thymus and spleen, and in liver, kidney and cerebral cells. Necrosis was also observed within 72 h post inoculation in all organs examined, except the cerebrum, and was characterized by cell swelling and collapsed plasma membrane. These results suggest that the recent outbreak of disease caused by DHAV-C virus is pantropic, causing apoptosis and necrosis of different organs. The apoptosis and necrosis caused by the DHAV-C field strain in this study is associated with pathogenesis and DHAV-C-induced lesions.

Development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of Duck hepatitis A virus type 1.

We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Duck hepatitis A virus type 1 (DHAV-1). The amplification could be finished in 1 h under isothermal conditions at 63 °C by employing a set of four primers targeting the 2C gene of DHAV-1. The RT-LAMP assay showed higher sensitivity than the RT-PCR with a detection limit of 0.1 ELD(50) 0.1 ml(-1) of DHAV-1. The RT-LAMP assay was highly specific; no cross-reactivity was observed from the samples of other related viruses, bacteria, allantoic fluid of normal chicken embryos, or the livers of uninfected ducks. Thirty clinical samples were subjected to detection by RT-LAMP, RT-PCR, and virus isolation, which obtained completely consistent, positive results. As a simple, rapid, and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of DHAV-1.

Complete genome comparison of duck hepatitis virus type 1 parental and attenuated strains.

Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5'UTR and 2 in 3'UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5'/3'UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone.