HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

mRNA and miRNA expression profiles reveal the potential roles of RLRs signaling pathway and mitophagy in duck hepatitis A virus type 1 infection.

Duck hepatitis A virus 1 (DHAV-1) is the primary cause of duck viral hepatitis, leading to sudden mortality in ducklings and significant economic losses in the duck industry. However, little is known about how DHAV-1 affects duckling liver at the molecular level. We conducted an analysis comparing the expression patterns of mRNAs and miRNAs in DHAV-1-infected duckling livers to understand the underlying mechanisms and dynamic changes. We identified 6,818 differentially expressed mRNAs (DEGs) and 144 differentially expressed microRNAs (DEMs) during DHAV-1 infection. Functional enrichment analysis of DEGs and miRNA target genes using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed their potential involvement in innate antiviral immunity, mitophagy, and pyroptosis. We constructed coexpression networks of mRNA-miRNA interactions and confirmed key DEMs (novel-mir333, novel-mir288, novel-mir197, and novel-mir71) using RT-qPCR. Further investigation demonstrated that DHAV-1 activates the RLRs signaling pathway, disrupts mitophagy, and induces pyroptosis. In conclusion, DHAV-1-induced antiviral immunity is closely linked to mitophagy, suggesting it could be a promising therapeutic target.

Whole-transcriptome sequencing revealed the role of noncoding RNAs in susceptibility and resistance of Pekin ducks to DHAV-3.

As the most prevalent pathogen of duck viral hepatitis (DVH), duck hepatitis A virus genotype 3 (DHAV-3) has caused huge economic losses to the duck industry in China. Herein, we obtained whole-transcriptome sequencing data of susceptible (S) and resistant (R) Pekin duckling samples at 0 h, 12 h, and 24 h after DHAV-3 infection. We found that DHAV-3 infection induces 5,396 differentially expressed genes (DEGs), 85 differentially expressed miRNAs (DEMs), and 727 differentially expressed lncRNAs (DELs) at 24 hpi in S vs. R ducks, those upregulated genes were enriched in inflammation and cell communications pathways and downregulated genes were related to metabolic processes. Upregulated genes showed high connectivity with the miR-33, miR-193, and miR-11591, and downregulated genes were mainly regulated by miR-2954, miR-125, and miR-146b. With the construction of lncRNA-miRNA-mRNA axis, we further identified a few aberrantly expressed lncRNAs (e.g., MSTRG.36194.1, MSTRG.50601.1, MSTRG.34328.7, and MSTRG.29445.1) that regulate expression of hub genes (e.g., THBD, CLIC2, IL8, ACOX2, GPHN, SMLR1, and HAO1) by sponging those highly connected miRNAs. Altogether, our findings defined a dual role of ncRNAs in immune and metabolic regulation during DHAV-3 infection, suggesting potential new targets for treating DHAV-3 infected ducks.

Duck hepatitis A virus type 1 infection induces hepatic metabolite and gut microbiota changes in ducklings.

Duck hepatitis A virus type 1 (DHAV-1) can cause severe liver damage in infected ducklings and is a fatal and contagious pathogen that endangers the Chinese duck industry. The objective of this study was to explore the correlation mechanism of liver metabolism-gut microbiota in DHAV-1 infection. Briefly, liquid chromatography-mass spectrometry and 16S rDNA sequencing combined with multivariate statistical analysis were used to evaluate the effects of DHAV-1 infection on liver metabolism, gut microbiota regulation, and other potential mechanisms in ducklings. In DHAV-1-infected ducklings at 72 h postinfection, changes were found in metabolites associated with key metabolic pathways such as lipid metabolism, sugar metabolism, and nucleotide metabolism, which participated in signaling networks and ultimately affecting the function of the liver. The abundance and composition of gut microbiota were also changed, and gut microbiota is significantly involved in lipid metabolism in the liver. The evident correlation between gut microbiota and liver metabolites indicates that DHAV-host gut microbiome interactions play important roles in the development of duck viral hepatitis (DVH).

The DHAV-1 protein VP1 interacts with PI3KC3 to induce autophagy through the PI3KC3 complex.

Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens responsible for death in ducklings. Autophagy is a catabolic process that maintains cellular homeostasis, and the PI3KC3 protein plays an important role in the initiation of autophagy. DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) but the molecular mechanism between it and autophagy has not been reported. First, we determined that DHAV-1 infection induces autophagy in DEFs and that autophagy induction is dependent on the integrity of viral proteins by infecting DEFs with UV-inactivated or heat-inactivated DHAV-1. Then, in experiments using the pharmacological autophagy inducer rapamycin and the autophagy inhibitor chloroquine, autophagy inhibition was shown to reduce intracellular and extracellular DHAV-1 genome copies and viral titres. These results suggest that autophagy activated by DHAV-1 infection in DEFs affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of the DHAV-1 structural proteins VP0, VP3, and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEFs but not the full autophagic flux. Finally, we found that VP1 promotes protein expression of PI3KC3 and Beclin1 by western blot experiments and that VP1 interacts with PI3KC3 by co-immunoprecipitation experiments; moreover, 3-MA-induced knockdown of PI3KC3 inhibited VP1 protein-induced autophagy in DEFs. In conclusion, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEFs.

NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

Evidence of possible vertical transmission of duck hepatitis A virus type 1 in ducks.

Duck hepatitis A virus (DHAV) causes a highly contagious and acute disease in ducklings younger than 3 weeks of age and spreads rapidly by horizontal transmission to all susceptible ducklings in the flock. To date, there is no evidence of vertical transmission of DHAV-1. In a previous study, we identified a novel DHAV type 1 (DHAV-1) isolate that could infect adult ducks and induce laying drop. In this study, 30 non-embryonated duck eggs and 60 17-day-old embryos were collected from three breeding duck flocks with egg drop syndrome caused by DHAV-1 in China, and 30 17-day-old embryos were randomly selected from the 60 embryos and allowed to hatch. DHAV-1 RNA was detected by RT-PCR in 10 of 30 non-embryonated eggs, 9 of 30 17-day-old embryos, 5 of 7 dead embryos and 5 of 23 newly hatched ducklings. Overall, 29 of 90 (32.2%) eggs and embryos were positive for DHAV-1. Three DHAV-1 strains were isolated from the dead duck embryos of the three breeding duck flocks, respectively. Pathogenicity studies showed that the three DHAV-1 isolates had median embryo lethal doses but were highly pathogenic to healthy ducklings. Compared with the DHAV reference strains, there were two specific amino acid mutation sites (F169 and S220 ) in VP1 of the three isolates. To the best of our knowledge, this is the first report that DHAV-1 is isolated from duck embryos. The findings provide evidence of possible vertical transmission of DHAV-1 from breeding ducks to ducklings.

The inhibitory effect of phosphorylated Codonopsis pilosula polysaccharide on autophagosomes formation contributes to the inhibition of duck hepatitis A virus replication.

Duck hepatitis A virus type 1 (DHAV) infection causes duck viral hepatitis and results in enormous loss to poultry farming industry. We reported that phosphorylated Codonopsis pilosula polysaccharide (pCPPS) inhibited DHAV genome replication. Here we further explored its underlying antiviral mechanisms. Autophagosomes formation is essential for the genome replication of picornaviruses. In this study, Western blot, confocal microscopy observation, and ELISA methods were performed to analyze polysaccharides' effects on autophagy by the in vitro and in vivo experiments. Results obtained from in vitro and in vivo experiments showed that Codonopsis pilosula polysaccharide did not play a role in regulating autophagy and had no therapeutic effects on infected ducklings. However, pCPPS treatment downregulated LC3-II expression level activated by DHAV and rapamycin, indicating the inhibition of autophagosomes formation. The interdiction of autophagosomes formation resulted in the inhibition of DHAV genome replication. Further study showed that pCPPS treatment reduced the concentration of phosphatidylinositol-3-phosphate (PI3P), an important component of membrane, in cells and serum, and consequently, autophagosomes formation was downregulated. In vivo experiments also verified the therapeutic effect of pCPPS. Phosphorylated Codonopsis pilosula polysaccharide treatment increased the infected ducklings' survival rate and alleviated hepatic injury. Our studies verified the effects of pCPPS against DHAV infection in duck embryo hepatocytes and ducklings and confirmed that phosphorylated modification enhanced the bioactivities of polysaccharides. The results also stated pCPPS's antiviral mechanisms, provided fundamental basis for the development of new anti-DHAV agents.

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.

Outbreaks of Duck Hepatitis A Virus in Egyptian Duckling Flocks.

During 2015, duck farms (n = 27) in Sharkia Province, Egypt, experienced several disease outbreaks leading to mortality and nervous manifestations. Upon necropsy, the affected ducklings showed liver lesions, such as hemorrhage or necrosis, suggestive of duck virus hepatitis (DVH). Reverse transcription-PCR (RT-PCR), on the basis of the 3D gene, found duck livers from 21 farms to be positive for duck hepatitis A virus serotype 1 (DHAV-1). All duck breeds (Pekin, Mallard, and Muscovy) were infected. The virus was isolated in embryonated chicken eggs, which showed embryonic mortality (40%-80%) within 5-7 days, stunting or dwarfing (69.6%), and necrotic liver foci (60.9%). The VP1 gene of 11 DHAV-1 strains was characterized by RT-PCR and Sanger sequencing. All study strains were clustered in a monophyletic branch within subclade B2 of Group 4 and were separated from the Egyptian vaccine strain. Several amino acid (aa) residues, such as V129, S142 (only in four strains), L181, G184, and K217, were related to virus attenuation. However, two aa residues (N193 and E205), found in virulent DHAV-1 strains, were also observed in our strains. This study confirms the circulation of DHAV-1 (subclade B2) in Lower Egypt and elucidates the phylogenetic characters of the VP1 genes, which will be useful in following the local trends of DHAV-1 infections. Further studies are indicated to determine the correlation between these mutations and the virulence of the Egyptian DHAV-1 isolates.

Brotes de virus de la hepatitis A del pato en parvadas de patitos en Egipto. Durante el año 2015, granjas de patos (n = 27) en la provincia de Sharkia en Egipto, experimentaron varios brotes de enfermedades que causaron mortalidad y manifestaciones nerviosas. Durante la necropsia, los patitos afectados mostraron lesiones hepáticas, como necrosis, hemorragia o ambas, sugestivas de la hepatitis viral del pato (con las siglas en inglés DVH). Un método de transcripción reversa y PCR (RT-PCR), sobre la base del gene 3D, encontró que los hígados de pato de 21 granjas eran positivos para el serotipo 1 del virus de la hepatitis A de pato (DHAV-1). Todas las razas de patos (Pekin, mallard y pato real) se infectaron. El virus se aisló en huevos de gallina embrionados, que mostraron una mortalidad embrionaria (40% -80%) en cinco a siete días, retraso del crecimiento o enanismo (69.6%) y focos hepáticos necróticos (60.9%). El gene VP1 de 11 cepas de DHAV-1 se caracterizó por RT-PCR y secuenciación por el método de Sanger. Todas las cepas del estudio se agruparon en una rama monofilética en el subclado B2 dentro del Grupo 4 y se separaron de la cepa de la vacuna egipcia. Varios residuos de aminoácidos, como V129, S142 (solo en cuatro cepas), L181, G184 y K217, se relacionaron con la atenuación del virus. Sin embargo, dos residuos de aminoácidos (N193 y E205), encontrados en cepas virulentas de DHAV-1, se observaron en las cepas descritas en este estudio. Este estudio confirma la circulación del virus de la hepatitis A del pato DHAV-1 (subclado B2) en el Bajo Egipto y aclara las características filogenéticas de los genes VP1, que serán útiles para seguir las tendencias locales de las infecciones por este virus. Se indican estudios adicionales para determinar la correlación entre estas mutaciones y la virulencia de los aislamientos egipcios del virus de la hepatitis A del pato.

Molecular cloning and mRNA expression of IFIT5 in tissues of ducklings infected with virulent duck hepatitis A virus type 3.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins strongly induced downstream of type I interferon signaling. The function of IFITs has been investigated extensively in mammals. IFIT5 is the sole protein in this family found in birds and little information is available about the function of avian IFIT5. In this study, duck IFIT5 was cloned from peripheral blood mononuclear cells. Multiple amino acid sequence alignment and phylogenetic analysis showed that duck IFIT5 is highly homologous to chicken IFIT5. Tissue specificity analysis demonstrated that duck IFIT5 was ubiquitously expressed in all examined tissues of five-day-old ducklings, with the highest expression levels in heart, followed by thymus, cerebrum, liver, and lung; kidney expressed the lowest. Quantitative real-time PCR (qRT-PCR) analysis revealed that duck IFIT5 expression rapidly increased both in vitro and in vivo after stimulation with polyinosinic:polycytidylic acid [poly (I:C)] and infection with virulent duck hepatitis A virus type 3 (DHAV-3), respectively. Altogether, these results indicate that the expression of duck IFIT5 is positively correlated with viral load and may play an important role in the immune response to DHAV-3 infection. This study lays a foundation for further research into the innate antiviral immune responses of ducklings.

Comparative liver transcriptome analysis in ducklings infected with duck hepatitis A virus 3 (DHAV-3) at 12 and 48 hours post-infection through RNA-seq.

Duck hepatitis A virus 3 (DHAV-3), the only member of the novel genus Avihepatovirus, in the family Picornaviridae, can cause significant economic losses for duck farms in China. Reports on the pathogenicity and the antiviral molecular mechanisms of the lethal DHAV-3 strain in ducklings are inadequate and remain poorly understood. We conducted global gene expression profiling and screened differentially expressed genes (DEG) of duckling liver tissues infected with lethal DHAV-3. There were 1643 DEG and 8979 DEG when compared with mock ducklings at 12 hours post-infection (hpi) and at 48 hpi, respectively. Gene pathway analysis of DEG highlighted mainly biological processes involved in metabolic pathways, host immune responses, and viral invasion. The results may provide valuable information for us to explore the pathogenicity of the virulent DHAV-3 strain and to improve our understanding of host-virus interactions.

Inhibition mechanisms of baicalin and its phospholipid complex against DHAV-1 replication.

Duck hepatitis A virus type 1 (DHAV-1) is a serious infectious virus of ducklings. Recent study showed baicalin (BA) and baicalin phospholipid complex (BAPC) possessed anti-DHAV-1 effect. However, the antiviral mechanism is not clear. Therefore, the aim of the present work is to study influences and mechanisms of BA and BAPC on DHAV-1. The effects of BA and BAPC on DHAV-1 replication were analyzed by CCK-8 and RT-qPCR methods. And the results showed BA inhibited the replication of DHAV-1, and BAPC was more effective. Then, the influences of BA and BAPC on DHAV-1 protein translation and RNA synthesis were detected by western blot and RT-qPCR. Both BA and BAPC inhibited the protein translation, and BAPC did better. Furthermore, BAPC also inhibited the RNA synthesis. Afterwards, DHAV-1 IRES activity, DHAV-1 3D protein stability, and cellular Hsp70 expression were studied to in-depth understand the inhibition effects of BA and BAPC on DHAV-1 replication. The results indicated BA and BAPC dropped the protein translation via suppressing DHAV-1 IRES activity. Additionally, BAPC dropped the RNA synthesis via reducing the 3D protein stability and inhibiting cellular Hsp70 expression.

RAW REHMANNIA RADIX POLYSACCHARIDE CAN EFFECTIVELY RELEASE PEROXIDATIVE INJURY INDUCED BY DUCK HEPATITIS A VIRUS.

BACKGROUND: Duck viral hepatitis (DVH), caused by duck hepatitis A virus (DHAV), is a fatal contagious infectious disease which spreads rapidly with high morbidity and high mortality, and there is no effective clinical drug against DVH. MATERIALS AND METHODS: Raw Rehmannia Radix Polysaccharide (RRRP), Lycii Fructus polysaccharides and Astragalus Radix polysaccharides were experimented in vitro and in vivo. Mortality rate, livers change, liver lesion scoring, peroxidative injury evaluation indexes in vitro and in vivo, and hepatic injury evaluation indexes of optimal one were detected and observed in this experiment. RESULTS: RRRP could reduce mortality with the protection rate about 20.0% compared with that of the viral control (VC) group, finding that RRRP was the most effective against DHAV. The average liver scoring of the VC, blank control (BC), RRRP groups were 3.5, 0, 2.1. Significant difference (P<0.05) appeared between any two groups, demonstrating that it can alleviate liver pathological change. RRRP could make the hepatic injury evaluation indexes similar to BC group while the levels of the VC group were higher than other two groups in general. The levels of SOD, GSH-Px, CAT of RRRP group showed significant higher than that of VC group while the levels of NOS and MDA showed the opposite tendency, thus, RRRP could release peroxidative injury. CONCLUSION: RRRP was the most effective against duck hepatitis A virus (DHAV). RRRP could reduce mortality, alleviate liver pathological change, down-regulate liver lesion score, release peroxidative injury and hepatic injury. The antiviral and peroxidative injury releasing activity of RRRP for DHAV provided a platform to test novel drug strategies for hepatitis A virus in human beings.

Comparative analysis of virus-host interactions caused by a virulent and an attenuated duck hepatitis A virus genotype 1.

Because of their better immunogenicity and the improved protection they afford, live attenuated vaccines derived from serial passaging in an abnormal host are widely used to protect humans or animals from certain pathogens. Here, we used a virulent and a chicken embryo-attenuated duck hepatitis A virus genotype 1 to compare the different regulated immune responses induced by viruses with differing virulence. In this study, the attenuated strains had lower protein expression levels than the virulent strains as identified by immunohistochemistry. This may be caused by apparent codon usage bias selected during passage. Furthermore, lower translation efficiency led to decreased viral replication, which is highly dependent on non-structural viral protein expression. Although the two strains had differing levels of virulence, both could induce strong innate immune responses and robust Tc or Th cell populations during the early stages of the immune response. However, due to fixed single nucleotide polymorphisms (SNPs) selected by passage, the virulent and attenuated strains may induce differing immune responses, with stronger Tc cell immunity induced by the attenuated strain in the spleen and thymus and stronger Tc cell immunity induced by the virulent strain in the liver, lung, bursa of Fabricius and Harderian gland. Four immune related genes (RIG-1, MDA5, IFN-ß, and IL-6) were highly differentially expressed in the Harderian gland, bursa of Fabricius and thymus. This study has provided further information about differences in virus-host interactions between duck hepatitis A viruses of differing virulence.

Effects of Chrysanthemum indicum polysaccharide and its phosphate on anti-duck hepatitis a virus and alleviating hepatic injury.

To explore new effective anti-duck hepatitis A virus drugs, Chrysanthemum indicum polysaccharide (CIPS) was phosphorylation modified using STMP-STPP method, and phosphorylated Chrysanthemum indicum polysaccharide (pCIPS) was obtained. Characteristic absorption peaks were observed in pCIPS using IR spectrum, suggested that CIPS was successfully modified. In addition, field emission scanning electron micro-scope (FE-SEM) was used to observe the polysaccharides' surface features. In vitro, we found that the survival rate of DHAV-infected hepatocytes increased after the two drugs treatment, indicated that the two drugs possess good anti-DHAV activity. The results of real-time PCR showed that pCIPS inhibited the virus gene replication more effectively than CIPS. Reed-Muench assay was used to observe the changes of the virulence, and the expression level of IFN-ß was observed to verify the changes of virulence. In vivo experiment, the blood virus content reduced after CIPS and pCIPS treatment. To evaluate the ducklings' hepatic injury, the serum ALT, AST, TP and ALB levels were detected. Results showed that both CIPS and pCIPS could alleviate the hepatic injury of ducklings infected DHAV, especially for pCIPS. All the results above mentioned demonstrated that the anti-DHAV activity of CIPS was enhanced after phosphorylation modification.

Phosphorylated Codonopsis pilosula polysaccharide could inhibit the virulence of duck hepatitis A virus compared with Codonopsis pilosula polysaccharide.

To screen effective anti-duck hepatitis A virus (DHAV) drugs, we applied STMP-STPP method to prepare phosphorylated Codonopsis pilosula polysaccharide (pCPPS), the phosphorylation-modified product of Codonopsis pilosula polysaccharide (CPPS). The IR spectrum and field emission scanning electron microscope (FE-SEM) were subsequently used to analyze the structure of pCPPS. Several tests were conducted to compare the anti-DHAV activities of CPPS and pCPPS. The MTT method was used to compare the effect of the drugs on DHAV-infected duck embryonic hepatocytes (DEHs), and the Reed-Muench assay was employed to observe changes in the virulence of DHAV. We also applied real-time PCR to examine the relationship between virus replication and the expression of IFN-ß. The results indicated that CPPS could not inhibit the replication of DHAV. In contrast, pCPPS increased the virus TCID50, inhibited viral replication and, accordingly, increased the survival rate of DEHs infected with DHAV. Because DHAV induced the expression of IFN-ß, and the IFN-ß expression level was positively associated with the number of DHAV, the reduction of IFN-ß expression levels after pCPPS treatment demonstrated a decrease in the number of virus particles. These results indicated that pCPPS, which reduces the number of DHAV, was more effective than CPPS in anti-DHAV activity.

Evidence of VP1 of duck hepatitis A type 1 virus as a target of neutralizing antibodies and involving receptor-binding activity.

The VP1 protein of the foot-and-mouth disease virus (FMDV) is a major target of neutralizing antibodies and is responsible for viral attachment to permissive cells via an RGD motif. VP1 of duck hepatitis A type 1 virus (DHAV-1) does not contain any RGD motif. To investigate the antibody and receptor-binding properties of DHAV-1, VP1 has been expressed as a His fusion protein (His-VP1) in baculovirus system. Sera against His-VP1 raised in rabbits effectively neutralized DHAV-1 infection in vitro and in vivo. A flow cytometry binding assay indicated that His-VP1 bound to duck embryo fibroblast cell (DEF) surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified DHAV-1 virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated His-VP1 in receptor binding. Importantly, anti-His-VP1 antibodies inhibited the binding of DHAV-1 virions to DEF cells, suggesting that these antibodies exert their neutralizing activity by blocking viral attachment. Similar to the counterpart of FMDV, DHAV-1 VP1 appears to be involved in receptor binding activity and a target of protective antibodies. This study confirms the potential of recombinant VP1 protein to serve as vaccine and diagnostic reagents for the control of DHAV-1 infections.

The 2A2 protein of Duck hepatitis A virus type 1 induces apoptosis in primary cell culture.

Duck hepatitis A virus type 1, (DHAV-1) 2A2pro, is one of the most highly conserved viral proteins within the DHAV serotypes. However, its effect on host cells is unclear. We predicted that DHAV-1 2A2pro was a GTPase-like protein based on the results of multiple sequence alignment and homologous modeling analysis. Upon transfection of a recombinant plasmid expressing DHAV-1 2A2, cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-sized DNA ladder, and positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining; hence, cell death has the characteristics of apoptosis. By staining cells with fluorescein Annexin V-FITC and PI, it is possible to distinguish and quantitatively analyze nonapoptotic cells, early apoptotic cells, late apoptotic/necrotic cells, and dead cells through flow cytometry and fluorescence microscopy. The percentage of apoptotic cells gradually increased and reached a maximum after 48 h of transfection. In conclusion, apoptosis induced by this GTPase-like protein may contribute to DHAV-1 pathogenesis.

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes.

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold were observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies.