Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1.

BACKGROUND: DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay. METHODS: A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 µg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 µg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings. RESULTS: Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest. CONCLUSION: We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.

Molecular epidemiology and genetic diversity of duck hepatitis A virus type 3 in Shandong province of China, 2012-2014.

 The infections with duck hepatitis A virus type 3 (DHAV-3) become common in eastern Asia. To better understand the molecular evolution and genetic variation of DHAV-3, a total of 482 dead Cherry Valley duckling liver samples collected from Shandong province of China during 2012-2014 were tested, and the complete P1 coding sequences of 18 DHAV-3 strains were analyzed. The detection rate of DHAV-3 was 64.5% (311/482) in clinical liver samples and 73.0% (92/126) in duckling flocks. The P1 genes of the 18 DHAV-3 isolates shared 91.9%-99.0% nucleotide similarity and 95.2%-100% amino acid similarity with those of the other 26 reference strains. Based on the P1 and VP1 gene sequences, phylogenetic analysis results indicated that the genotyping of DHAV-3 strains presented a distinct geographical distribution. Except B63 strain, all Chinese strains isolated from different host species (duck or goose) at different time were classed into the CH genotype. All Korean and Vietnamese strains belonged to the KV genotype, and all the Korean strains were clustered into KV1 subgenotype, while B63 strain and the Vietnamese strains from different host species (duck or goose) were clustered into KV2 subgenotype. Ten variable amino acid residues were highly conserved within genotypes or subgenotypes in the VP0, VP3 and VP1, respectively, which were possibly the geographic molecular markers of DHAV-3. To the best of our knowledge, this is the first study about the genetic variation of the P1 gene of different DHAV-3 strains, which will be helpful for understanding of the molecular epidemiology of DHAV-3.

Circulation and in vivo distribution of duck hepatitis A virus types 1 and 3 in infected ducklings.

The circulation of duck hepatitis A virus types 1 (DHAV-1) and 3 (DHAV-3) in Southeast Asia has resulted in a continuously changing epidemiological scenario. In this study, a duplex real-time PCR assay for simultaneous quantitative detection of DHAV-1 and DHAV-3 was established, and 200 liver samples from dead ducklings collected from 31 different flocks in Shandong province, China, were tested. Fifty-eight (29.0 %) samples from 13 flocks were positive for DHAV-1 single infection, 113 (56.5 %) samples from 13 other flocks were positive for DHAV-3 single infection, and 24 samples (12.0 %) from four flocks were positive for both viruses. DHAV-1 and DHAV-3 were detected with high viral loads in all of the organs tested (liver, spleen, pancreas, kidney, heart, thymus, bursa of Fabricius and brain). No significant difference in DHAV-1 and DHAV-3 viral loads was found between singly infected and coinfected samples, and there was no correlation between the viral loads of the two viruses and the age of dead ducklings. To the best of our knowledge, this is the first report about the in vivo distribution of DHAV-1 and DHAV-3 in clinically infected ducklings.

The prevalence of duck hepatitis A virus types 1 and 3 on Korean duck farms.

This study reports the prevalence of duck hepatitis A virus (DHAV) types 1 and 3 on Korean duck farms. By RT-nested PCR assays specific for DHAV-1 or DHAV-3, DHAV-1 was detected in 9 of 157 liver samples (5.7 %) from 2 of 30 farms (6.7 %), and DHAV-3 was positive in 104 of 157 liver samples (66.2 %) from 23 of 30 farms (76.7 %). Dual infections with DHAV-1 and DHAV-3 were detected in 23 of 157 samples (14.6 %) from 5 of 30 farms (16.7 %). The data indicate that DHAV-3 infections are prevalent and that DHAV-1 reemerged in Korea, resulting in dual infections on several farms. Our data will help to establish a vaccination policy against DHAV-1 and DHAV-3 in Korea.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

OBJECTIVE: To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. METHODS: We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. RESULTS: DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. CONCLUSION: The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

Molecular cloning and expression analysis of ferritin, heavy polypeptide 1 gene from duck (Anas platyrhynchos).

H-ferritin is a core subunit of the iron storage protein ferritin, and is related to the pathogenesis of malignant diseases. A differential expressed sequence tag of the ferritin, heavy polypeptide 1 gene (FTH1) was obtained from our previously constructed suppression subtractive cDNA library from 3-day-old ducklings challenged with duck hepatitis virus type I (DHV-1). The expression and function of FTH1 in immune defense against infection remains largely unknown in ducks. In this study, the full-length duFTH1 cDNA was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. It consisted of 153 basepairs (bp) 5'untranslated region (UTR), 183 bp 3'UTR, and 546 bp open reading frame that encodes a single protein of 181 amino acid residues. duFTH1 shares high similarity with FTH1 genes from other vertebrates. The amino acid sequence possesses the conserved domain of typical ferritin H subunits, including seven metal ligands in the ferroxidase center, one iron binding region signature, and a potential bio-mineralization residue (Thy(29)). Moreover, in agreement with a previously reported ferritin H subunit, we identified an iron response element in the 5'UTR. RT-PCR analyses revealed duFTH1 mRNA is widely expressed in various tissues. Real-time quantitative polymerase chain reaction analyses suggested that duFTH1 mRNA is significantly up-regulated in the liver after DHV-1 injection or polyriboinosinic polyribocytidylic acid (polyI:C) treatment, reaching a peak 4 h post-infection, and dropping progressively and returning to normal after 24 h. Our findings suggest that duFTH1 functions as an iron chelating protein subunit in duck and contributes to the innate immune responses against viral infections.

Apoptosis induction in duck tissues during duck hepatitis A virus type 1 infection.

To investigate the role of apoptosis in duck viral hepatitis pathogenesis, 4- and 21-d-old ducks were inoculated with duck hepatitis A virus serotype 1 and killed at 2, 6, 12, 24, and 48 h postinfection. TdT-mediated dUTP nick-end labeling was used to detect apoptosis cells. Expression profiles of apoptosis-related genes including caspase-3, -8, -9, and Bcl-2 in spleen, bursa of Fabricius, liver, and the quantity of virus in blood were examined using real-time PCR. The TdT-mediated dUTP nick-end labeling analysis indicated there was a significant difference of apoptotic cells between treatments and controls. The same difference also appeared in virus amount variation in blood during infection. Gene expression analysis revealed that the apoptosis-related gene expression profile was different in the 2 groups, and also different between various organs. This study suggested that apoptosis may play an important role in duck hepatitis A virus serotype 1 infection, and apoptosis suppression might facilitate virus multiplication, resulting in the highest virus concentration in the host.

[Molecular characteristic of duck hepatitis A virus type 1 causing pancreatitis ].

[OBJECTIVE] We studied the molecular characteristics of the full-length genome of duck hepatitis A virus type 1 causing pancreatitis in Muscovy ducklings. [METHODS] We determined the entire genomic sequence of duck hepatitis A virus type 1 strain MPZJ1206 using reverse transcription polymerase chain reaction assay and analyzed the bioinformatics of the viral genome sequence. [ RESULTS] The genome length of strain MPZJ1206 comprised 7703 bases, with a G + C content of 43.05%. The genome of MPZJ1206 contains a single, long open reading frame encoding a polypeptide of 2249 amino acids, with a genomic orgariization similar to those of other isolates of duck hepatitis A virus type 1. MPZJ1206 is identical with previously isolates by 93. 5% - 99. 6% in nucleotide sequence and 97. 9% - 99. 6% in amino acid sequence and shares genetic distance no more than 7%. Phylogenetic analysis based on genome sequence indicates that MPZJ1206 shares a close genetic relationship with two strains isolated in 2011. [CONCLUSION] Although pathotype caused by MPZJ1206 strain is significantly distinct from those induced by classical isolates of duck hepatitis A virus type 1, the genome of MPZJ1206 shares high homology with those of previous isolates. The change of pathotype may result from an alteration in viral tissue tropism of MPZJ1206.

A rapid simultaneous detection of duck hepatitis A virus 3 and novel duck reovirus based on RPA CRISPR Cas12a/Cas13a.

The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 100 copy/µL, while DRC-LFA achieved limit of 101 copies/µL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection.

Complete genome sequence of a novel duck hepatitis A virus discovered in southern China.

We report here the complete genomic sequence of a novel duck hepatitis A virus (DHAV) isolated from mixed infections with DHAV type 1 (DHAV-1) and DHAV-3 in ducklings in Southern China. The whole nucleotide sequence had the highest homology with the sequence of DHAV-3 (GenBank accession number DQ812093) (96.2%). To our knowledge, this is the first report of gene rearrangement between DHAV-1 and DHAV-3, and it will help to understand the epidemiology and molecular characteristics of duck hepatitis A virus in Southern China.

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.

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.

Establishment of Duplex SYBR Green I-based Real-time PCR Assay for Simultaneous Detection of Duck Hepatitis A Virus-1 and Duck Astrovirus-3.

Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR® Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 101 copies/µl and 3.78 × 101 copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.

Artículo regular­Establecimiento de un ensayo dúplex de PCR en tiempo real basado en SYBR Green I para la detección simultánea del virus de la hepatitis A del pato-1 y del astrovirus del pato tipo 3. La hepatitis viral del pato (DVH) afecta principalmente a los patitos menores de 1 mes de edad, causa necrosis hepática, agrandamiento y hemorragia, y es altamente letal, lo que pone en grave peligro la industria del pato. La prevalencia del virus de la hepatitis A del pato (DHAV-1) y del astrovirus del pato tipo 3 (DAstV-3) está aumentando y la coinfección es común. Además, las características clínicas similares de las infecciones por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3 así como la alta frecuencia de coinfección dificultan el diagnóstico. En este estudio, para establecer un método para la detección rápida y simultánea por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3, se diseñaron dos pares de iniciadores específicos según sus regiones génicas conservadas. Se estableció con éxito un ensayo cuantitativo de PCR basado en SYBR® Green I que pudo detectar rápida y diferencialmente los dos virus. Además, el ensayo es muy específico y no muestró reacción cruzada con otros virus comunes. El límite de detección del método fue de 7.34 × 101 copias/µl y de 3.78 × 101 copias/µl para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3, respectivamente, lo que indica una alta sensibilidad. Se analizaron un total de 34 muestras clínicas utilizando el método establecido; las tasas positivas para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3 fueron del 14.71% y 8.82%, respectivamente y la de coinfección fue del 2.94% (1/34), que fue mejor que la obtenida con el método de PCR convencional. En resumen, el ensayo cuantitativo de PCR basado en SYBR Green I establecido en este estudio tiene alta especificidad, buena sensibilidad y precisión, alta viabilidad y es rápido. Por lo tanto, puede ser una herramienta poderosa para la detección de coinfecciones con el virus de la hepatitis A del pato y astrovirus del pato tipo 3 y para futuros estudios epidemiológicos.

First Report of Duck Hepatitis A Virus 3 from Duckling Flocks of Egypt.

Duck hepatitis A viruses (DHAV-1, DHAV-2, and DHAV-3) are the predominant causes of duck virus hepatitis (DVH), a disease of ducklings that leads to massive morbidities, mortalities, and economic losses. As a duck-producing country, Egypt suffered lately from several attacks of DVH, despite the regular vaccination of birds. Between Spring 2016 and Summer 2018, 54 duckling flocks in the Sharkia province of Egypt were tested using the reverse-transcription PCR (RT-PCR) based on the DHAV-3D targeting primers. Of them, 27.8% (15/54) were positive. Upon retesting of positive samples using RT-PCR and duck hepatitis A virus (DHAV)-3 VP1-based primers, 33.3% (5/15) contained DHAV-3 RNA. For further analysis at the molecular level, the VP1 and the 3D genes were sequenced using the same primer sets used earlier. The phylogenetic trees confirmed that study sequences belonged to DHAV-3. However, they were displayed as a separate cluster following a geographically dependent distribution. They were also completely unrelated to the Egyptian DHAV-1-based vaccine. This was further confirmed by low nucleotide and amino acid identities in relation to this vaccine. In addition, the VP1 and 3D genes had the same phylogenetic topography. The study VP1 sequences had three unique amino acid substitutions (L59, V208 only in one strain, and C219). As far as we know, this is the first report on DHAV-3 outside Asia, particularly in Egypt. Accordingly, the vaccination strategy against DHAV should be quickly updated to avoid further dissemination of the virus. The epidemiology, pathogenicity, and evolution of DHAV-3 should be carefully monitored in Egypt.

Development and application of a reverse transcriptase polymerase chain reaction to detect Chinese isolates of duck hepatitis virus type 1.

We developed a reverse transcriptase polymerase chain reaction (RT-PCR) method for the detection of duck hepatitis virus type 1 (DHV-1) in the tissues of infected and clinically affected ducks and in chick and duck embryos. We found the assay to be effective in detecting the virus in China, where it is being used in studies on the epidemiology of the disease. We applied this simple and rapid diagnostic method to the detection of DHV isolates grown in chick and duck embryos and in tissues obtained from infected birds. The assay also proved useful for the differentiation of DVH from the duck plague virus (DPV), muscovy parvovirus (MPV), gosling parvovirus (GPV), avian influenza virus (AIV/H5N1), Pasteurella multocida (PA/5:A), Riemerella anatipestifer (RA/serotype 1), and Salmonella enteritidis (SE). The limit of the sensitivity of this method for the detection of DHV-1 RNA was 3 pg/10 microl. As compared to ELISA and virus isolation, the rate of agreement for the detection of experimentally infected livers was 100%; moreover, the RT-PCR method was also capable of detecting DHV-1 RNA from the livers that had been infected and stored at -20 degrees C for 22 years; in contrast, ELISA and virus isolation method could only detect DHV-1 from the livers that had been infected and stored at -20 degrees C for 13 and 11 years, respectively. The rate of positivity in 185 clinically suspected diseased livers subjected to detection by RT-PCR, ELISA, and virus isolation was 89.2%, 69.2%, and 55.7%, respectively. These results indicated that the RT-PCR approach is rapid, sensitive, and reliable for the detection and differentiation of DHV-1 from the other clinical samples and suspected isolates.

Detection of chicken astrovirus by reverse transcriptase-polymerase chain reaction.

The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting chicken astroviruses (CAstV) is described. Primers, which amplified a fragment of 510 base pairs, were located in conserved regions within the ORF 1b (RNA polymerase) gene. The limit of detection of the test was estimated to be approximately 60 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative CAstV samples, some of which were not detected by a previously described RT-PCR test for detecting CAstV, but other avian astroviruses including avian nephritis virus and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples and swabs from UK and German broiler flocks with growth problems, CAstVs were detected by RT-PCR in 50/52 (96%) samples. CAstVs were detected in between 30% and 72.5% pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performances. Whereas all day 0 samples were CAstV-negative, high detection rates were observed when the surveyed birds were aged 4, 5 and 7 days. Based on partial ORF 1b sequences, a phylogenetic analysis of 20 CAstVs indicated the existence of two groups. One group comprised four CAstV isolates, including FP3 and 11672, and two field CAstVs, which shared >94% nucleotide identity. The remaining 14 CAstVs, comprising the first characterized CAstV and 612 isolates and 12 field CAstVs, shared 85% to 99% nucleotide identity and displayed 76% to 79% nucleotide identity with the 11672-like and FP3-like CAstVs.

An investigation of duck circovirus and co-infection in Cherry Valley ducks in Shandong Province, China.

The co-infection of duck circovirus (DuCV) with Riemerella anatipestifer (RA) or/and Escherichia coli (E. coli) or/and duck hepatitis virus I (DHV-I) in Cherry Valley ducks in China's Shandong Province was investigated by using polymerase-chain-reaction (PCR)-based methods. For this study, 742 ducks sampled at random from 70 duck farms during 2006-2007 were examined using PCR and dot-blot hybridisation (DBH) tests. Overall the DuCV infection rate was 33.29%. Compared with those at 2 weeks of age, the ducks at 3-4 weeks of age were more susceptible to DuCV infection. Compared with the DuCV-negative ones, the DuCV-positive ducks had a higher rate of infection by DHV-I (25.5% vs. 7.475%), RA (23.48% vs. 8.28%) and E. coli (16.19% vs. 4.85%). This investigation shows that DuCV infection is common in Cherry Valley ducks on some farms in Shandong Province.

Development and evaluation of reverse transcription-insulated isothermal PCR assay to detect duck hepatitis A virus type A in liver samples using the POCKITTM system.

Duck hepatitis A virus (DHAV) infection is characterized by severe hepatitis. In recent years, DHAV-A has become widespread in Asia and has led to economic losses. Conventional methods of DHAV-A detection must often be performed in the laboratory with inconvenience equipment. We have developed a rapid reverse transcription insulated isothermal (RT-iiPCR) technique for the on-site detection of DHAV-A based on the POCKITTM system in a convenient minitype device. We optimized the PCR primers and probes for the amplification of the DHAV-A 3C/3D genes, and successfully amplified a specific fragment of DHAV-A, but no fragment from 18 other duck pathogens. The limit of detection for viral RNA was 49 copies per reaction, and the sensitivity and specificity were each 100% in the analysis of 60 liver samples. By comparison, the sensitivities of RT-iiPCR was comparable in sensitivity to existing rRT-PCR. Furthermore, the RT-iiPCR results were 98.3% in agreement with those of the rRT-PCR, with a kappa value of 0.938. In conclusion, this new method not only offers a higher sensitivity and specificity than existing techniques, but also time-saving and better suited to field diagnoses because device is portable.

A reverse transcription-insulated isothermal PCR assay for the detection of duck hepatitis A virus type 3 based on the POCKIT™ system.

Duck hepatitis A virus type 3 (DHAV-3) infection is characterized by severe hepatitis. In recent years, DHAV-3 has become widespread in Asia and has led to economic losses. Conventional methods for DHAV-3 detection usually depend on the use of larger equipment that is not portable and is not fit for on-site diagnoses. In this study, a rapid reverse transcription insulated isothermal (RT-iiPCR) technique was developed for the on-site detection of DHAV-3 based on the POCKIT™ system in a convenient device. The concentration of primer pairs and probes were optimized for amplification of the DHAV-3 VP3 gene of DHAV-3, with no amplification of 12 other duck pathogens. The detection limit of viral RNA was 3.85 × 101 copies/µL, and the analytical sensitivity and specificity levels were both 100% in the detection of 40 liver samples. Furthermore, 97.5% of the RT-iiPCR results were in agreement with those of rRT-PCR, with a kappa value of 0.93. This method is time-saving and better suited to field diagnoses because of its portable device.

Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3.

BACKGROUND: The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. METHODS AND RESULTS: Based on multiple 5' noncoding region (5'-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. CONCLUSIONS AND SIGNIFICANCE: This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening.