Anatid Herpesvirus Research Articles

Metaviromic reveals the dynamics and diversity of the virosphere in wastewater samples from Natal, Brazil

The COVID-19 pandemic underscored the significance of omics technology and Wastewater-Based Epidemiology for epidemic preparedness. This study investigates the virosphere in wastewater samples from Natal (Brazil), aiming to understand its structure, relationships, and potential. Metaviromic analysis was used on DNA and RNA from weekly samples collected over a year (June/2021 to May/2022) from three wastewater treatment plants. The virosphere showed stability, particularly in viruses infecting microorganisms and plants. However, an alternation of representatives of viruses that infect animals has been observed. Among the most abundant viruses infecting microorganisms are genera associated with the bacterial genera Escherichia, Pseudomonas, and Caulobacte. Regarding the viruses infecting plants, Sobemovirus and Tobamovirus are the most abundant genera. Odontoglossum ringspot virus was identified as a possible RNA virus biomarker. Among DNA viruses infecting animals, genera Bocaparvovirus and Mastadenovirus are the most prevalent. Intriguingly, some Poxviridae family members were observed in the samples. Co-occurrence network analysis identified potential biomarkers like Volepox virus, Anatid herpesvirus 1, and Caviid herpesvirus 2. Among RNA viruses affecting animals, Mamastrovirus, Rotavirus, and Norovirus genera were the most abundant pathogens. Furthermore, members of the Coronaviridae family exhibited a high degree of centrality values in the co-occurrence network, even connecting with unclassified viruses. The study emphasizes the importance of research in understanding the roles of unclassified viruses. In addition, we observed an association between Coronaviridae reads, rainfall, and the number of reported COVID-19 cases. Our study highlights the diversity and complexity of the viral community in wastewater and the need for research to understand better the ecological roles unclassified viruses play. Such advances will significantly contribute to our preparedness and response to future viral threats. Furthermore, our study contributes to knowledge of virosphere dynamics, offering insights that can contribute to the direction of future public health policies and interventions.

An alpha-herpesvirus employs host HEXIM1 to promote viral transcription.

Although it is widely accepted that herpesviruses utilize host RNA polymerase II (RNAPII) to transcribe viral genes, the mechanism of utilization varies significantly among herpesviruses. With the exception of herpes simplex virus 1 (HSV-1) in alpha-herpesviruses, the mechanism by which RNAPII transcribes viral genes in the remaining alpha-herpesviruses has not been reported. In this study, we investigated the transcriptional mechanism of an avian alpha-herpesvirus, Anatid herpesvirus 1 (AnHV-1). We discovered for the first time that hexamethylene-bis-acetamide-inducing protein 1 (HEXIM1), a major inhibitor of positive elongation factor B (P-TEFb), was significantly upregulated during AnHV-1 infection, and its expression was dynamically regulated throughout the progression of the disease. However, the expression level of HEXIM1 remained stable before and after HSV-1 infection. Excessive HEXIM1 assists AnHV-1 in progeny virus production, gene expression, and RNA polymerase II recruitment by promoting the formation of more inactive P-TEFb and the loss of RNAPII S2 phosphorylation. Conversely, the expression of some host survival-related genes, such as SOX8, CDK1, MYC, and ID2, was suppressed by HEXIM1 overexpression. Further investigation revealed that the C-terminus of the AnHV-1 US1 gene is responsible for the upregulation of HEXIM1 by activating its promoter but not by interacting with P-TEFb, which is the mechanism adopted by its homologs, HSV-1 ICP22. Additionally, the virus proliferation deficiency caused by US1 deletion during the early infection stage could be partially rescued by HEXIM1 overexpression, suggesting that HEXIM1 is responsible for AnHV-1 gaining transcription advantages when competing with cells. Taken together, this study revealed a novel HEXIM1-dependent AnHV-1 transcription mechanism, which has not been previously reported in herpesvirus or even DNA virus studies.IMPORTANCEHexamethylene-bis-acetamide-inducing protein 1 (HEXIM1) has been identified as an inhibitor of positive transcriptional elongation factor b associated with cancer, AIDS, myocardial hypertrophy, and inflammation. Surprisingly, no previous reports have explored the role of HEXIM1 in herpesvirus transcription. This study reveals a mechanism distinct from the currently known herpesvirus utilization of RNA polymerase II, highlighting the dependence on high HEXIM1 expression, which may be a previously unrecognized facet of the host shutoff manifested by many DNA viruses. Moreover, this discovery expands the significance of HEXIM1 in pathogen infection. It raises intriguing questions about whether other herpesviruses employ similar mechanisms to manipulate HEXIM1 and if this molecular target can be exploited to limit productive replication. Thus, this discovery not only contributes to our understanding of herpesvirus infection regulation but also holds implications for broader research on other herpesviruses, even DNA viruses.

Duck plague virus pUL15 performs a nonspecial cleavage activity through its C terminal nuclease domain in vitro

Duck plague virus (DPV), also known as anatid herpesvirus, is a double-stranded DNA virus and a member of α herpesvirus. DPV pUL15 is a homolog of herpes simplex virus 1 (HSV-1) pUL15, a terminase large subunit, and plays a key role in the cleavage and packaging of the viral concatemeric genome. However, the sequence similarity between DPV pUL15 and its homologs is low, and it is not sure if DPV pUL15 has the potential to cleave the concatemeric genome as same as its homologs. Here, we expressed the C terminal domain of DPV pUL15 to explore the nuclease function of DPV pUL15. The main results showed that (Ⅰ) DPV pUL15 C-terminal domain possesses nonspecific nuclease activity and lacks the DNA binding ability. (Ⅱ) DPV pUL15 nuclease activity needs to coordinate with divalent metal ions and tends to be more active at high temperatures. (Ⅲ) Even though the structure of DPV pUL15 nuclease domain is relatively conserved, the mutations of conserved amino acids on the nuclease domain do not significantly inhibit the nuclease activity.

Molecular Characterization of Duck Plague Virus for Determination of TCID<sub>50</sub>

Duck plague is an enveloped DNA virus that belongs to the Anatid Herpes Virus; the Herpesviridae family is an acute and highly infectious duck, geese, and swan disease that causes tremendous economic losses of duck rearing in Bangladesh and other duck rearing countries. Therefore, we decided to isolate duck plague virus from recent fields’ outbreaks area and performed molecular detection and phylogenetic analysis to find out the similarities between our findings and other isolates around the world. Visceral organs of 13 suspected ducks from recent outbreaks area were collected by post-mortem examination for inoculum preparation. Several passages were performed to harvest into 9-11 old embryonated eggs Chorioallantoic membrane (CAM) route and duck embryo fibroblast (DEF) primary cell culture. DNA polymerase (446bp) and DNA polymerase (UL, 602bp) genes were used for molecular detection by Polymerase chain reaction (PCR). Pathogenicity was done with duckling and TCID50 on DEF. Molecular characterization was performed from extracted DNA of duckling and 2 Positive PCR products were partially sequenced for phylogenetic analysis of their origin and nucleotide variations. Sequenced data was analyzed to reveal genetic relationships among constructed phylogenetic tree for understanding potential transmission with origin of virus and data was then submitted to gene bank and got accession number for DPV-BR1-MN937272 and DPV-BR-2-MN937273. Among 13 samples, 4(30.77%) were found positive by PCR using DNA polymerase at 446 bp and UL at 602 bp gene. Chorioallantoic membrane (CAM) was observed hemorrhagic after 72 days and duck embryo fibroblast (DEF) become round as showed cytopathic characteristics after 48h of infection .Duckling showed that isolated virus was highly pathogenic as characteristics signs of post-mortem examination. Therefore, this has found that recent isolates have similarity with Bangladesh, India and China isolates. Moreover, TCID50 has confirmed the isolates have accepted titer to be a vaccine strain.
 Res. Agric., Livest. Fish.8(1): 125-133, April 2021

Adaptation and characterization of Anatid herpesvirus 1 in different permissible cell lines

Duck viral enteritis is an acute, contagious infection of Anatidae family members. The disease is caused by Anatid herpesvirus 1 (AnHV-1). The infection of AnHV-1 is controlled by vaccination to the flock with chick embryo adapted attenuated vaccine in developed countries. However, its economic impact in developing countries is substantial and there is a need to understand the cell culture spectrum of the virus to produce its vaccine on a mass scale. In the present study, the permissivity of AnHV-1 for different cells was analyzed. The AnHV-1 showed enhanced replication following its serial passage in CEF, DF-1, Vero, MDCK, and QT-35 cells. The characteristic cytopathic effect (CPE) of rounding and clumping of cells were observed in CEF, DF-1, Vero, and QT-35 cell lines. The infectivity and viral replication were highest in CEF, DF-1, Vero, and QT-35 cells. In contrast, the results suggested that MDCK cells are less permissive for AnHV-1 infection with negligible CPE and reduced viral replication. Heterologous cell culture systems other than chicken embryo fibroblasts to adapted live vaccine viruses will provide a system devoid of other avian infectious agents. Moreover, it can be used for the propagation and cultivation of AnHV-1 vaccine strain for developing cell culture-based vaccines with high titer and could be an economical alternative for the existing options.

Role of cholesterol in anatid herpesvirus 1 infections in vitro

Cholesterol is an essential constituent of the cell membrane that modulates several physiological events, including virus entry into the host. Duck virus enteritis (DVE) is a contagious and lethal infection that attacks several species of waterfowl. Anatid herpesvirus 1 (AnHV-1) is the causative agent of duck viral enteritis and classified under subfamily Alphaherpesvirinae. In this study, the effect of cholesterol depletion in both host cell membrane and viral envelope on the infectivity of AnHV-1 was explored. Cholesterol depletion of chicken embryo fibroblast cells (DF-1) by methyl-β-cyclodextrin (MβCD) inhibited the infectivity of AnHV-1. This inhibitory effect was moderately reversed by the exogenous replenishment of cholesterol in the cells. Furthermore, the inhibition of endogenous cholesterol synthesis by a statin drug also inhibited the infectivity of AnHV-1. Presumably, the removal of cholesterol from AnHV-1 envelope might be disrupting the viral envelope resulting in its diminished infectivity. The presence of a relatively hydrophobic cavity in MβCD can be used to extract cholesterol from the cell membrane. Loss of infectivity of the virus might be due to the effects of MβCD mediated cholesterol depletion from the cell membrane. The results implicate that the cell membrane cholesterol is vital for the infectivity of AnHV-1 in DF-1 cells, and its depletion from virion curtails the infectivity by destabilizing the envelope.

Active virological surveillance in backyard ducks in Bangladesh: detection of avian influenza and gammacoronaviruses

ABSTRACT Domestic waterfowl play an important role in the perpetuation and transmission of avian pathogens including avian influenza viruses (AIV) of low and high pathogenicity, which pose severe economic and public health concerns in Bangladesh. This study focused on active surveillance of several avian viral pathogens with a special reference to AIV in selected backyard duck populations in Bangladesh. A total of 500 pooled oropharyngeal and cloacal samples from individual ducks of four districts were tested by real time PCRs for the presence of AIV, avian avulavirus-1, anatid herpesvirus-1, avian parvovirus, avian bornavirus and avian coronavirus. The investigation identified 27 (5.4%) ducks positive for AIV and 12 (2.4%) positive for avian coronavirus. In 13 samples, RNA specific for AIV H4N6 was detected. Phylogenetic analysis of the AIV haemagglutinin H4 and neuraminidase N6 genes suggested a clustering of Bangladeshi AIV H4N6 in Eurasian lineage group 2. Other AIV positive samples had very low virus loads (Cq > 36) and were not subtyped. Coronaviral sequences of a fragment of the polymerase gene were related to Eurasian-Australian duck gamma-coronaviruses. Our current active surveillance in free-range domestic backyard ducks in Bangladesh failed to detect highly pathogenic (HP) AIV in contrast to our previous passive monitoring study. Nevertheless, active monitoring of domestic duck populations may be important to highlight presence and transmission dynamics of economically less important AIV that still may serve as reassortment partners for the generation of new HP and zoonotic AIV. RESEARCH HIGHLIGHTS Active surveillance for viral pathogens in domestic free-range backyard ducks. Detection of avian influenza virus subtype H4N6. First identification of avian gammacoronavirus in ducks in Bangladesh.

Role of vaccine virus in interfering the pathogenesis of wild strain of duck plague virus (DPV)

Duck plague or duck viral enteritis is an acute highly contagious disease of duck, geese and swan of all ages caused by Anatid Herpesvirus-1 and characterized by high morbidity and mortality and sudden death. In the present study, revival, pathogenesis and pathology of wild strain (DP/As-Km/0019) of duck plague virus (DPV) were studied in 2 month old ducklings. In order to study the role of vaccine virus in interfering the pathogenesis of wild strain of DPV in ducklings, a total of 24 numbers of ducklings were divided into four groups. Group I was vaccinated followed by challenge after 24 hours, Group II was challenged 48 hours prior to vaccination, Group III was kept as positive control, whereas Group IV was kept as negative control without vaccination or challenge. Clinical signs in inoculated ducklings were weakness, lethargy, depression, loss of appetite, ruffled feathers, nasal discharge, lacri- mation, pasted eyelids with periorbital ring formation and greenish diarrhoea. On necropsy, gross lesions were observed in brain, oesophagus, liver, spleen, heart and intestine. Microscopically, vascular and degenerative changes like congestion, haemorrhages, necrosis and formation of diphtheritic membrane were recorded, whereas fatty changes, emphysema and coagulative necrosis were seen in all parenchymatous organs. Presence of viral DNA was detected in clinical samples as well as in tissue samples by molecular assay and confirmation of duck plague viral antigen in tissue samples was done by sandwich ELISA. In conclusion, vaccination could fully protect the ducks in endemic areas, however, near protection could be found after 48 hours of challenge.

Detection and isolation of Duck Plague virus from field outbreaks in Assam, India

Duck plague is an acute, contagious and lethal disease of ducks caused by an enveloped DNA virus, belonging to Anatid Herpes Virus 1 under genus Mardivirus, subfamily Alphaherpesvirinae, family Herpesviridae. The disease is characterized by sudden death, high mortality, hemorrhages and necrosis in the internal organs. Duck farming plays an important role in the livelihood of the farmers of Assam. Duck Plague being the major killer disease among duck population of Assam, posing a threat to the growth of duck farming in the state. The present report investigates various outbreaks of Duck plague in Assam, detection and isolation of the virus. A total of 29 outbreaks suspected for Duck Plague were attended in different districts of Assam and 380 numbers of samples comprising of both clinical (n=107) as well as post mortem (n=273) were collected. Presence of DPV in samples was detected by Sandwich-ELISA (S-ELISA) and amplification of UL 44.5 as well as DNA polymerase gene of DPV by PCR. Out of 380 samples collected from 29 outbreaks, 213 (56.05%) and 299 (78.68%) samples were found positive for presence of DPV by S-ELISA and PCR, respectively. Pooled positive samples from various outbreaks were processed for isolation of duck plague virus in DEF primary cell culture. DPV field strain produced CPE comprising of rounding of cells, formation of syncytia, marked cytoplasmic granulations and intracellular vacuoles.

Detection of Low Pathogenicity Influenza A(H7N3) Virus during Duck Mortality Event, Cambodia, 2017

In January 2017, an estimated 3,700 (93%) of 4,000 Khaki Campbell ducks (Anas platyrhynchos domesticus) died in Kampong Thom Province, Cambodia. We detected low pathogenicity avian influenza A(H7N3) virus and anatid herpesvirus 1 (duck plague) in the affected flock; however, the exact cause of the mortality event remains unclear.

Potential disease transmission from wild geese and swans to livestock, poultry and humans: a review of the scientific literature from a One Health perspective

ABSTRACTThere are more herbivorous waterfowl (swans and geese) close to humans, livestock and poultry than ever before. This creates widespread conflict with agriculture and other human interests, but also debate about the role of swans and geese as potential vectors of disease of relevance for human and animal health. Using a One Health perspective, we provide the first comprehensive review of the scientific literature about the most relevant viral, bacterial, and unicellular pathogens occurring in wild geese and swans. Research thus far suggests that these birds may play a role in transmission of avian influenza virus, Salmonella, Campylobacter, and antibiotic resistance. On the other hand, at present there is no evidence that geese and swans play a role in transmission of Newcastle disease, duck plague, West Nile virus, Vibrio, Yersinia, Clostridium, Chlamydophila, and Borrelia. Finally, based on present knowledge it is not possible to say if geese and swans play a role in transmission of Escherichia coli, Pasteurella, Helicobacter, Brachyspira, Cryptosporidium, Giardia, and Microsporidia. This is largely due to changes in classification and taxonomy, rapid development of identification methods and lack of knowledge about host specificity. Previous research tends to overrate the role of geese and swans as disease vectors; we do not find any evidence that they are significant transmitters to humans or livestock of any of the pathogens considered in this review. Nevertheless, it is wise to keep poultry and livestock separated from small volume waters used by many wild waterfowl, but there is no need to discourage livestock grazing in nature reserves or pastures where geese and swans are present. Under some circumstances it is warranted to discourage swans and geese from using wastewater ponds, drinking water reservoirs, and public beaches. Intensified screening of swans and geese for AIV, West Nile virus and anatid herpesvirus is warranted.

Molecular characterization of the complete genome of falconid herpesvirus strain S-18

Falconid herpesvirus type 1 (FaHV-1) is the causative agent of falcon inclusion body disease, an acute, highly contagious disease of raptors. The complete nucleotide sequence of the genome of FaHV-1 has been determined using Illumina MiSeq sequencing. The genome is 204,054 nucleotides in length and has a class E organization. The genome encodes approximately 130 putative protein-coding genes, of which 70 are orthologs of conserved alphaherpesvirus and Mardivirus proteins. Three FaHV-1 genes (UL3.5, UL44.5 and CIRC) were identified that encode protein homologues unique to Mardivirus and Varicellovirus. The genome also encodes homologues to the Mardivirus genes LORF2, LORF3, LORF4, LORF5, SORF3 and SORF4. An opal mutation resulting in premature termination was identified in the FaHV-1 UL43 gene. Phylogenetically, FaHV-1 resides in a monophyletic group with the other Mardiviruses but, along with anatid herpesvirus 1, represents a more distant divergence from the rest of the Mardivirus genus.

First survey of the occurrence of duck enteritis virus (DEV) in free-ranging Polish water birds

Duck plague (DP) caused by anatid herpesvirus 1, also called duck enteritis virus (DEV), presents one of the most important concerns in mass waterfowl production. Apart from geese and ducks, free-ranging water birds are the most notorious infection carriers. The epidemiology of DEV in Western Europe remains unknown. Therefore, it was reasonable to conduct a study on its occurrence using modern but simple real-time loop-mediated isothermal amplification (LAMP). Analysis of 132 field isolates showed the presence of DEV in 96 birds (72.7 %), and it was found predominantly in wild ducks (Anas platyrhynchos) and mute swans (Cygnus olor). This virus was also found in graylag geese (Anser anser), tundra bean geese (Anser fabalis), and grey herons (Ardea cinerea). The results were recorded as green colour of positive samples, fluorescence under ultraviolet light, and florescent curves in a real-time PCR system. This study indicates the high prevalence of DEV among free-ranging water birds in Poland and the possible transmission to other birds settling in the water environment. This is the first report of DEV detection among free-ranging water birds in Poland.

THE EPIZOOTIOLOGY OF ANATID HERPESVIRUS 1 INFECTION IN FREE-FLYING WATERFOWL: A COMPARISON OF LATENT AND ACTIVE INFECTIONS AMONG NATIVE WATERFOWL, CAPTIVE-REARED RELEASED DUCKS, AND PERIDOMESTIC OR FERAL DUCKS

The epizootiology of anatid herpesvirus 1 (AHV-1) infection in waterfowl is poorly understood but apparently involves persistence of the virus in latently infected birds. Epornitics have often occurred in captive waterfowl or semiwild ducks in parklike settings, and many wildlife professionals conclude that such ducks may be the source of infection for wild waterfowl. We assessed the prevalence of latent infection and viral shedding from four groups of waterfowl: naturally occurring populations of native waterfowl, captive-reared waterfowl released for shooting, introduced nonmigratory waterfowl (e.g., resident, wild Mallards; Anas platyrhynchos), and semiwild peridomestic waterfowl (e.g., park ducks) in North Carolina and Florida, USA from 2004 to 2009. A nested PCR assay was used to detect viral DNA in trigeminal ganglia and cloacal swabs. Detection of viral DNA in trigeminal ganglia, but not cloacal swabs, was assumed to indicate latent infection, whereas PCR-positive cloacal swabs indicated active shedding of the virus. We collected 2,045 samples from 23 species of native, wild waterfowl, and detected latent infections in nine species. Wild Northern Pintails (Anas acuta), a species reportedly resistant to the virus, had the highest prevalence (8.1%). However, low prevalences were identified in other waterfowl from various families. Cloacal shedding was rarely detected (0.1% prevalence) among native waterfowl and was observed in one Blue-winged Teal (Anas discors) and one Mottled Duck (Anas fulvigula). All captive-reared, released waterfowl (n=13) collected were Mallards and one was latently infected, suggesting that these birds could also serve as a source of AHV-1 for naive waterfowl. All nonmigratory waterfowl sampled (n=90) were also Mallards. None of the resident Mallards were shedding virus, but one was latently infected. The peridomestic waterfowl sampled included breeds of domestic Mallard (n=6) and Muscovy Ducks (Cairina moschata; n=73). One peridomestic Mallard and four Muscovy Ducks were shedding virus at the time they were sampled, but no latently infected, asymptomatic carriers were identified.

Distribution characteristics of DNA vaccine encoded with glycoprotein C from Anatid herpesvirus 1 with chitosan and liposome as deliver carrier in ducks

BackgroundA eukaryotic expression plasmid encoding glycoprotein C (gC) of Anatid herpesvirus 1 (AnHV-1) (pcDNA3.1-gC) was constructed and validated. The tissue distribution of chitosan/DNA complexes, liposome/DNA complexes and pcDNA3.1-gC alone were evaluated using a quantitative real-time PCR based TaqMan™ probe following intramuscular administration in ducklings.ResultsCompared with pcDNA3.1-gC alone, liposomes universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain, bursa of Fabricius, and especially in the enteron (esophagus, duodenum, rectum, and cecum). Chitosan also universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain and esophagus. Compared with lipoplex-gC, higher chitosan-gC plasmid DNA copy numbers were detected at the injection sites, liver, spleen, heart, brain and esophagus. In contrast, compared with lipoplex-gC, lower copy numbers of chitosan-gC plasmid DNA were detected in the duodenum, rectum and cecum.ConclusionsThe results of this study demonstrated that chitosan and liposomes mediated rapid and extensive plasmid distribution in duck tissues, with low levels maintained from 1 d after DNA vaccination.

Computational identification of microRNAs in Anatid herpesvirus 1 genome.

BackgroundMicroRNAs (miRNAs) are a group of short (~22 nt) noncoding RNAs that specifically regulate gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of ~70 nt, are processed into mature miRNAs by cellular RNases III. To date, thousands of miRNAs have been identified in different organisms. Several viruses have been reported to encode miRNAs.FindingsHere, we extended the analysis of miRNA-encoding potential to the Anatid herpesvirus 1 (AHV-1). Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs. We then compared the 12 mature miRNAs candidates with the all known miRNAs of the herpesvirus family. Interestingly, the “seed sequences” (nt 2 to 8) of 2 miRNAs were predicted to have the high conservation in position and/or sequence with the 2 miRNAs of Marek’s disease virus type 1 (MDV-1). Additionally, we searched the targets from viral mRNAs.ConclusionsUsing computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs and 2 miRNAs have the high conservation with the 2 miRNAs of MDV-1. The result suggested that AHV-1 and MDV-1 should have closed evolutionary relation, which provides a valuable evidence of classification of AHV-1. Additionally, seven viral gene targets were found, which suggested that AHV-1 miRNAs could affect its own gene expression.

Detection of anatid herpesvirus 1 gC gene by TaqMan™ fluorescent quantitative real-time PCR with specific primers and probe

BackgroundAnatid herpesvirus 1 (AHV-1) is known for the difficulty of monitoring and controlling, because it has a long period of asymptomatic carrier state in waterfowls. Furthermore, as a significant essential agent for viral attachment, release, stability and virulence, gC (UL44) gene and its protein product (glycoprotein C) may play a key role in the epidemiological screening. The objectives of this study were to rapidly, sensitively, quantitatively detect gC gene of AHV-1 and provide the underlying basis for further investigating pcDNA3.1-gC DNA vaccine in infected ducks by TaqMan™ fluorescent quantitative real-time PCR assay (FQ-PCR) with pcDNA3.1-gC plasmid.ResultsThe repeatable and reproducible quantitative assay was established by the standard curve with a wide dynamic range (eight logarithmic units of concentration) and very good correlation values (1.000). This protocol was able to detect as little as 1.0 × 101 DNA copies per reaction and it was highly specific to AHV-1. The TaqMan™ FQ-PCR assay successfully detected the gC gene in tissue samples from pcDNA3.1-gC and AHV-1 attenuated vaccine (AHV-1 Cha) strain inoculated ducks respectively.ConclusionsThe assay offers an attractive method for the detection of AHV-1, the investigation of distribution pattern of AHV-1 in vivo and molecular epidemiological screening. Meanwhile, this method could expedite related AHV-1 and gC DNA vaccine research.

Development of TaqMan® MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1

BackgroundAnatid herpesvirus 1 (AHV-1) is an alphaherpesvirus associated with latent infection and mortality in ducks and geese and is currently affecting the world-wide waterfowl production severely. Here we describe a fluorescent quantitative real-time PCR (FQ-PCR) method developed for fast measurement of AHV-1 DNA based on TaqMan MGB technology.ResultsThe detection limit of the assay was 1 × 101 standard DNA copies, with a sensitivity of 2 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation.ConclusionThe high sensitivity, specificity, simplicity and reproducibility of the AHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method suitable for a broad spectrum of AHV-1 etiologically related application.

Anatid herpesvirus 1 CH virulent strain induces syncytium and apoptosis in duck embryo fibroblast cultures

Anatid herpesvirus 1 (AHV-1) CH virulent strain was first isolated from an infected duck and it was found that this virus strain could induce cytopathic effect (CPE) in duck embryo fibroblast (DEF). Following AHV-1 infection, DEF showed morphological changes such as cell rounding, improved refractivity and detachment from the culture surface. However, its pathological characteristics were not adequately known. Related studies were performed and the results showed that syncytium formation could be observed as the other type of CPE in AHV-1 infection. Hematoxylin-eosin staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining of infected DEF were each used to visualize the shape and distribution of chromatin within nuclei and nuclear fragmentation was observed. Chromatin condensation and margination, as well as formation of apoptotic bodies were observed by transmission electron microscopy (TEM). DNA ladder formation was detected in AHV-1 infected cells and apoptosis of the infected DEF was also detected by flow cytometry analysis of Annexin V-FITC/PI staining method. Therefore, it was suggested that AHV-1 virulent strain can induce syncytium and apoptosis in DEF. Syncytium formation and apoptosis observed in this study may contribute to the elucidation of AHV-1 pathogenesis.

Purification of anatid herpesvirus 1 particles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation

Anatid herpesvirus 1 (AHV-1) infection causes substantial economic losses to the world-wide waterfowl production. However, little is known about the efficient method used to study the purification of AHV-1 and the negative staining morphology of the purified virus particles. This lack of knowledge is one of the important factors that have affected the progress of research studies on AHV-1 molecular virology to such an extent that they are lagging far behind those on other members of the same family Herpesviridae. Therefore, an efficient method for purifying AHV-1 from cell-culture medium has been developed. Abundant AHV-1 particles, whose morphological features match those of herpesvirus, were obtained by using the following procedures: (1) conventional differential centrifugation for removal of debris after cell disruption, (2) tangential-flow ultrafiltration coupled with sucrose density gradient ultracentrifugation for isolation of the virus, and (3) conventional differential ultracentrifugation for virus concentration. The purified AHV-1 particles were subjected to transmission electron microscopy (TEM), infectivity and recovery tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting assay, and agar gel diffusion test (AGDT). The results of examinations revealed that purified AHV-1 particles were free of visible contamination or degradation. The purified AHV-1 particles were biologically active and were successful in initiating infection upon inoculation into susceptible duck embryo fibroblast. The procedures are reliable technically and feasible for purification of large volumes of viruses.