Genus Mardivirus Research Articles

Herpesviruses in migrating procellariforms, northeastern Brazil

Seabirds are one of the most threatened avian groups. Viruses, including herpesvirus, represent considerable threats to marine avifauna. Herein, our goal was to survey herpesvirus in Procellariiformes that stranded in Brazil between June and July 2021. We analyzed 12 Cory's shearwaters (Calonectris borealis), two Great Shearwaters (Ardenna gravis, syn. Puffinus gravis) and one Yellow-nosed Albatross (Thalassarche chlororynchos) found in an unusual mortality event in Bahía state, northeastern Brazil. After necropsy, selected tissue samples were tested for herpesvirus using a broad-range nested PCR. Overall, 20% (3/15) of the birds were herpesvirus-positive, i.e., two Cory's Shearwaters and one Great Shearwater. One alphaherpesvirus sequence type was identified in each shearwater species, classified into the genus Mardivirus. This study describes two likely novel herpesviruses in shearwaters, contributing to the currently very scarce data regarding infectious agents in Procellariiformes. Further studies are necessary to evaluate the presence and characteristics of herpesvirus in Procellariiformes, and the presence (or not) of related disease in order to understand the epidemiology of this infectious agent and eventually contribute to the conservation of this endangered seabird group.

Detection of a Novel Alphaherpesvirus and Avihepadnavirus in a Plantar Papilloma from a Rainbow Lorikeet (Trichoglosis moluccanus).

Cutaneous plantar papillomas are a relatively common lesion of wild psittacine birds in Australia. Next-generation sequencing technology was used to investigate the potential aetiologic agent(s) for a plantar cutaneous papilloma in a wild rainbow lorikeet (Trichoglosis moluccanus). In the DNA from this lesion, two novel viral sequences were detected. The first was the partial sequence of a herpesvirus with the proposed name, psittacid alphaherpesvirus 6, from the Mardivirus genus of the family alphaherpesviruses. This represents the first mardivirus to be detected in a psittacine bird, the first mardivirus to be detected in a wild bird in Australia, and the second mardivirus to be found in a biopsy of an avian cutaneous papilloma. The second virus sequence was a complete sequence of a hepadnavirus, proposed as parrot hepatitis B genotype H (PHBV-H). PHBV-H is the first hepadnavirus to be detected in a wild psittacine bird in Australia. Whether other similar viruses are circulating in wild birds in Australia and whether either of these viruses play a role in the development of the plantar papilloma will require testing of biopsies from similar lesions and normal skin from other wild psittacine birds.

A Special Issue on Marek’s Disease Virus—The Editors’ View

Marek's disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide [...].

Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek's Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1.

ABSTRACTMarek’s disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek’s disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens.IMPORTANCE Marek’s disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.

The Requirement of Glycoprotein C for Interindividual Spread Is Functionally Conserved within the Alphaherpesvirus Genus (Mardivirus), but Not the Host (Gallid).

Marek’s disease (MD) in chickens is caused by Gallid alphaherpesvirus 2, better known as MD herpesvirus (MDV). Current vaccines do not block interindividual spread from chicken-to-chicken, therefore, understanding MDV interindividual spread provides important information for the development of potential therapies to protect against MD, while also providing a natural host to study herpesvirus dissemination. It has long been thought that glycoprotein C (gC) of alphaherpesviruses evolved with their host based on their ability to bind and inhibit complement in a species-selective manner. Here, we tested the functional importance of gC during interindividual spread and host specificity using the natural model system of MDV in chickens through classical compensation experiments. By exchanging MDV gC with another chicken alphaherpesvirus (Gallid alphaherpesvirus 1 or infectious laryngotracheitis virus; ILTV) gC, we determined that ILTV gC could not compensate for MDV gC during interindividual spread. In contrast, exchanging turkey herpesvirus (Meleagrid alphaherpesvirus 1 or HVT) gC could compensate for chicken MDV gC. Both ILTV and MDV are Gallid alphaherpesviruses; however, ILTV is a member of the Iltovirus genus, while MDV is classified as a Mardivirus along with HVT. These results suggest that gC is functionally conserved based on the virus genera (Mardivirus vs. Iltovirus) and not the host (Gallid vs. Meleagrid).

US3 Serine/Threonine Protein Kinase from MDV-1, MDV-2, and HVT Differentially Regulate Viral Gene Expression and Replication.

Gallid alphaherpesvirus 2 (GaHV-2), commonly known as Marek’s disease virus type 1 (MDV-1), is an oncogenic avian alphaherpesvirus, and along with its close relatives—Gallid alphaherpesvirus 3 (GaHV-3) or MDV-2 and Meleagrid alphaherpesvirus 1 (MeHV-1) or turkey herpesvirus (HVT)—belongs to the Mardivirus genus. We and others previously showed that MDV-1 US3 protein kinase plays an important role in viral replication and pathogenesis, which could be partially compensated by MDV-2 and HVT US3. In this study, we further studied the differential roles of MDV-1, MDV-2 and HVT US3 in regulating viral gene expression and replication. Our results showed that MDV-2 and HVT US3 could differentially compensate MDV-1 US3 regulation of viral gene expression in vitro. MDV-2 and HVT US3 could also partially rescue the replication deficiency of MDV-1 US3 null virus in the spleen and thymus, as determined by immunohistochemistry analysis of MDV-1 pp38 protein. Importantly, using immunohistochemistry and dual immunofluorescence assays, we found that MDV-2 US3, but not HVT US3, fully compensated MDV-1 US3 regulation of MDV-1 replication in bursal B lymphocytes. In conclusion, our study provides the first comparative analysis of US3 from MDV-1, MDV-2 and HVT in regulating viral gene expression in cell culture and MDV-1 replication in lymphocytes.

Adaptation of wild strain of duck plague virus in cell culture systems

Duck virus enteritis (DVE) is an acute, contagious herpes viral disease of ducks, geese and swans and members of the family Anatidae under the order Anseriformes. As per the recent taxonomic classification by ICTV, DEV has been classified into the genus Mardivirus, subfamily Alpha-herpesvirinae of the family Herpesviridae. Vaccination is the only option for the prevention and control of Duck plague. In India vaccination is done with chicken embryo adapted live virus which has many shortcomings. So, in this present situation a safe and potent vaccine development is the need of the hour. The use of chicken embryo fibroblast and a certified cell line may, probably, be the best option to achieve this. Therefore, the present study was undertaken for adaptation and propagation of local strain of duck plague virus in various cell culture systems. During the study a wild strain of DPV (DP/As-Km/0019) which was isolated in Duck Embryo Fibroblast, available in the Department of Microbiology was selected. The selected wild strain was used for adaptation in various cell culture systems viz. Chicken embryo fibroblast cell culture (CEF), Vero cell line and QT-35 cell line. The virus was passaged up to 12th passage levels and the presence of viral antigen was demonstrated by appearance of cytopathic effect (CPE), by Polymerase Chain Reaction (PCR) and by Sandwich ELISA. PCR was able to detect virus from all the cell cultures from 5th passage onwards and virus titre was detected at 10th and 12th passage level by S-ELISA. It was observed that various cell culture systems can be a good candidate for propagation of DPV and further study is required to study its immunogenicity and its feasibility as a vaccine candidate.

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.

First molecular detection and characterization of Marek\u2019s disease virus in red-crowned cranes (Grus japonensis): a case report

BackgroundMarek’s disease virus (MDV) resides in the genus Mardivirus in the family Herpesviridae. MDV is a highly contagious virus that can cause neurological lesions, lymphocytic proliferation, immune suppression, and death in avian species, including Galliformes (chickens, quails, partridges, and pheasants), Strigiformes (owls), Anseriformes (ducks, geese, and swans), and Falconiformes (kestrels).Case presentationIn 2015, two red-crowned cranes died in Nanjing (Jiangsu, China). It was determined that the birds were infected with Marek’s disease virus by histopathological examination, polymerase chain reaction (PCR), gene sequencing and sequence analysis of tissue samples from two cranes. Gross lesions included diffuse nodules in the skin, muscle, liver, spleen, kidney, gizzard and heart, along with liver enlargement and gizzard mucosa hemorrhage. Histopathological assay showed that infiltrative lymphocytes and mitotic figures existed in liver and heart. The presence of MDV was confirmed by PCR. The sequence analysis of the Meq gene showed 100% identity with Md5, while the VP22 gene showed the highest homology with CVI988. Furthermore, the phylogenetic analysis of the VP22 and Meq genes suggested that the MDV (from cranes) belongs to MDV serotype 1.ConclusionWe describe the first molecular detection of Marek’s disease in red-crowned cranes based on the findings previously described. To our knowledge, this is also the first molecular identification of Marek’s disease virus in the order Gruiformes and represents detection of a novel MDV strain.

Molecular characterization of Marek's disease virus in a poultry layer farm from Colombia

Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies.

A novel alphaherpesvirus associated with fatal diseases in banded Penguins.

A novel avian alphaherpesvirus, preliminarily designated sphenicid alphaherpesvirus 1 (SpAHV-1), has been independently isolated from juvenile Humboldt and African penguins (Spheniscus humboldti and Spheniscus demersus) kept in German zoos suffering from diphtheroid oropharyngitis/laryngotracheitis and necrotizing enteritis (collectively designated as penguin-diphtheria-like disease). High-throughput sequencing was used to determine the complete genome sequences of the first two SpAHV-1 isolates. SpAHV-1 comprises a class D genome with a length of about 164 kbp, a G+C content of 45.6 mol% and encodes 86 predicted ORFs. Taxonomic association of SpAHV-1 to the genus Mardivirus was supported by gene content clustering and phylogenetic analysis of herpesvirus core genes. The presented results imply that SpAHV-1 could be the primary causative agent of penguin-diphtheria-like fatal diseases in banded penguins. These results may serve as a basis for the development of diagnostic tools in order to investigate similar cases of penguin diphtheria in wild and captive penguins.

Isolation and characterization of a herpesvirus from feral pigeons in China

A herpesvirus was isolated during a diagnostic investigation of severe cases of conjunctivitis in feral pigeons (Columba livia f. domestica). Isolates of the virus were recovered from throat swabs of the pigeons followed by inoculation of the swab samples in chicken embryo fibroblasts. Pigeons inoculated with the isolated virus had similar clinical signs to those observed in naturally infected birds. Transmission electron microscopy revealed viral structures with typical herpesvirus morphology. Polymerase chain reaction amplification, using herpesvirus-identifying primers resulted in an amplicon of the expected size for herpesvirus. Sequencing of these amplicons and database comparisons identified the herpesvirus UL30 homologue. Phylogenetic reconstructions suggested that the isolated herpesvirus belongs to the Mardivirus genus of Alphaherpesvirinae. Using the current herpesvirus nomenclature conventions, the authors propose that the herpesvirus be named Columbid herpesvirus-1 Heilongjiang.

Chicken skin virome analyzed by high-throughput sequencing shows a composition highly different from human skin.

Recent studies show that human skin at homeostasis is a complex ecosystem whose virome include circular DNA viruses, especially papillomaviruses and polyomaviruses. To determine the chicken skin virome in comparison with human skin virome, a chicken swabs pool sample from fifteen indoor healthy chickens of five genetic backgrounds was examined for the presence of DNA viruses by high-throughput sequencing (HTS). The results indicate a predominance of herpesviruses from the Mardivirus genus, coming from either vaccinal origin or presumably asymptomatic infection. Despite the high sensitivity of the HTS method used herein to detect small circular DNA viruses, we did not detect any papillomaviruses, polyomaviruses, or circoviruses, indicating that these viruses may not be resident of the chicken skin. The results suggest that the turkey herpesvirus is a resident of chicken skin in vaccinated chickens. This study indicates major differences between the skin viromes of chickens and humans. The origin of this difference remains to be further studied in relation with skin physiology, environment, or virus population dynamics.

Characterization of the first columbid herpesvirus 1 isolate from a hybrid meat-type pigeon flock in China.

Infections of pigeons with herpesviruses have been described in several species of domestic and wild birds. In July 2012, increased mortality was observed in a hybrid meat-type pigeon flock in Beijing, China. Diagnostic tests led to the isolation of a virus designated columbid herpesvirus 1 BJ strain (CoHV-1BJ). Sequence analysis of the viral DNA polymerase catalytic subunit gene revealed a single open reading frame of 3753nt encoding 1250amino acids. Phylogenetic analysis revealed that the CoHV-1BJ is closely related to the members of the genus Mardivirus within the subfamily Alphaherpesvirinae. An experimental infection demonstrated that CoHV-1BJ is pathogenic to young pigeons, resulting in systemic infection with scattered focal necrosis in the liver and spleen. The results provide an initial assessment of herpesvirus infection in domestic pigeons in China.

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.

Marek's disease outbreak among vaccinated commercial layer flocks in the mining area of Karnataka, India

Marek's disease (MD) of chicken is caused by Gallid herpesvirus 2 belonging to the genus Mardivirus and family Herpesviridae . Annual losses due to MD the world over have been...

Bioinformatics Analysis of the Complete Nucleotide Sequence of UL17 Gene from Duck Enteritis Virus

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. Our laboratory had identified and sequenced the DEV-UL17 gene, one of the conservative genes of this viral genome. It is a 2055bp segment, GenBank accession NO. 643567. Here in this article we used kinds of bioinformatics softwares and online web servers to analyze the nucleotide sequence of this gene for further research. The phylogenetic tree of the DEV-UL17 protein and its homologs of other 19 reference herpesviruses revealed that DEV had close evolutionary relationship with Meleagrid herpesvirus1, Gallid herpesvirus 2 and Gallid herpesvirus 3, the members of genus Mardivirus. In addition, multiple epitopes and potential motifs were found in DEV-UL17 protein and they may play an important role in in the function of immune system, it may greatly influence the antibodies and genetic vaccine, or in the clinical diagnosis of DEV.

Marek΄s Disease: rapid progress in research with unclear biological implementations

Here we would like to provide a brief overview of the modern history of Marek΄s disease (MD) research with a focus on the most recent developments in experimental work and we will try to sum up their impact on the understanding of the biological properties of Marek΄s disease type 1 (MDV-1), the only representative of the Mardivirus genus causing fatal lymphoproliferative disease in poultry. We will also compare MDV-1 with other serologically-related poultry herpesviruses, Marek΄s disease virus type 2 (MDV-2) and herpesvirus of turkeys (HVT). Although MD was first described at the beginning of the last century, proper characterization of its biological impact on poultry production and utilization of molecular biology methods for detailed characterization of causative agent MDV-1 were introduced only in recent decades. However, many characteristics of MD infection, pathogenesis and vaccine protection mechanisms remain unclarified, though novel methods bring a challenge for better understanding of these unanswered questions.

Bioinformatics Analysis of UL39 Gene from Duck Plague Virus

The structures and characteristics of the large subunit of ribonucleotide reductase (R1), encoded by UL39 gene from duck plague virus (DPV) were analyzed and predicted by using a series of free bioinformatics software packages and bioinformatics tools. The DPV UL39 gene has a base composition of 681 adenine (27.99%), 503 cytosine (20.67%), 593 guanine (24.37%) and 656 thymine (26.96%). The online analysis of the physico-chemical properties demonstrates that the protein has 40 potential phosphorylation sites and 4 N-glycosylation sites when the threshold of prediction score is above 0.5; without the signal peptide and the transmembrance region. The phylogenetic tree proved that DPV R1 protein had a close evolutionary relationship with the Mardivirus genus of the Alphaherpesviruses. In conclusion, all those results will provide some valuable information for the further research of UL39 gene.