Marek's disease virus in vaccinated poultry flocks in Turkey: its first isolation with molecular characterization.

Marek's disease (MD) is an important disease of avian species and a potential threat to the poultry industry worldwide. In this study, 16 dead commercial chickens from flocks with suspected MD were necropsied immediately after death. Pathological findings were compatible with MD, and gallid alphaherpesvirus 2 was identified in PCR of spleen samples. Virus isolation was performed in primary cell culture, and partial sequencing of the meq gene of the isolate revealed >99% nucleotide sequence identity to virulent and very virulent plus strains from a number of European countries, placing it in the same subclade of clade III as two virulent Italian strains and a very virulent plus Polish strain as well as virulent strains of geese and ducks. The data reported here indicate that a virulent strain of Marek's disease virus is circulating in Turkey and has not been stopped by the current national vaccination programme.

Comparison of three filter paper-based devices for safety and stability of viral sample collection in poultry.

General diagnosis of poultry viruses primarily relies on detection of viruses in samples, but many farms are located in remote areas requiring logistic transportation. Filter paper cards are a useful technology that offer an alternative for collecting and preserving samples without hazardous exposure. The goal of this study was to compare three filter papers: the Flinders Technology Associates filter (FTA®) card, dried blood spot (DBS) card and qualitative filter paper (FP) grade 2 to collect poultry samples. In particular, we have used Newcastle disease virus (NDV) to evaluate safety and a Marek's disease virus (MDV) attenuated vaccine (CVI988) to evaluate stability of viral DNA. This experiment was divided into two parts. The first part was to determine the DNA stability and detection limit of CVI988 in samples collected in different paper supports after four storage times (3, 7, 14 and 30 days post spot). The second part was to determine the safety of papers by evaluating the viral inactivation efficacy using NDV as a representative virus. Results showed that all papers could preserve CVI988 DNA at all times, with a detection limit of 0.5 PFU/5 µl for FTA® and DBS cards, and 5 PFU/5 µl for FP. Our results showed that the NDV remained viable and infectious on the DBS card and FP, while no viable virus was detected on the FTA® card, suggesting that the FTA® card was safest to use. Therefore, the use of the DBS card and FP for infectious sample collection should be discouraged and reconsidered. RESEARCH HIGHLIGHTS The detection limits of the FTA® card, DBS card and FP for CVI988 detection were 0.5, 0.5 and 5 PFU/5 µl, respectively. All three filter papers could preserve viral DNA for at least 30 days of post spot. The DBS card and FP are not suitable for collecting NDV samples, which is one of the major economical threats for the poultry industry worldwide.

Gut microbiota profiles of commercial laying hens infected with tumorigenic viruses.

BACKGROUND: Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection on the intestinal microflora of chickens. RESULTS: A 29-week commercial layer flock positive for avian leukosis virus-J (ALV-J), Marek's disease virus (MDV) and avian reticuloendotheliosis virus (REV) was selected, and fresh fecal samples were collected and examined for the composition of the gut microflora by Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The operational taxonomic units (OTUs) of the fecal microbiota differentiated the chickens infected with only ALV-J and those coinfected with ALV-J and MDV or REV from infection-negative chickens. The enrichment and diversity of cloacal microflora in chickens infected with ALV-J alone were slightly different from those in the infection-negative chickens. However, the diversity of cloacal microflora was significantly increased in chickens coinfected with both ALV-J and MDV or REV. CONCLUSIONS: The intestinal microbiota was more strongly disturbed in chickens after coinfection with ALV-J and MDV or REV than after infection with ALV-J alone, and there may be underlying mechanisms by which the capacity for the stabilization of the intestinal flora was impaired due to viral infection and tumorigenesis.

UL36 Encoded by Marek's Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity.

(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek's disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480)H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.

Identification of Marek's disease virus genes associated with virulence of US strains.

Marek's disease virus (MDV) is the most well-cited example of vaccine-driven virulence evolution. MDV induces a lymphoproliferative disease in chickens, which is currently controlled by widespread vaccination of flocks. Unfortunately, Marek's disease (MD) vaccines, while effective in preventing tumours, do not prevent viral replication and mutation, which has been hypothesized as the major driving force for increased MDV virulence of field strains during the past 40 years in US commercial flocks. To limit future virulence increases, there is interest in characterizing MDV strain genomes collected over the years and associating genetic variations with variation in virulence. In this study, we characterized 70 MDV genomes with known virulence by complete or targeted DNA sequencing, and identified genetic variants that showed association with virulence. Our results revealed a number of MDV genes as would be expected for a complex trait. In addition, phylogenetic analysis revealed a clear separation of strains that varied by virulence. Interestingly, high virulence isolates from the same farms persisted over years despite eradication attempts, which has implications on control efforts. Given the growing ability to bioengineer the MDV genome, it should be feasible to experimentally test whether these individual variants influence virulence markers alone or combinations. Once validated, these markers may provide an alternative to live bird testing for evaluating virulence of new MDV field strains.

Molecular characterization and phylogenetic analysis of oncogenes from virulent serotype-1 Marek's disease virus in India.

Marek's disease (MD) is one of the most important neoplastic diseases of poultry caused by Marek's disease virus (MDV), an oncogenic avian herpes virus, which is responsible for great economic losses to the poultry industry worldwide. Inspite of the usage of HVT and bivalent vaccines in the poultry flocks, MD continues to be a major threat to the poultry industry in India. In the present study, MD outbreaks were reported in poultry farms from different regions of Andhra Pradesh, India. The postmortem examination of dead birds showed presence of lymphomas in different visceral organs suggestive of virulent oncogenic MDVs. Histopathological examination revealed infiltration of pleomorphic lymphoblastoid cells typical of MD. The blood and tissue samples were collected and PCR was standardized targeting a 132 bp tandem repeat region specific for serotype-1 MD viruses. Further, the characterization of the oncogenes i.e. Meq and viral interleukin 8 (vIL-8) was carried out by PCR and nucleotide sequencing. The sequence analysis of Meq gene of different clinical cases from India revealed >99 % homology with RB1B (very virulent) and GA (virulent) strains and that of vIL-8 gene showed >99 % identity with virulent strains LS and LMS. Phylogenetic analysis of oncogenes was carried out with other available sequences in the GenBank. Finally, we conclude that MDV strains obtained in the present outbreaks in India could be designated as virulent or very virulent pathotypes based on nucleotide, amino acid and phylogenetic analysis of the viruses.

Differential amplification of Marek's disease CVI988 vaccine and of wild-type isolates from organs of commercial chickens using single or duplexed probes in real-time PCR.

The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay's applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.

Severe necrotic dermatitis in the combs of line 63 chickens infected with Marek's disease virus.

Marek's disease virus (MDV), the aetiological agent of Mareks' disease (MD), is a highly cell-associated oncogenic α-herpesvirus that replicates in chicken lymphocytes and establishes a latent infection within CD4(+) T cells. We investigated the possible effect of MDV infection on the exacerbation of necrotic dermatitis in the combs of MD-susceptible (72) and MD-resistant (63) chicken lines at 21 days post infection. MDV-infected birds of line 63 are relatively resistant to tumour development but exhibit an unusual necrosis of combs, wattles, and footpads that is intensified when infected with MDV. Chickens from line 72, on the other hand, are highly susceptible to MDV infection and tumour development. Real-Time PCR analysis revealed that IL-6, IL-8, IL-12, IL-18, iNOS, and IFNγ were all up regulated in the comb tissues of MDV-infected susceptible line 72 with no visible necrotic damage. With the exception of IL-8 and iNOS, the expression of all the other tested genes was barely detected in the necrotic combs of the resistant line 63. Real-Time PCR analysis revealed the MDV meq oncogene transcripts in the spleen tissues of both infected lines but in the comb tissues of only the susceptible line 72. A significant infiltration of macrophages and lymphocytes was detected in the comb tissues of both resistant and susceptible lines. Histopathological analysis also showed thinning and erosion of epidermis and inflammation, lympho-plasmocytic infiltration, heterophilic, and histocytic cellulitis within the connective tissues of the necrotic combs. Gram stain of the sectioned frozen comb samples exposed the presence of Gram-positive micrococcus.

Isolation and full-genome sequence of two reticuloendotheliosis virus strains from mixed infections with Marek's disease virus in China.

Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China.

Marek's disease virus associated ocular lymphoma in Roulroul partridges (Rollulus rouloul).

Two 1-year old Roulroul partridges (Rollulus rouloul), one male and one female, were presented because of eye problems and anorexia. Twenty of the 30 Roulroul partridges in the owner's collection had already died. The affected birds stopped eating, became thinner, and eventually died. Antibiotic treatment, which started because of the suspicion of a septicaemic process, was unsuccessful. At clinical examination of the two partridges it was found that in both birds, one eye ball was filled with a whitish yellow amorphous material and the other eye ball of the female showed a distinct corneal opacity. Both presented birds were euthanized. Necropsy revealed no significant abnormalities in addition to the eye lesions. Histology and immunohistochemistry of the female's eye revealed an infiltrate of T-lymphocytes corresponding to ocular lymphoma. Herpesvirus genus-specific PCR, followed by Sanger sequencing confirmed the presumptive diagnosis of Marek's disease in both birds. To our knowledge, this is the first confirmed case of infection with Gallid Herpesvirus 2 (Marek's disease virus) in partridges and the first case in this specific species.

Common occurrence of Gallid herpesvirus-2 with reticuloendotheliosis virus in chickens caused by possible contamination of vaccine stocks.

AIMS: The aim of this study was to investigate the common occurrence of reticuloendotheliosis virus (REV) among Gallid herpesvirus 2 (GaHV-2) infected chickens. The possible cause of this co-occurrence may be linked to contaminated vaccine stocks, which were also examined. METHODS AND RESULTS: The study was conducted on 25 field isolates of GaHV-2 collected between 2007 and 2013 from vaccinated chickens. Additionally, 10 commercial Marek's Disease vaccine stocks manufactured between 1993 and 2013, comprising of FC126 HVT, CVI988/Rispens and bivalent HVT + Rispens vaccines were examined. Chicken isolates were collected from the liver. Due to difficulties in differentiation between GaHV-2 and REV, by observation of clinical signs or lesions presented in liver or spleen, loop-mediated isothermal amplification (LAMP and RT-LAMP) as well as PCR-based methods were applied. CONCLUSIONS: The co-occurrence of GaHV-2 and REV genetic material was shown in 24 (96%) of 25 examined isolates. A marginal REV contamination was detected in three out 10 (30%) commercial vaccine stocks, mainly in bivalent HVT + Rispens vaccines produced between 2009 and 2012. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicated the common occurrence of GaHV-2 and REV in Polish chicken flocks, which is probably linked to contaminated HVT + Rispens vaccine stocks. Reasons for the detection of a marginal REV contamination need to be further elucidated.

Absolute quantification of a very virulent Marek's disease virus dynamic quantity and distributions in different tissues.

Chickens infected with Marek's disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors and increases its potential for horizontal transmission. There is a positive correlation between very virulent (vv) MDV quantity and the pathology. The purpose of this study was to determine the vvMDV loads dynamics in different phases, and the correlation between the viral quantity and tumor development. We used a SYBR Green duplex real-time quantitative PCR (q-PCR) assay to detect and quantify MDV loads and distributions in different tissues, targeting the Eco-Q protein gene (meq) of the virus and the house-keeping ovotransferrin (ovo) gene of chickens. The q-PCR was performed using different tissue DNA preparations derived from chickens which were infected with 1,000 pfu of the SDWJ1302 strain and tissue samples were collected from control and MDV-infected birds on 7, 10, 15, 21, 28, 40, 60, and 90 d post-infection (DPI). The data indicated that the MDV genome was almost quantifiable in immune organs of infected chickens as early as 7 DPI, and the number of MDV genome copies in the blood and different organs peaked by 28 DPI, but then gradually decreased by 40 DPI. The levels of viral quantity in the lymphocytes, liver, and spleen were all higher than those in other organs, and that in the feather follicles was the highest among different phases of MDV infection. The vvMDV could still be detected in peripheral blood and tissues by 90 DPI, and the vast existence of virus will stimulate tissue destruction. The data provided further evidence of viral infection involving multi-organ distribution and mainly involving immune organ proliferation, resulting in immunosuppression.

Pathotyping of recent Indian field isolates of Marek's disease virus serotype 1.

A study was undertaken to assess the virulence of Marek's disease virus (MDV) serotype 1 field isolates obtained from poultry flocks of southern part of India. Five representative MDV serotype 1 strains were isolated from eighty-six blood samples collected from fifteen farms. Three out of five isolates which were free from avian leukosis virus (ALV) and reticuloendotheliosis virus (REV) were adapted in chicken embryo fibroblast (CEF) culture and designated as Ind/TN/11/01, Ind/KA/12/02 and Ind/TN/12/03. Pathotyping assay was conducted in two trials. In the first trial, non-vaccinated chickens were challenged (trial I), while in second trial, two types of vaccinated chickens along with non-vaccinated controls were challenged (trial II). Birds inoculated with field isolate Ind/TN/12/03 had very low body (75.34 ± 3.04 g 15 days post infection (dpi)) and bursa Fabricii weight (1.64 ± 0.06 at 15 dpi) when compared to those inoculated with the other two isolates (Ind/TN/11/01 and Ind/KA/12/02) and uninoculated controls (body weight 111.33 ± 1.30 g and bursa Fabricii weight 4.33 ± 0.11 15 dpi). Incidence of early mortality syndrome (53%) and lymphoma (86%) induced by Ind/TN/12/03 was comparable with very virulent strains published elsewhere. In protection test, the percentage of Marek's disease (MD) incidence induced by Ind/TN/12/03 was 57.5% and 25% in monovalent and bivalent vaccine inoculated birds respectively compared to uninoculated control (100%). Based on the above findings in pathotyping experimental trials with a supportive evidence of histopathological observations, isolate Ind/TN/12/03 was considered as very virulent MDV and other two isolates were considered as virulent MDVs.

Direct detection of Marek's disease virus in poultry dust by loop-mediated isothermal amplification.

Marek's disease virus (MDV) is a serious concern for poultry production and represents a unique herpesvirus model. MDV can be shed by doubly infected chickens despite vaccination. The fully infectious MDV particles are produced in the feather follicle epithelium (FFE), and MDV remains infectious for many months in fine skin particles and feather debris. Molecular biology methods including PCR and real-time PCR have been shown to be valuable for the detection of MDV DNA in farm dust. Recently, loop-mediated isothermal amplification (LAMP) was found to be useful in the detection of MDV in feathers and internal organs of infected chickens. LAMP is also less affected by the inhibitors present in DNA samples. Taking into account the advantages of LAMP, direct detection of MDV DNA in poultry dust has been conducted in this research. The detection of MDV DNA was possible in 11 out of the 12 examined dust samples without DNA extraction. The DNA was retrieved from dust samples by dilution and incubation at 95 °C for 5 min. The direct detection of MDV DNA in the dust was possible within 30 min using a water bath and UV light. The results were confirmed by electrophoresis and melting curve analysis of the LAMP products. Our results show that LAMP may be used to test for the presence of virulent MDV in poultry farm dust without DNA extraction.

[Isolation and characterization of a non-pathogenic Marek's disease virus with a naturally inserted REV LTR sequence].

OBJECTIVE: Marek's disease virus (MDV), GD06 strain, was isolated from healthy Three-Yellow chickens without MD vaccine. In this study, we characterized GD06 strain. METHODS: Meq gene and repeat short region of GD06 strain were amplified and analyzed, and the serotype was determined by indirect immunofluorescence assay. The growth characteristics of GD06 stain were evaluated in Specific Pathogen Free (SPF) chickens and chicken embryo fibroblasts. The pathogenicity of GD06 strain was assessed by challenge test using SPF chickens. RESULTS: GD06 strain was MDV-1 serotype with a naturally inserted reticuloendotheliosis virus long terminal repeat sequence. Similar to the vaccinated strains CVI988/Rispens and 814, the Meq gene of strain GD06 contained a 59-aa insertion in the proline-rich domain compared with very virulent strain Md5. On 96, 120, 144, 168 and 192 hours post infection, the virus titers of GD06 were higher than that of CVI988/Rispens in vitro (P < 0.05), and the viremia levels in GD06-infected group were significantly higher than that of CVI988/Rispens group on 21 and 42 days post inoculation (P < 0.05). The result of challenge test demonstrated that GD06 remained nonpathogenic for chickens and induced little immunosuppressive effects. CONCLUSION: GD06 is an MDV-1 non-pathogenic strain naturally integrated with reticuloendotheliosis virus and long terminal repeat sequence.

Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt.

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.

Sequence analysis of the whole genome of a recombinant Marek's disease virus strain, GX0101, with a reticuloendotheliosis virus LTR insert.

Marek's disease virus Chinese strain GX0101, isolated in 2001, is the first reported recombinant gallid herpesvirus type 2 (GaHV-2) field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. We constructed an infectious bacterial artificial chromosome (BAC) clone of GX0101, which showed characteristics very similar to those of the parental virus in replication and pathogenicity. Using the GX0101 BAC clone, the complete genome of GX0101 was sequenced and analyzed. The length of the GX0101 genome is 178,101 bp, and it contains only one REV-LTR insert at a site 267 bp upstream of the sorf2 gene.

Genome sequence determination and analysis of a Chinese virulent strain, LMS, of Gallid herpesvirus type 2.

Marek's disease (MD) is a neoplastic and neurodegenerative disease of chickens, which is caused by the Gallid herpesvirus type 2 (GaHV-2). Although vaccination has been used widely in China, MD still occurs frequently. Some molecular epidemiologic studies have shown that Chinese GaHV-2 isolates seem to constitute a separate clade from strains isolated from other regions. However, more of a genomic background of the Chinese strains is necessary. In 2007, a virulent GaHV-2 field strain, named LMS, was isolated from diseased chicken flocks in the southwest of China. The whole genome sequence of LMS was determined to evaluate its genetic property. The genome of LMS is 177,526 bp long, and 197 open reading frames (ORFs) were predicted. Most of the ORFs have high sequence identity with homologous ORFs of reference strains. Two regions in the LMS genome are grossly different from other strains: the α-like region and the latency-associated transcripts (LATs) promoters. Evolutionary analysis demonstrated that LMS has a larger phylogenetic distance from most American isolated strains but a closer relationship with 648Ap80 and the European pC12/130 strain. The characterised genome of LMS provides further insight into the genetics of the Chinese GaHV-2 field strains, which is useful for the control of MD in China.

Genotypic characterization of two bacterial artificial chromosome clones derived from a single DNA source of the very virulent gallid herpesvirus-2 strain C12/130.

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

Deletion of the Meq gene significantly decreases immunosuppression in chickens caused by pathogenic Marek's disease virus.

BACKGROUND: Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in immunosuppression, which is considered to be an integral aspect of the pathogenesis of Marek's disease (MD). A recent study showed that deletion of the Meq gene resulted in loss of transformation of T-cells in chickens and a Meq-null virus, rMd5ΔMeq, could provide protection superior to CVI988/Rispens. RESULTS: In the present study, to investigate whether the Meq-null virus could be a safe vaccine candidate, we constructed a Meq deletion strain, GX0101ΔMeq, by deleting both copies of the Meq gene from a pathogenic MDV, GX0101 strain, which was isolated in China. Pathogenesis experiments showed that the GX0101ΔMeq virus was fully attenuated in specific pathogen-free chickens because none of the infected chickens developed Marek's disease-associated lymphomas. The study also evaluated the effects of GX0101ΔMeq on the immune system in chickens after infection with GX0101ΔMeq virus. Immune system variables, including relative lymphoid organ weight, blood lymphocytes and antibody production following vaccination against AIV and NDV were used to assess the immune status of chickens. Experimental infection with GX0101ΔMeq showed that deletion of the Meq gene significantly decreased immunosuppression in chickens caused by pathogenic MDV. CONCLUSION: These findings suggested that the Meq gene played an important role not only in tumor formation but also in inducing immunosuppressive effects in MDV-infected chickens.