Marek's Disease Virus Serotype Research Articles

Retrospective analysis of Marek's disease virus outbreak in 2013 on chicken farms in North Sumatra, Indonesia

<p class="MDPI17abstract"><strong>Objective:</strong> Marek's disease is lymphoma in chicken caused by Marek's disease virus serotype 1 (MDV-1), characterized by paralysis and tumour formation. The disease has caused enormous economic loss worldwide, including in Indonesia. Unfortunately, molecular data related to MDV-1 from Indonesia is limited. Therefore, this study aimed to identify genetic characteristics of MDV-1 causing outbreaks in commercial farms in North Sumatra, Indonesia, 2013.</p><p class="MDPI17abstract"><strong>Methods:</strong> The amplification of Meq, pp38, gC, and gE genes of the strain MDN2013 was carried out using the PCR protocol. Fragment gene sequencing was accomplished using the Sanger method. Subsequently, the bioinformatic analyses of BLASTN and phylogenetic tree were applied to analyze the molecular characteristics of these gene sequences.</p><p class="MDPI17abstract"><strong>Results:</strong> The Meq, pp38, gC, and gE genes of the strain MDN2013 were successfully sequenced and submitted to the NCBI GenBank database. The Meq genes share a high identity with Asian viruses, mainly those originating from Indonesia, India, and China. The pp38, gC, and gE genes share similarities with other viruses in more diverse regions in Asia, Africa, Europe, and America. The phylogenetic tree analysis of the Meq gene demonstrated specific patterns for pathotype biodiversity where the strain MDN2013 has been categorized as virulent up to very virulent virus.</p><p class="MDPI17abstract"><strong>Conclusion:</strong> In conclusion, the molecular approach is an effective tool for investigating the MDV-1 biodiversity in the field, especially the Meq gene sequence analysis.</p>

Retrospective analysis of Marek's disease virus outbreak in 2013 on chicken farms in North Sumatra, Indonesia

<p class="MDPI17abstract"><strong>Objective:</strong> Marek's disease is lymphoma in chicken caused by Marek's disease virus serotype 1 (MDV-1), characterized by paralysis and tumour formation. The disease has caused enormous economic loss worldwide, including in Indonesia. Unfortunately, molecular data related to MDV-1 from Indonesia is limited. Therefore, this study aimed to identify genetic characteristics of MDV-1 causing outbreaks in commercial farms in North Sumatra, Indonesia, 2013.</p><p class="MDPI17abstract"><strong>Methods:</strong> The amplification of Meq, pp38, gC, and gE genes of the strain MDN2013 was carried out using the PCR protocol. Fragment gene sequencing was accomplished using the Sanger method. Subsequently, the bioinformatic analyses of BLASTN and phylogenetic tree were applied to analyze the molecular characteristics of these gene sequences.</p><p class="MDPI17abstract"><strong>Results:</strong> The Meq, pp38, gC, and gE genes of the strain MDN2013 were successfully sequenced and submitted to the NCBI GenBank database. The Meq genes share a high identity with Asian viruses, mainly those originating from Indonesia, India, and China. The pp38, gC, and gE genes share similarities with other viruses in more diverse regions in Asia, Africa, Europe, and America. The phylogenetic tree analysis of the Meq gene demonstrated specific patterns for pathotype biodiversity where the strain MDN2013 has been categorized as virulent up to very virulent virus.</p><p class="MDPI17abstract"><strong>Conclusion:</strong> In conclusion, the molecular approach is an effective tool for investigating the MDV-1 biodiversity in the field, especially the Meq gene sequence analysis.</p>

Occurrence of Marek's Disease in Backyard Chicken Flocks in Vietnam.

Marek's disease (MD) is a common lymphomatous and neuropathic disease of birds, especially chickens, and has caused significant losses to chicken production as a result of high mortality and morbidity. This study aims to determine the status of MD in backyard flocks in the Mekong Delta of Vietnam and to analyze clinical cases occurring during a year. The study was carried out from August 2018 to July 2019, during which time 16 suspected cases of chicken flocks with MD were observed, 40 chickens were subjected to anatomopathological examination, and PCR was performed for diagnosis of MD and determination of Marek's disease virus (MDV) serotypes. The results showed that all of the examined flocks were confirmed as experiencing MD. Nearly all cases were in an acute form with typical lesions of visceral lymphomas. Besides the presence of Marek's disease virus serotype 1 (MDV-1) from 100.0% of tested chicken flocks (16/16), nononcogenic turkey herpesvirus serotype 3 (MDV-3) was also found from 8 of 16 (50.0%) of examined chicken flocks. Morbidity and mortality at sampling time varied from 1% to 42.11% and 0.6% to 10%, respectively. Chicken flocks with MD vaccination had lower morbidity and mortality. These first findings confirm endemic MD in backyard chicken populations in Vietnam and confirm it continues to be a threat to chickens in backyard flocks and poultry production in general.

Marek's Disease in an Indian Peafowl (Pavo cristatus) with Clinical Ocular Disease and Paraparesis

Marek's disease (MD) is caused by virulent strains of Gallid alphaherpesvirus type 2 (MD virus serotype 1; MDV 1) and frequently causes a lymphoproliferative disorder in poultry and other galliform birds worldwide. However, within the peafowl (Phasianinae) subfamily, there are only rare confirmed reports of MD. Here we report MD in an Indian peafowl (Pavo cristatus), which clinically presented with hindlimb paraparesis and intraocular swelling of the right eye. Soft, off-white to tan masses within the right eye, sciatic nerves and coelomic cavity were identified at post-mortem examination which effaced the cranial pole of the kidneys and diffusely effaced the testes. Lymphoid neoplasia was identified histologically at all of these sites and there was extensive hepatic lymphoid cell infiltration, which had not been grossly evident. The T-cell origin of the lymphoid cells was confirmed by immunohistochemistry for CD3 antigen. A virulent strain of MDV 1 was detected by real-time polymerase chain reaction in DNA samples extracted from the kidney and testes. As MD is rare in peafowl it should be considered as a differential diagnosis for intraocular and coelomic masses with associated clinical signs.

Vaccinal Efficacy of Recombinant Marek's Disease Vaccine 301B/1 Expressing Chicken Interleukin-15.

Marek's disease (MD) vaccine does not provide sterilizing immunity that prevents subsequent MD virus (MDV) replication and shedding in vaccinated birds. It is hypothesized that cell-mediated immunity is critical to control the virus replication in chickens because MDV exists in cell-associated forms in the host. To improve the MD vaccine efficacy, particularly cell-mediated immunity, we constructed recombinant v301B/1-IL-15, an MDV serotype 2 vaccine strain 301B/1 expressing chicken interleukin-15 (IL-15), a cytokine which promotes T-cell proliferation and enhances T-cell responses. We examined the vaccine efficacy of v301B/1-IL-15 given as a bivalent MD vaccine in combination with turkey herpesvirus (HVT) against a very virulent MDV challenge. The expression of IL-15 did not interfere with virus stability and the growth of recombinant v301B/1-IL-15. However, the protective efficacy of v301B/1-IL-15 was not significantly different from that of v301B/1, the parental virus used to construct v301B/1-IL-15. Shedding of challenge virus was slightly reduced at Day 21 (16 days postchallenge) in the v301B/1-IL-15 plus HVT vaccinated group, with no statistically significant difference to that of the v301B/1 plus HVT vaccinated group, and thymus atrophy was observed to be less severe in the v301B/1-IL-15 plus HVT vaccinated group. Overall, the protection of v301B/1-IL-15 was not differentiable from v301B/1 against very virulent MDV challenge, but there is no interference with bivalent MD vaccine efficacy.

Establishment of an antigen-capture enzyme-linked immunosorbent assay for detecting avian reticuloendotheliosis virus

In this study, an antigen-capturing enzyme-linked immunosorbent assay (AC-ELISA) was established for the detection of avian reticuloendotheliosis virus (REV) using monoclonal and polyclonal antibodies against gp90. New Zealand white rabbits were immunized with recombinant REV-gp90 protein, and polyclonal antibodies were obtained after purification and used as the capture antibody. Mice monoclonal antibody 1A12D against REV-gp90 protein previously prepared in our laboratory was used as the detection antibody. The specificity of the AC-ELISA was confirmed with REV, avian leukosis virus subgroup J, Marek's disease virus serotype Ⅰ, avian hepatitis E virus and Fowl adenovirus serotype 4. The results showed that the AC-ELISA had specific binding reaction with REV, and did not react with other viruses. The detection limit of this assay was 195 TCID50 units of REV. Furthermore, commercial vaccine artificially contaminated with REV was detected by three methods: AC-ELISA, the TaqMan probe fluorescence real-time quantitative RT-PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The results showed that the positive coincidence rate of RT-qPCR and AC-ELISA was 90.63 %, and the positive coincidence rate of RT-qPCR and IFA was 96.88%, indicating that the AC-ELISA established in this study was effective and feasible. This method simplified the detection process for REV contamination in poultry attenuated vaccines, and provide necessary technical tools for high-throughput detection of REV.

Detection and molecular characterization of a highly oncogenic Marek's disease virus from vaccinated hens in Turkey

Marek's disease (MD) is a highly contagious neoplastic disease of chickens associated with economic losses, often due to visceral lymphomas. The etiological agent is MD virus serotype1 (MDV-1), also called Gallid alphaherpesvirus 2 (GaHV-2). Despite intensive vaccination, MDV is constantly evolving and maintaining its presence in the world. The aim of this study was to genetically analyze a highly oncogenic MDV/Tur/2019 strain obtained from a poultry farm in Turkey's Elazig province in 2019. Genes associated with viral pathogenicity and oncogenicity Marek's EcoRI-Q-encoded protein (MEQ), phosphoprotein-38 (pp38), and viral interleukin 8 (vIL-8) were selected for this purpose. The vIL-8 nucleotide sequence showed high similarity (100% identity) to some European (EU-1, Polen 5) and Asian (03 India, GADVASU-M2) MDV strains. The pp38 nucleotide sequence showed high similarity (100% identity) to some American (CU-2, JM/102W, RB1B) and European (MD70/13, ATE2539) MDV strains. There were no disrupted four-proline molecules (PPPP) within the transactivation domain of the MEQ. However, according to phylogenetic results, the MDV/Tur/2019 strain was included in cluster 2a alongside European MDV strains (Polish, Hungarian, Italian) with very virulent and very virulent plus pathotypes. In conclusion, we believe that the MDV/Tur/2019 strain obtained from turkey herpesvirus (HVT)-vaccinated chickens has a very virulent or very virulent plus pathotype. Although this result provides some clues regarding the virulence of this strain, in vivo studies are needed to achieve exact pathotyping. Further, combination of HVT with the CVI988 strain should be used for vaccination to provide the best protection, as highly pathogenic MDV strains can break sterile immunity against the HVT vaccine. Keywords: GaHV-2; Marek's disease; oncogenes; Turkey.

Marek's Disease Virus (Gallid alphaherpesvirus 2)-Encoded miR-M2-5p Simultaneously Promotes Cell Proliferation and Suppresses Apoptosis Through RBM24 and MYOD1-Mediated Signaling Pathways.

MicroRNAs (miRNAs) have been demonstrated for their involvement in virus biology and pathogenesis, including functioning as key determinants of virally-induced cancers. As an important oncogenic α-herpesvirus affecting poultry health, Marek’s disease virus serotype 1 [Gallid alphaherpesvirus 2 (GaHV-2)] induces rapid-onset T-cell lymphomatous disease commonly referred to as Marek’s disease (MD), an excellent biological model for the study of virally-induced cancer in the natural hosts. Previously, we have demonstrated that GaHV-2-encoded miRNAs (especially those within the Meq-cluster) have the potential to act as critical regulators of multiple processes such as virus replication, latency, pathogenesis, and/or oncogenesis. In addition to miR-M4-5p (miR-155 homolog) and miR-M3-5p, we have recently found that miR-M2-5p possibly participate in inducing MD lymphomagenesis. Here, we report the identification of two tumor suppressors, the RNA-binding protein 24 (RBM24) and myogenic differentiation 1 (MYOD1), being two biological targets for miR-M2-5p. Our experiments revealed that as a critical miRNA, miR-M2-5p promotes cell proliferation via regulating the RBM24-mediated p63 overexpression and MYOD1-mediated IGF2 signaling and suppresses apoptosis by targeting the MYOD1-mediated Caspase-3 signaling pathway. Our data present a new strategy of a single viral miRNA exerting dual role to potentially participate in the virally-induced T-cell lymphomagenesis by simultaneously promoting the cell proliferation and suppressing apoptosis.

Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/μL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Alterations in cellular and viral microRNA and cellular gene expression in Marek's disease virus-transformed T-cell lines treated with sodium butyrate.

A shared feature of herpesviruses is their ability to enter a latent state following an initially lytic infection. Marek's disease virus serotype 1 (MDV-1) is an oncogenic avian herpesvirus. Small RNA profiling studies have suggested that microRNAs (miRNAs) are involved in viral latency. Sodium butyrate treatment is known to induce herpesvirus reactivation. The present study was undertaken to determine transcriptome and miRNome changes induced by sodium butyrate in 2 MDV-transformed cell lines, RP2 and CU115. In the first 24 h post-treatment, microarray analysis of transcriptional changes in cell lines RP2 and CU115 identified 137 and 114 differentially expressed genes, respectively. Small RNA deep-sequencing analysis identified 17 cellular miRNAs that were differentially expressed. The expression of MDV-encoded miRNAs was also altered upon treatment. Many of the genes and miRNAs that are differentially expressed are involved in regulation of the cell cycle, mitosis, DNA metabolism, and lymphocyte differentiation.

Molecular detection of Marek's disease virus in feather and blood samples from young laying hens in Colombia.

Marek's disease virus (MDV) is an immunosuppressive pathogen that can cause low production efficiency and high mortality rates in chickens. There is no current information on the MDV serotypes and pathotypes circulating in vaccinated commercial farms in Colombia where the birds are vaccinated in the incubator with Gallid herpesvirus (GaHV-2) and Meleagrid herpesvirus 1 (MeHV-1). Based on that, the main focus of this study was to understand the MDV's infection dynamics for the three known serotypes and to detect wild-virus pathogenic strains in 4-layer poultry farms in Antioquia. Samples of blood, feathers and spleens were collected from three randomly chosen animals according to age category: 1, 15, 30, 60, 90, and 120 days. Quantitative real-time PCR (qPCR) that differentiates between the three serotypes of MDV was used to assess viral loads over time, and phylogenetic analysis of the Meq oncogene was done to compare the strains of MDV with those of known pathogenicity. Meleagrid herpesvirus 1 (MeHV-1) was detected in all blood and feather follicle samples with an average number of genome copies (per 10,000 cells) of 31.44 in blood as expected as a result of vaccination. GaHV-2 was also detected in almost 100% of the blood and feather follicle samples throughout all defined age categories, with an average of 10.65 genome copies in blood samples. Gallid herpesvirus 3 (GaHV-3) was detected in 72% of blood and 84.61% of feather samples, with less than 1 copy per 10,000 cells. Based on the number of 132 bp repeats of the BamHI-H and BamHI-D regions in pooled feather samples, there were 70% (8/25) of attenuated MDV and 30% (17/25) of virulent MDV strains circulating in the farms. Virus isolation was performed successfully from every farm. In conclusion, different strains of MDV are circulating for up to 120 days in layers in Antioquia-Colombia and could be of major impact in poultry health. Keywords: Marek's disease virus (MDV); Antioquia-Colombia; qPCR; PCR; Meq gene phylogeny.

Design of miRNA sponges for MDV-1 as a therapeutic strategy against lymphomas.

Lymphomas are solid-type tumors containing lymphoid cells. Some of latent herpesvirus infections established in B and/or T-lymphocytes could result in the formation of lymphomas. Marek's disease virus serotype 1 (MDV-1) is an avian herpes virus causing to lymphoproliferative tumors in birds, known as Marek’s disease (MD). MD has often been used as an ideal biological model for studying the pathogenesis of lymphoma diseases caused by viruses. Therefore, we used it as a research subject to study the effect of miRNA sponges on its tumorigenicity, and to develop the theoretical basis for a new anti-tumor small molecule. The miRNA sponges designed in this study specifically bind to and degrade the miRNAs of meq gene cluster of MDV-1, including miR-M2-3p, miR-M3-5p, miR-M5-3p, miR-M9-5p and miR-M12-3p.qPCR results showed that the knockdown efficiency was 85.03%, 74.97%, 47.06%, 75.33% and 62.55%, respectively. EDU staining and CCK-8 results showed that miRNA sponges inhibited the proliferation of MDV-1 transformed MSB-1 cells in vitro, and the proliferation rate of miRNA sponges-treated cells was about 50% of the control group. DAPI staining and Annxin V-FITC/PI double staining showed that miRNA sponges induced apoptosis in MSB-1 cells, and the apoptotic rate was increased by about 27.87% compared with the control group. The results of transwell showed that miRNA sponges could inhibit the invasion of MSB-1 cells in vitro, and the inhibitory rate was about 64.52%. The soft agar assay showed that miRNA sponges could inhibit the tumorigenic ability of MSB-1 cells in vitro, and the inhibitory rate was about 66.44%.The 60-days animal study showed that miRNA sponges could alleviate the growth inhibition of MSB-1 cells (about 14.78%) and reduce the mortality (about 16.00%). In addition, the tumor formation rate was 0 (8–12% in the control group).This study suggests that miRNA sponges can serve as an effective anti-tumor small molecule for the tumors caused by herpesvirus, with potential clinical implications.

Application of loop-mediated isothermal amplification assay in the detection of herpesvirus of turkey (FC 126 strain) from chicken samples in Nigeria.

Aim:This study was designed to optimize and apply the use of loop-mediated isothermal amplification (LAMP) as an alternative to conventional polymerase chain reaction (PCR) for the detection of herpesvirus of turkeys (HVT) (FC 126 strain) in vaccinated and non-vaccinated poultry in Nigeria.Materials and Methods:HVT positive control (vaccine) was used for optimization of LAMP using six primers that target the HVT070 gene sequence of the virus. These primers can differentiate HVT, a Marek’s disease virus (MDV) serotype 3 from MDV serotypes 1 and 2. Samples were collected from clinical cases of Marek’s disease (MD) in chickens, processed and subjected to LAMP and PCR.Results:LAMP assay for HVT was optimized. HVT was detected in 60% (3/5) and 100% (5/5) of the samples analyzed by PCR and LAMP, respectively. HVT was detected in the feathers, liver, skin, and spleen with average DNA purity of 3.05-4.52 μg DNA/mg (A260/A280) using LAMP. Conventional PCR detected HVT in two vaccinated and one unvaccinated chicken samples, while LAMP detected HVT in two vaccinated and three unvaccinated corresponding chicken samples. However, LAMP was a faster and simpler technique to carry out than PCR.Conclusion:LAMP assay for the detection of HVT was optimized. LAMP and PCR detected HVT in clinical samples collected. LAMP assay can be a very good alternative to PCR for detection of HVT and other viruses. This is the first report of the use of LAMP for the detection of viruses of veterinary importance in Nigeria. LAMP should be optimized as a diagnostic and research tool for investigation of poultry diseases such as MD in Nigeria.

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.

Turkey herpesvirus with an insertion in the UL3-4 region displays an appropriate balance between growth activity and antibody-eliciting capacity and is suitable for the establishment of a recombinant vaccine.

We constructed turkey herpesvirus (HVT) vector vaccines in which the VP2 gene of infectious bursal disease virus (IBDV) was inserted into the HVT genome in the following regions: UL3-4, UL22-23, UL45-46, and US10-SORF3. We then evaluated the relationship between the gene insertion site and the capacity of the virus to elicit antibodies. rHVT/IBD (US10) showed good growth activity in vitro, with growth comparable to that of the parent HVT. On the other hand, rHVT/IBD (UL3-4), rHVT/IBD (UL22-23), and rHVT/IBD (UL45-46) exhibited decreased growth activity in chicken embryo fibroblast (CEF) cells compared to the parent HVT. However, the rHVT/IBD (US10) elicited lower levels of virus-neutralizing (VN) antibodies compared to the other constructs. rHVT/IBD (UL3-4) and rHVT/IBD (UL45-46) appeared to be similar in their ability to elicit VN antibodies. Based on the results of in vitro and in vivo assays, rHVT/IBD (UL3-4) was selected for further testing. In a challenge assay, rHVT/IBD (UL3-4) protected chickens from challenge with virulent Marek's disease virus serotype 1 and IBDV. In conclusion, the site of gene insertion may have a strong effect on the growth of the vector virus in vitro and its antibody-eliciting capacity. Insertions in the UL3-4 region permitted a balance between growth activity and VN-antibody-eliciting capacity, and this region might therefore be an appropriate insertion site for IBDV VP2.

Evaluation of two strains of Marek's disease virus serotype 1 for the development of recombinant vaccines against very virulent infectious bursal disease virus

Attenuated strains of Marek's disease virus serotype 1 (MDV1), and the closely related herpesvirus of turkeys, are among the most potent vectors for development of recombinant vaccines for poultry. To investigate the effects of MDV1 strain characteristics on the protective efficacy of the recombinant vaccines, we developed two recombinant MDV1 vaccines for expressing the VP2 gene of infectious bursal disease virus (IBDV) based on two different MDV1 strains, the attenuated strain 814 and the Meq gene-deleted recombinant MDV1 strain rLMS△Meq, as the viral vectors. The r814-VP2 virus based on the 814 strain exhibited higher replication efficiency in cell culture while lower viral titers in chickens, compared to rLMS△Meq-VP2 derived from the rLMS△Meq strain. Further studies indicated that r814-VP2 produced higher levels of VP2 protein in cells and elicited stronger immune responses against IBDV in chickens than rLMS△Meq-VP2. After IBDV challenge, rLMS△Meq-VP2 provided 50% protection against mortality, and the birds that survived developed bursal atrophy and gross lesions. In contrast, r814-VP2 conferred complete protection not only against development of clinical signs and mortality, but also against the formation of bursal lesions. The results indicate that different MDV1 vector influences the protective efficacy of recombinant MDV1 vaccines. The r814-VP2 has the potential to serve as a bivalent vaccine against two important lethal pathogens of chickens.

Combination of Two Marek's Disease Virus Vectors Shows Effective Vaccination Against Marek's Disease, Infectious Bursal Disease, and Newcastle Disease.

Herpesvirus of turkeys (HVT) is a widely used vector for poultry vaccines. However, different HVTs expressing different foreign antigens cannot always be used simultaneously because of the risk of recombination and interference. In this study, we inoculated a mixture of an HVT-expressing the antigen of Newcastle disease virus (NDV; HVT/ND) and Marek's disease virus (MDV) serotype 1 Rispens virus expressing the antigen of infectious bursal disease virus (IBD; Ripens/IBD) into chickens. This mixture showed 94%, 100%, or 94% protection against MDV, IBDV, or NDV challenge, respectively. In conclusion, the combination of Rispens/IBD and HVT/ND is effective for vaccination against MDV, IBDV, and NDV without significant interference.

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.

Comparison of Loop-Mediated Isothermal Amplification and PCR for the Detection and Differentiation of Marek's Disease Virus Serotypes 1, 2, and 3

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.

Establishment of an immortal chicken embryo liver-derived cell line

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.