Avian Reticuloendotheliosis Virus Research Articles

Molecular characterization and immunopathological investigation of Avian reticuloendotheliosis virus in breeder flocks in Egypt

BackgroundReticuloendotheliosis virus (REV) is an oncogenic immunosuppressive retrovirus that infects different kinds of avian species; posing significant economic losses to the poultry industry worldwide.MethodsIn Egypt, there is an unidentified disease associated with the runting-stunting syndrome with neoplasia, suspected to be REV, that has been continuously monitored in several breeder flocks. To diagnose and analyze REV by cell cultures, enzyme-linked immunosorbent assay (ELISA), histopathological investigation, the polymerase chain reaction (PCR) test, and sequencing analysis, 200 blood samples, and 50 tissue specimens were collected. The current study targets the occurrence and genetic characteristics of a viral neoplastic disease, resembling REV infection, circulating in breeder flocks from 2022 to 2023 in the Ismailia, El-Sharqia, and El-Dakahliya governorates.ResultHere, REV was isolated on chicken embryo fibroblast cell culture; exhibiting cell aggregation, rounding, and cell detachments. Collectively, only 70 serum samples were positive for anti‐REV antibodies with seroprevalence rates of 35% based on the ELISA test. The histopathological observation demonstrated lymphoreticular tumors in the liver, spleen, and other examined organs. The immunohistochemical staining method confirmed the REV-positive signals in all examined organs (liver, kidney, spleen, bursa, ovaries) except for the heart. The PCR assay of the LTR gene assessed 370 base pairs with only 5 positive samples with a percentage of 16.6%. Three positive samples were further sequenced and submitted to the Genbank under accession numbers (PP763709, PP763710, PP763711). Phylogenetic analysis of the REV-LTR gene showed that our three isolates (Sharquia-1-REV, Ismilia-2-REV, Mansoura-3-REV) are REV subtype III which predominantly circulated in breeders in Egypt. These three isolates are highest similar to American, Chinese, and Taiwanese REV reference strains, and other Egyptian strains with nucleotide identity percentages of 100%, 99%, and 99%; respectively, and on the amino acid identity level were with (99–100%), (98%, 99%), (99%, 100%); respectively.ConclusionsThis study established that REV infection was extensively distributed in the breeders and became one of the causes of the clinical outbreaks of tumors, raising awareness of REV as the causative agent of avian oncogenic disease in Egypt.

A New Dual Fluorescence Method for Rapid Detection of Infectious Bronchitis Virus at Constant Temperature.

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I-V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/μL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I-V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype.

Molecular Method of Marek’s Disease Virus Serotype 2 Detection using Polymerase Chain Reaction in Chicken

Background: Marek’s disease is an oncogenic virus that produces malignant lymphomas in chickens. In this study Marek’s disease virus serotype 2 identification using polymerase chain reaction with unique gene. Methods: DNA isolated from MDV infected feather follicles, liver, spleen and commercially available serotype 2 vaccines (SB 1 and HVT combined bivalent, HVT monovalent) by phenol-chloroform DNA isolation method. Serotype 2 specific primers were designed by using primer 3.0 software targeting Long Open Reading Frame (LORF) gene-specific to serotype 2 SB 1 strain. DNA template from vaccines showed amplification of the LORF gene. PCR parameters were optimized and the analysis of sensitivity and specificity of primers was carried out. Result: MDV serotype 2 with an optimized annealing temperature of 53°C with primer concentration of 3 pmol/µl. LORF gene of SB1 of MDV did not cross-react with HVT and MDV 1 strains and also with other oncogenic viruses of Avian Leucosis Virus (ALV) and Reticuloendotheliosis Virus (REV). The sensitivity was analyzed based on the different DNA concentrations and amplification up to 0.5 ng/µl. Screening of field samples of 260 along with three MDV serotype 2 vaccine SB1 strain samples revealed that all the filed samples did not show positivity for MDV serotype 2 whereas in the vaccine strain samples of MDV serotype 2 vaccine SB1 showed positivity by amplification at 270bp and these results indicated that MDV serotype 2 SB1 strain did not cause an outbreak in chicken and might be inferred that no incidence of spontaneous Lymphoid Leucosis-like lymphomas with augmentation property of MDV serotype 2. This serotype 2 specific assay was used to detect Marek’s disease caused by serotype 2 which is commonly used as a vaccine.

Mannose-modified erythrocyte membrane-encapsulated chitovanic nanoparticles as a DNA vaccine carrier against reticuloendothelial tissue hyperplasia virus.

The erythrocyte membranes used in nanovaccines include high membrane stability, long circulation life, adaptability and extremely good bio compatibility. Nanoparticles encapsulated by erythrocyte membranes are widely used as ideal drug delivery vehicles because of their high drug loading, long circulation time, and excellent biocompatibility. The mannose modification of delivery materials can help target mannose receptors (MRs) to deliver antigens to antigen-presenting cells (APCs). In this study, the antigen gene gp90 of avian reticuloendotheliosis virus (REV) was encapsulated with carboxymethyl chitosan (CS) to obtain CSgp90 nanoparticles, which were coated with mannose-modied fowl erythrocyte membranes to yield CS-gp90@M-M nanoparticles. The physicochemical characterization and immune response of the CS-gp90@M-M nanoparticles were investigated in vitro and in vivo. CS-gp90@M-M nanoparticles were rapidly phagocytized in vitro by macrophages to induce the production of cytokines and nitric oxide. In vivo, CS-gp90@M-M nanoparticles increased cytokine levels, the CD4+/8+ ratio, REV-specific antibodies in the peripheral blood of chicks, and the mRNA levels of immune-related genes in the spleen and bursa of immunized chicks. CS-gp90@M-M nanoparticles could be targeted to lymphoid organs to prolong the retention time of the nanoparticles at the injection site and lymphatic organs, leading to a strong, sustained immune response. Moreover, the CS-gp90@M-M nano-vaccine showed a lasting immunoprotective effect and improved the body weight of chicks after the challenge. Overall, CS-gp90@M-M nanoparticles can be used in vaccine designs as an effective delivery carrier with immune response-enhancing effects.

An Investigation of the Cause of Wild Turkey Mortality in Mississippi.

Lymphoproliferative disease virus (LPDV) is an exogenous alpharetrovirus that sporadically causes fatal lymphoid neoplasia in affected turkeys. Previous studies of wild turkeys (Meleagridis gallopavo) in the United States have demonstrated geographically widespread LPDV infection and frequent coinfection with avian poxvirus (APV) and reticuloendotheliosis virus (REV). This study was conducted to better understand health risks to Mississippi wild turkeys, including the relative importance of LPDV, APV, and REV in contributing to mortality. Thirteen wild turkeys, which died naturally or were euthanized due to illness, were submitted to Mississippi State University's Poultry Research and Diagnostic Laboratory for postmortem examinations. Birds originated from nine counties across the state over the past 5 yr. Carcasses were submitted as fresh (nonfrozen) or frozen. At autopsy, 9 of 13 turkeys had severe, proliferative cutaneous lesions on the head and neck, with diphtheritic or proliferative oral and esophageal lesions. Samples were collected for molecular diagnostic testing (LPDV and REV PCR), histopathology, and bacterial culture and isolation. External and internal parasites were preserved in formalin for identification. APV (cutaneous and/or diphtheritic forms) was diagnosed in 9 of 13 birds by identification of pathognomonic histologic lesions (including intracytoplasmic inclusion bodies). Interestingly, all birds with APV were also REV PCR positive. Furthermore, eight turkeys were positive for LPDV, and LPDV was commonly associated with coinfections with APV and REV.

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.

An optimized secretory expression system and immunogenicity evaluation for glycosylated gp90 of avian reticuloendotheliosis virus

Reticuloendotheliosis is an important immunosuppressive disease, associated with avian reticuloendotheliosis virus (REV) infection, and causes notable economic losses worldwide. Glycoprotein gp90 is an important structural protein of REV, and considered to be the most important immunogenic antigen, which can induce neutralizing antibodies against REV. In this study, an optimized suspension culture system was developed and applied to secretory express the immunogenic surface antigen gp90. To achieve an optimal glycosylation, the gp90 was designed to secretory expressed into the supernatant of the cell culture, which also occurs in the natural protein maturation procedure of REV. Serum-free culture medium was introduced to simplify the purification process and reduce the production costs. Based on the purified glycosylated gp90, an oil-emulsion subunit REV vaccine candidate was developed and evaluated in chickens. The subunit gp90-based vaccine induced fast immune responses, high levels of antibodies (REV-specific antibody, gp90-specific antibody, and neutralizing antibody against REV), and preferential T helper 2 (Th2) (interleukin-4 secretion) not Th1 (interferon-γ secretion) response. Furthermore, the viremia induced by REV infection was significantly reduced in chickens immunized with the glycosylated gp90. Overall, an optimized secretory expression system for glycosylated gp90 was developed, and the glycosylated gp90 obtained in this study retained good immunogenicity and could be an attractive vaccine candidate to protect chickens against REV horizonal infection.

Oncogenic Viruses and Their Molecular Diagnosis in Poultry

Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock and human populations, limit productivity and causes significant economic losses for each. Avian oncogenic viruses are one of the most dangerous pathogenic microorganisms that threaten the poultry industry and cause damage of over billions dollars annually worldwide. These viruses include a highly contagious herpesvirus Marek’s disease virus (MDV), as well as retroviruses such as avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). Each group is distinguished by its nucleic acid type, antigenicity, epidemiology, host range and molecular characteristics. These viruses are cells associated with tumors and are in all organs except in the feather follicle where enveloped infectious virions egress from the body. It is difficult to detect diseases caused by these viruses and at the same time, vaccines that can provide sterile immunity against these diseases and prevent infection are incomplete. In order to be able to fight oncogenic viruses more effectively in the future, it is vital to learn more about the host immunity-oncovirus interaction and to determine powerful diagnostic techniques. In this review, oncogenic viruses and effective diagnostic techniques for these viruses are emphasized in poultry.

Safety and Efficacy Evaluation of Recombinant Marek's Disease Virus with REV-LTR.

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek’s disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.

Gut microbiota profiles of commercial laying hens infected with tumorigenic viruses

BackgroundStudies 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.ResultsA 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.ConclusionsThe 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.

Isolation and pathogenicity testing of avian reticuloendotheliosis virus from layer chickens in China.

Reticuloendotheliosis virus (REV) can cause runting, immunosuppression, acute reticulum cell neoplasia, and chronic lymphoid tumors in a variety of domestic and wild birds. We diagnosed a case of reticuloendotheliosis with obvious tumors in liver and kidney. We isolated and sequenced the virus and performed pathogenicity testing of the REV strain. Immunohistochemistry and PCR confirmed that the diseased layer chickens were infected with REV. The strain, named BJ1503, was successfully isolated from the case by inoculation of tissue homogenates onto chicken embryo fibroblasts. The length of the proviral REV genome is 8,293 nucleotides. The isolate had 99.7% identity with REV-HA9901 (AY842951.1), which was isolated from Jiangsu, China, in 1999. The chickens infected with REV-BJ1503 had depressed weight gain and lymphoid atrophy. Our findings suggest that REV isolate BJ1503 was phylogenetically close to the earlier strain found in China, with minor variations, and the virus was associated with severe production problems.

Integrated Analysis of miRNA and mRNA Expression Profiles in Spleen of Specific Pathogen-Free Chicken Infected with Avian Reticuloendotheliosis Virus Strain SNV.

The Reticuloendotheliosis virus (REV) primarily causes avian severe immunosuppression, in addition to other symptoms, which include avian dwarfing syndrome and chronic tumors in lymphoid and other tissue. To date, REV’s molecular mechanisms leading to immunosuppression have not been fully elucidated. In the current study, we aimed to elucidate the role of microRNAs (miRNA) in regulating gene expression during REV infections. Therefore, we used a high-dose spleen necrosis virus (SNV) model of REV to inoculate one-day-old specific pathogen-free (SPF) chickens, thereby inducing congenital infections. We analyzed miRNA and mRNA expression profiles using Next Generation Sequencing (NGS) in a total of 19 spleen samples that were collected at 7, 14, and 21 days post infection (dpi). The results showed that 63 differentially expressed miRNAs (DEmiRNAs) (30 known miRNAs and 33 novel miRNAs) and 482 differentially expressed target genes (DETGs) were identified. Integration analysis identified 886 known miRNA–mRNA and 580 novel miRNA–mRNA interaction pairs, which involved miRNAs that were inversely correlated with the above DETGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DETGs were considerably enriched in the immune-relevant pathways category, such as immune system, cell growth and death, signaling molecules and interaction, signal transduction, etc. We further verified selected immune-relevant miRNA and their DETGs while using quantitative RT-PCR (qRT-PCR). Overall, our data revealed valuable immune-related miRNA–mRNA interaction information that occurred during REV infections, thereby broadening our understanding of the REV-induced immunosuppression.

Epidemiological investigation of the novel genotype avian hepatitis E virus and co-infected immunosuppressive viruses in farms with hepatic rupture haemorrhage syndrome, recently emerged in China.

Since 2016, hepatic rupture haemorrhage syndrome (HRHS) appeared in chickens of China and caused huge economic loss. To assess the infection status of the avian hepatitis E virus (HEV) and co-infected viruses, including avian leukosis virus (ALV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV), and chicken infectious anaemia virus (CIAV), in farms with HRHS, 180 liver samples were collected from 24 farms in different provinces and detected by strict molecular virology methods. Results showed that the positive rates of HEV, ALV, REV, FAdV, and CIAV were 74.44%, 20.00%, 27.78%, 31.11%, and 12.22%, respectively, whereas there are also 112 samples with co-infection, for a rate of 58%. Meanwhile, the positive rate of HEV decreased gradually with age; the lowest positive rate of ALV (5.76%) and REV (19.23%) appeared in 25-35weeks age, during which the positive rate of CIAV was the highest (19.23%); the positive rate of HEV in layers (64.00%) was lower than that of broilers (83.33%), but the positive rates of ALV (38.46%) and CIAV (15.38%) in layers were higher than that of broilers (5.88%, 9.80%); the positive rates of HEV (75.88%) and CIAV (15.60%) in parental generation (PG) were higher than that of commodity generation (CG, 64.10%, 0.00%), whereas the positive rate of ALV showed inverse relationship (PG: 14.89%; CG: 38.46%). Additionally, phylogenetic analysis showed that all the avian HEV identified this study belong to a novel genotype, and found the close relationship between the wild strains (REV and CIAV) and corresponding isolates from contaminated vaccine. The data presented in this report will enhance the current understanding of the epidemiology characteristics in farms with HRHS in China.

Avian Reticuloendotheliosis in Chickens - An Update on Disease Occurrence and Clinical Course.

Avian reticuloendotheliosis (RE) represents an important immunosuppressive disease of poultry. The occurrence of RE in both chickens and turkeys has an immunosuppressive effect and may lead to vaccination failures. Avian reticuloendotheliosis virus (REV) is widely distributed in different kinds of birds, causing subclinical infections. Another important issue adhering to this disease is contamination of vaccines against fowl pox (FP) and Marek’s disease (MD) with REV. The capability of REV to integrate into the genome of other larger DNA viruses complicates its diagnosis and prevention. There are no efficient vaccines against RE nor treatment, which also complicates how to limit its impact on poultry farming. This paper reviews the current state of knowledge of this important immunosuppressive agent of poultry emphasising the importance of this problem in terms of diagnosis of RE.

Detection of Fowlpox virus carrying distinct genome segments of Reticuloendotheliosis virus

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam’s wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.

Molecular detection and phylogenetic analysis of avian reticuloendotheliosis virus from formalin fixed tissue by PCR in fancy chicken

The present study was carried out to identify avian neoplastic viruses in the formalin fixed tissues of fancy chicken. The tissue samples collected during necropsy were examined by histopathology. It showed lymphoid and reticular cell infiltration in kidney, liver and lungs. For identification and differentiation of avian neoplastic viruses, PCR was performed using primer sets specific for Mareks disease virus, avian leukosis complex and reticuloendotheliosis virus. It was found that tumors were REV originated. Further confirmation, purified PCR product was subjected to sequencing. It showed 99% homology with other REV isolates available in the NCBI database. The present communication describes infection of a fancy chicken with REV on the basis of histopathological findings as well as molecular methods.

Development of reliable techniques for the differential diagnosis of avian tumour viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections

ABSTRACTA variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek’s disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek’s disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analysed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek’s disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.

Protection induced by a gp90 protein-based vaccine derived from a Reticuloendotheliosis virus strain isolated from a contaminated IBD vaccine

BackgroundReticuloendotheliosis is an immunosuppressive disease caused by avian reticuloendotheliosis virus (REV). It is commonly found in poultry farms and has caused a notable economic loss worldwide. Despite this, there is currently no effective vaccine available to protect against REV infection.MethodIn this study, gp90 protein derived from an REV isolated from a contaminated vaccine was co-administered with cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) adjuvant to hens to determine if it protects their chicks against REV infection. To synthesize the gp90 protein, the gp90 gene was amplified using polymerase chain reaction, expressed in Escherichia coli, and purified. The resulting recombinant protein was injected intramuscularly into breeder hens along with CpG-ODN adjuvant and then serum antibody levels were regularly evaluated. After the fertilized eggs from these vaccinated hens had hatched, the resulting chicks were challenged with a 102.7 50% tissue culture infectious dose (TCID50) of REV at 1 day old and the REV antibody levels in these hatched chickens were evaluated before and after the challenge. Viremia and growth rate were measured weekly and statistically analyzed.ResultsThe results suggest that the gp90 recombinant protein was successfully prepared and, when used with CpG-ODN adjuvant to immunize breeder hens, induced serological antibody production against REV in both hens and their hatched chicks. In addition, the maternal antibodies induced by the gp90 protein vaccine effectively protected majority of the chicks from REV infection.ConclusionsOverall, we found the gp90 protein obtained in this study may be a potential vaccine candidate that had good immunogenicity and could be an auxiliary measure to accelerate the eradication of REV.

Naturally Occurring Co-infection of Avian Leukosis Virus (subgroups A-E) and Reticuloendotheliosis Virus in Green Peafowls (Pavo muticus)

ABSTRACT Most of the neoplastic diseases affecting poultry have a viral etiology, such as Marek’s disease, avian leukosis/sarcoma and reticuloendotheliosis. In the present paper, histopathology and molecular methods were performed, in addition of necropsy and gross morphology evaluation, for the differential diagnosis of these diseases in two green peafowls (Pavo muticus). The affected birds showed non-specific signs, such as depression, anorexia, and diarrhea. At necropsy, diffuse enlargement by several times the normal size of the liver and the spleen was observed. In one of the birds, the liver presented white to grayish discoloration was present, and focal and nodular growths in the other bird. Grossly, the other visceral organs and the peripheral nerves were normal. Microscopically, most of the tumor cells were large and uniform with pleomorphic nuclei. The PCR analysis for Marek’s disease virus (MDV), avian leukosis virus (ALV), lymphoproliferative disease virus (LPDV), and reticuloendotheliosis virus (REV)based on their specific sequences showed the presence of ALV subgroups A-E and REV. According to these results, definitive diagnosis was co-infection of ALV and REV in naturally-infected green peafowls.

Molecular characterization and phylogenetic analysis of a virulent Marek’s disease virus field strain in broiler chickens in Japan

ABSTRACTMarek’s disease is a lymphoproliferative disease causing a serious threat in poultry production. Field strains of Marek’s disease virus (MDVs) are continuously re-emerging, causing great economical losses to the poultry industry worldwide in spite of the intensive vaccination and restrictive management policy used. Histopathological and molecular characterizations of MDVs are essential for monitoring the changes of viruses and evaluating the effectiveness of existing vaccines. During 2016, 190 visceral tumour tissues representing 30 vaccinated chicken flocks from the Gifu prefecture, Japan, were analysed. A pathological examination revealed the presence of lymphoproliferative lesions in the visceral organs. Polymerase chain reaction screening of tissue specimens using specific primers for avian leucosis virus, reticuloendotheliosis virus, and MDV was positive only for MDV. The polymerase chain reaction products of meq, pp38, virus-induced IL-8 homology, and glycoprotein MDV genes were sequenced and used for homology, phylogenetic, and similarity level analysis with the published reference of MDVs in the database. The results revealed high similarity between the field isolates, vv and vv+ strains of MDV from the USA and China. Several point mutations in the nucleotide sequence of the field isolates and their deduced amino acid sequences were detected in those genes. The present molecular analyses indicated that nucleotide and amino acid changes could be valuable criteria for differentiation and determination of the pathogenicity and oncogenicity of MDVs according to the Avian Disease and Oncology Laboratory pathotyping in vivo studies. Furthermore, the results suggest that development of a new vaccine must be considered to overcome this devastating avian oncogenic viral disease.