Complete genome sequence analysis of Reticuloendotheliosis virus integrated in nonhomologous Avipoxvirus.

Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirus（APV） has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus.

Isolation and identification of a pigeonpox virus strain and study on the integration of reticuloendotheliosis virus sequence.

In order to study the integration of reticuloendotheliosis virus (REV) in pigeonpox virus (PPV), we collected suspected pigeonpox disease material, amplified the 4b core protein gene of PPV, the gp90 gene of REV, and the integrated sequence fragments from the end of the ORF201 segment of PPV to the beginning of the LTR of REV, and sequenced these genes. The results showed that a 4b core protein fragment of 332 bp was amplified and identified as pigeonpox virus, which was named SX/TY/LTR 01/2023. Sequence analysis showed that the pigeonpox virus isolate belonged to genotype A2, which was the closest to the domestic CVL strain, with a identity of 99.4%. A band of 1191 bp was amplified from the gp90 gene of REV, named SX/TY/PPV-REV01/2023, and sequence analysis indicated that REV belonged to genotype III. The sequence analysis showed that REV belonged to genotype III, and belonged to the same large branch as the domestic isolates JSRD0701 and LNR0801, with 99.3% identity. The integrated sequence fragment was amplified to a band of 637 bp, which determined that the REV sequence was integrated in the PPV rather than a mixed infection of the two viruses. This indicates that REV was integrated in this isolation of PPV, suggesting that pigeon farms need to prevent reticuloendotheliosis at the same time when preventing pigeonpox.

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.

Isolation of Reticuloendotheliosis Virus from Chorio-allantoic Membrane of SPF Chicken Eggs inoculated with Fowl Pox Virus.

Fowl Pox Viruses (FPV) infect chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth etc. The birds, affected with FPV, also show anemia and ruffled appearance which are clinical symptoms of Reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of Reticuloendotheliosis Virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the Chorio-allantoic Membrane (CAM) of 10 days old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. But the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV virus, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

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.

Emergence of spontaneously occurring neoplastic disease caused by reticuloendotheliosis virus in breeding Muscovy ducks in China, 2019.

Reticuloendotheliosis virus (REV) has a wide range of avian hosts leading to reticuloendotheliosis, and its characteristic of vertical transmission makes it to be one of the most important diseases in breeder avian populations. Up to date, reports on neoplastic disease caused by REV in breeding ducks are few. Here, spontaneously occurring neoplastic disease caused by REV in breeding Muscovy ducks was reported in Guangdong province, China. The most significant gross lesions of sick ducks were tumour-bearing liver and enlarged spleen. Histopathological examination found proliferation of malignant lymphoreticular cells in the liver and reticuloendothelial cells in the spleen. REV strain, CH-GD2019, was successfully isolated using DF-1 cells, and the presence of REV was confirmed by PCR detection and transmission electron microscopy. The length of complete proviral genome is 8,238 nucleotides. Genetic and phylogenetic analyses revealed that CH-GD2019 was closely related to chicken-origin REV strains circulating in China. The results will provide a basic data for better understanding of REV in breeding ducks and suggest that REV from chickens may be a threat to ducks.

A high frequency of Gallid herpesvirus-2 co-infection with Reticuloendotheliosis virusis associated with high tumor rates in Chinese chicken farms.

The prevalence of Marek's disease (MD) caused by Gallid herpesvirus-2 (GaHV-2) has been increasing in chickens in China despite universal vaccination. Among the possible reasons for this trend, of Reticuloendotheliosis virus (REV) contamination in vaccines could lead to co-infection and reduce the vaccine efficacy. Here, we report the epidemiological findings of our continuous surveillance of MD, and an examination of the effects of REV and/or GaHV-2 co-infection. A total of 1230 samples were collected between 2011 and 2015 from 305 flocks covering many of the chicken-raising regions of China. Among these, 606 samples were determined to be GaHV-2-positive, 13.0% of which were found to be co-infected with REV from 18.8% of the flocks. One GaHV-2 strain (HS/1412), a REV strain (HS/1412R), and a GaHV-2 and REV-co-infected strain (HS/1412 GR) were isolated from different chickens of a GaHV-2 and REV co-infected flock. Pathogenicity tests showed that HS/1412 and HS/1412 GR caused disease in all chickens and that HS/1412R induced morbidity in 84.6% of the infected chickens. HS/1412 GR induced 100% mortality and 76.9% tumor formation, which were significantly higher frequencies than those observed with strain HS/1412 (38.5% and 15.4%, respectively) and HS/1412R (0% and 0%). These results indicate that co-infection with GaHV-2 and REV might explain the persistent, sporadic outbreaks of neoplastic disease in some commercial flocks, resulting in a significant economic burden to the poultry industry of China.

Assessment on reticuloendotheliosis virus infection in specific-pathogen-free chickens based on detection of yolk antibody.

Reticuloendotheliosis virus (REV) is the most frequent exogenous virus that contaminates attenuated vaccines. Therefore, it is extremely important to select REV-free specific-pathogen-free (SPF) chicken embryos. Generally, REV infection is assessed by detecting REV antibodies in SPF chickens. This present study seeks to evaluate REV infection by replacing serum antibody detection with yolk antibody detection. A cohort of 40 nineteen-week-old SPF chickens were artificially inoculated with REV, with 32 SPF chickens raised in another isolation environment served as a blank control. Eggs and serum from 23-week-old chickens were sampled, and yolks were diluted separately to ratios of 1:150, 1:200, 1:300 and 1:400, which were detected together with serum. We found that the yolk antibody detection findings at a dilution of 1:300 had the highest coincidence rate compared with that based on serum antibody measurements. At a dilution ratio of 1:300 for yolk antibody, 72 chickens were continuously observed for 10 weeks from 25- to 34-weeks-old. Our findings were based on serum antibody or yolk antibody detection, and the evaluation results were completely consistent. Therefore, all serum antibody-positive chickens were yolk antibody-positive, and vice versa. Accordingly, vaccine producers can estimate REV cleanliness in a poultry farm by sampling yolk antibody titers.

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

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.

A 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.

The experimental transmission of reticuloendotheliosis virus by cock semen.

Following artificial insemination, the egg-laying rate of a large-scale breeder chicken flock declined by10-15 %. Real-time quantitative polymerase chain reaction (qPCR) analysis detected the presence of reticuloendotheliosis virus (REV) in semen from the breeder cocks used. Six REV strains were successfully isolated from semen randomly extracted from those cocks. Additionally, the whole sequence of SDAUR-S1 was sequenced and analysed. Cock models with continuous production of REV-positive semen were established by intravenous injection with SDAUR-S1. Eggs were then collected from hens after artificial insemination with REV-positive semen, for virus detection. The positive REV antibody rate for egg albumen was 58.3 % and the REV-positive rate for hatched embryos was 8.3 %, which suggested not only that REV can infect cock semen, but can also infect the offspring. In conclusion, the present study is the first to report on the isolation, genome analysis and transmission of REV in cock semen.

Wild Birds, a Source of Reticuloendotheliosis Virus Infection for the Endangered Attwater's Prairie-Chicken ( Tympanuchus cupido attwateri)?

Reticuloendotheliosis virus (REV) infects a wide range of avian species. Since 1998, when it was first reported in a captive flock of the endangered Attwater's Prairie-chicken ( Tympanuchus cupido attwateri; APC), REV has plagued APC recovery efforts. While REV frequently occurs in captive bird flocks throughout the world, including commercial poultry, the reservoir for initial infection of flocks is poorly understood. From 2008-16, 412 blood samples and 216 liver samples collected from 32 species of birds on or near Attwater Prairie Chicken National Wildlife Refuge in Colorado County, Texas, US, and 89 blood samples obtained from a Texas game farm that provides thousands of Northern Bobwhites ( Colinus virginianus ) and Ring-necked Pheasants ( Phasianus colchicus ) for hunting throughout Texas, were tested for REV by real-time PCR. Of the 717 samples, one liver sample from a Savannah Sparrow ( Passerculus sandwichensis ) and three blood samples from game farm Ring-necked Pheasants tested positive for REV. These data, although limited, indicate a low prevalence of REV in birds sharing or in close proximity to APC habitat. More-extensive surveillance testing is warranted to determine the spatial and temporal dynamics of REV in wild bird populations and the relative role these birds may play as potential reservoirs for maintaining REV infections in both the wild and captive setting.

Gp85 genetic diversity of avian leukosis virus subgroup J among different individual chickens from a native flock.

To compare the genetic diversity and quasispecies evolution of avian leukosis virus (ALV) among different individuals, 5 chickens, raised in Shandong Provice of China, were randomly selected from a local chicken flock associated with serious tumor cases. Blood samples were collected and inoculated into chicken embryo fibroblast and DF-1 cell lines for virus isolation and identification, respectively, of Marek's disease virus (MDV), reticuloendotheliosis virus (REV), and ALV. Five strains of ALV subgroup J (ALV-J) were identified, and the gp85 gene from each strain was amplified and cloned. For each strain, about 20 positive clones of gp85 gene were selected for sequence analyses and the variability of the quasispecies of the 5 strains was compared. The results showed that the nuclear acid length of gp85 gene of 5 ALV-J isolates is 921 bp, 921 bp, 924 bp, 918 bp, and 912 bp respectively, and amino acid homologies of different gp85 clones from the 5 ALV-J strains were 99.3 to 100%, 99.3 to 100%, 99.4 to 100%, 98.4 to 100%, 99.0 to 100%, respectively. The proportions of dominant quasispecies were 65.0%, 85.0%, 85.0%, 50.0%, 84.2%, respectively, and homology of the gp85 among these dominant quasispecies was 89.2 to 92.5%. These data demonstrated the composition of the ALV-J quasispecies varied among infected individuals even within the same flock, and the dominant quasispecies continued to evolve both for their proportion and gene mutation.

Genome analysis and pathogenicity of reticuloendotheliosis virus isolated from a contaminated vaccine seed against infectious bursal disease virus: first report in China.

Specific-pathogen-free (SPF) chickens were inoculated with the virus seed of an infectious bursal disease virus (IBDV)-attenuated vaccine, and positive reticuloendotheliosis virus (REV) antibody levels were subsequently detected in the chicken sera, indicating potential REV contamination of the vaccine. After neutralization with IBDV-positive blood serum, the vaccine was inoculated into DF-1 cells for REV isolation and identification. An REV strain, designated IBD-C1605, was identified using an immunofluorescence assay test. Three pairs of primers were employed for the amplification, cloning and sequencing of three overlapping fragments of the IBD-C1605 genome, and the whole-genome sequence of this isolate was obtained after gene assembly. The genome was 8362 base pairs (nt) in length and its homology with the nucleotide sequences of different reference strains varied between 94.2 and 99.2 %. Isolate IBD-C1605 was inoculated into 1-day-old SPF chickens to observe its pathogenicity. Infection with this organism slowed down the weight gain of SPF chickens and caused atrophy of their immune organs, such as the bursa of Fabricius and thymus gland. Furthermore, the chicken antibody levels decreased significantly after Newcastle disease virus and avian influenza virus subtype H9 vaccine immunization. This is the first report on the isolation and identification of REV from attenuated vaccine virus seeds in China, and is also the first study on the pathogenicity of REV from a contaminated vaccine in China. Our findings contribute towards a better understanding of the detrimental effects of vaccine contamination with exogenous viruses such as REV.

Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).

Emergence of reticuloendotheliosis virus in pigeons in Guangdong Province, Southern China.

Reticuloendotheliosis virus (REV), an important immunosuppressive pathogen, has many hosts, including chickens, ducks, geese, turkeys, and wild birds. Clinically, REV may lead to increased susceptibility to other pathogens, resulting in serious tissue damage (especially tumors) and the death of its host. In this study, we encountered a disease outbreak resulting in a large number of deaths of pigeons in Guangdong Province, Southern China. Histopathological analysis revealed apparent tumor-like lesions in multiple organs of pigeons. PCR assays for detection of tumor-associated pathogens (REV, avian leukosis virus, and Marek's disease virus) in poultry revealed the presence of REV sequences only. Moreover, fowlpox virus (FPV) with an insertion of REV long terminal repeat (LTR) sequences was also considered, but it was excluded using a specific PCR assay. To gain more genetic information, two full-length REV genome sequences were determined and found to have the highest nucleotide sequence similarity (99.9 %) and the closest genetic relationship to a vaccine strain (MD-2) and had a more distant genetic relationship (94.3 %) to a duck-origin strain (ATCC-VR775). To confirm the presence of REVs in pigeons, specific-pathogen-free (SPF) chickens and healthy pigeons were inoculated with microfiltered tumor tissue homogenates and were found to be susceptible to infection with REV. To our knowledge, this is the first report of REV in pigeons, and the data suggest that pigeons may be the natural host of REV.

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.

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.

Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray.

Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions.

Synergetic effects of subgroup J avian leukosis virus and reticuloendotheliosis virus co-infection on growth retardation and immunosuppression in SPF chickens.

To further understand the effect of co-infection of subgroup J avian leukosis virus (ALV-J) and reticuloendotheliosis virus (REV) in specific-pathogen-free (SPF) white leghorn chickens, the experiment was made to study the pathogenicity, the weight of body and immune organs, response to newcastle disease virus (NDV) and avian influenza virus subtype H9 (AIV-H9) vaccination. Chickens were randomly divided into four groups, which includes injection groups (REV, ALV-J, REV plus ALV-J), and negative control group. The pathogenesis experiments indicated that chickens co-infected with REV and ALV-J had significantly higher mortality rate than those of the chickens infected with REV or ALV-J alone (P<0.05). Chickens inoculated with REV and ALV-J had significantly lower weights than chickens in all other groups (P<0.05). There were no significant differences between the two single infection groups and co-infection group (P>0.05) on bursa and thymus over body wt ratios, however, chickens co-infected with REV and ALV-J had significantly lower titers than REV-infected chickens and ALV-J-infected chickens on HI antibody titers to ND and AIV-H9 after vaccination (P<0.05). These findings suggested that the co-infection of REV and ALV-J caused more serious growth retardation and immunosuppression in SPF chickens.