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.

Molecular characterization of the meq oncogene of Marek's disease virus in vaccinated Brazilian poultry farms reveals selective pressure on prevalent strains.

Marek's disease virus (MDV) has become an increasingly virulent pathogen in the poultry industry despite vaccination efforts to control it. Brazil has experienced a significant rise of Marek's disease (MD) outbreaks in recent years. Our study aimed to analyze the complete meq gene sequences to understand the molecular epidemiological basis of MD outbreaks in Brazilian vaccinated layer farms. We detected a high incidence rate of visceral MD (67.74%) and multiple circulating MDV strains. The most prevalent and geographically widespread genotype presented several clinical and molecular characteristics of a highly virulent strain and evolving under positive selective pressure. Phylogenetic and phylogeographic analysis revealed a closer relationship with strains from the USA and Japan. This study sheds light on the circulation of MDV strains capable of infecting vaccinated birds. We emphasize the urgency of adopting preventive measures to manage MDV outbreaks threatening the poultry farming industry.

Differences in Pathogenicity and Vaccine Resistance Discovered between Two Epidemic Strains of Marek's Disease Virus in China.

Despite highly effective vaccines, Marek's disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 and DH/18) with clinically different pathotypes. We studied each strain's infection process and pathogenicity and observed differences in immunosuppression and vaccine resistance. Specific pathogen-free chickens, unvaccinated or vaccinated with CVI988, were challenged with AH/1807 or DH/18. Both infections induced MD damage; however, differences were observed in terms of mortality (AH/1807: 77.8%, DH/18: 50%) and tumor rates (AH/1807: 50%, DH/18: 33.3%). The immune protection indices of the vaccine also differed (AH/1807: 94.1, DH/18: 61.1). Additionally, while both strains caused interferon-ß and interferon-γ expression to decline, DH/18 infection caused stronger immunosuppression than AH/1807. This inhibition persisted even after vaccination, leading to increased replication of DH/18 that ultimately broke through vaccine immune protection. These results indicate that both strains have different characteristics, and that strains such as DH/18, which cause weaker pathogenic damage but can break through vaccine immune protection, require further attention. Our findings increase the understanding of the differences between epidemic strains and factors underlying MD vaccination failure in China.

Marek's disease in chicken farms from Northwest Ethiopia: gross pathology, virus isolation, and molecular characterization.

Marek's disease virus (MDV) is a highly contagious, immunosuppressive, and oncogenic chicken pathogen causing marek's disease (MD). In this outbreak-based study, 70 dual-purpose chickens that originated from poultry farms in Northwest Ethiopia and suspected of MD were sampled for pathological and virological study from January 2020 to June 2020. Clinically, affected chickens showed inappetence, dyspnea, depression, shrunken combs, and paralysis of legs, wings, and neck, and death. Pathologically, single or multiple greyish white to yellow tumor-like nodular lesions of various size were appreciated in visceral organs. In addition, splenomegaly, hepatomegaly, renomegaly, and sciatic nerve enlargement were observed. Twenty-seven (27) pooled clinical samples i.e. 7 pooled spleen samples and 20 pooled feathers samples were aseptically collected. Confluent monolayer of Chicken Embryo Fibroblast cells was inoculated with a suspension of pathological samples. Of this, MDV-suggestive cytopathic effects were recorded in 5 (71.42%) and 17 (85%) pooled spleen and feather samples respectively. Molecular confirmation of pathogenic MDV was conducted using conventional PCR amplifying 318 bp of ICP4 gene of MDV-1, of which, 40.9% (9/22) tested positive. In addition, 5 PCR-positive samples from various farms were sequenced further confirming the identity of MDV. The ICP4 partial gene sequences were submitted to GenBank with the following accession numbers: OP485106, OP485107, OP485108, OP485109, and OP485110. Comparative phylogenetics showed, two of the isolates from the same site, Metema, seem to be clonal complexes forming distinct cluster. The other three isolates, two from Merawi and one from Debretabor, appear to represent distinct genotypes although the isolate from Debretabor is closer to the Metema clonal complex. On the other hand, the isolates from Merawi appeared genetically far related to the rest of the 3 isolates and clustered with Indian MDV strains included in the analysis. This study presented the first molecular evidence of MDV in chicken farms from Northwest Ethiopia. Biosecurity measures should strictly be implemented to hinder the spread of the virus. Nationwide studies on molecular characteristics of MDV isolates, their pathotypes, and estimation of the economic impact associated with the disease may help justify production and use of MD vaccines within the country.

Investigation of a herpesvirus outbreak in mixed breeds of adult domestic ducks using next generation sequencing.

This report characterizes the first lethal outbreak of Marek's disease on a large farm of mixed-breed adult ducks (>18,000) and identifies the pathogen that resulted in high mortality (35%). Clinical signs included inappetence, respiratory distress, depression, muscle weakness, and ataxia. Post mortem revealed enlarged fragile liver mottled with miliary whitish spots and an enlarged spleen. Histopathology revealed hepatocellular necrosis with eosinophilic intra-nuclear inclusion bodies, necrosis of splenic follicles and degeneration/necrosis of renal tubules. The disease was tentatively diagnosed as a herpesvirus infection, confirmed by virus isolation from the liver. DNA was isolated from 15-year-old archival formalin-fixed tissues from infected ducks and subjected to next generation sequencing (NGS). Despite highly degraded DNA, short stretches of G- and C-rich repeats (TTAGGG and TAACCC) were identified as telomeric repeats frequently found in herpesviruses. Megablast and further investigative bioinformatics identified presence of Marek's disease virus (MDV), a Gallid alphaherpesvirus type 2 (GAHV-2), as the cause of the acute fatal infection. The source of infection may be attributed to a dead migratory flamingo found close to the duck enclosures three days prior to the outbreak; hence, GAHV-2 may also be responsible for the fatal infection of the flamingo accentuated by heat stress. Considering the possible spread of this highly contagious and lethal virus from a flamingo to the ducks, and the increasing zoonosis of animal viruses into humans, such as monkey B alphaherpesvirus transmission from macaques to humans with ~80% fatality, this observation has important ramifications for human health and safety of the poultry industry.

Current Epidemiology and Co-Infections of Avian Immunosuppressive and Neoplastic Diseases in Chicken Flocks in Central China.

The avian immunosuppressive and neoplastic diseases caused by Marek's disease virus (MDV), avian leucosis virus (ALV), and reticuloendotheliosis virus (REV) are seriously harmful to the global poultry industry. In recent years, particularly in 2020-2022, outbreaks of such diseases in chicken flocks frequently occurred in China. Herein, we collected live diseased birds from 30 poultry farms, out of 42 farms with tumour-bearing chicken flocks distributed in central China, to investigate the current epidemiology and co-infections of these viruses. The results showed that in individual diseased birds, the positive infection rates of MDV, ALV, and REV were 69.5% (203/292), 14.4% (42/292), and 4.7% (13/277), respectively, while for the flocks, the positive infection rates were 96.7% (29/30), 36.7% (11/30), and 20% (6/30), respectively. For chicken flocks, monoinfection of MDV, ALV, or REV was 53.3% (16/30), 3.3% (1/30), and 0% (0/30), respectively, but a total of 43.3% (13/30) co-infections was observed, which includes 23.3% (7/30) of MDV+ALV, 10.0% (3/30) of MDV+REV, and 10.0% (3/30) of MDV+ALV+REV co-infections. Interestingly, no ALV+REV co-infection or REV monoinfection was observed in the selected poultry farms. Our data indicate that the prevalence of virulent MDV strains, partially accompanied with ALV and/or REV co-infections, is the main reason for current outbreaks of avian neoplastic diseases in central China, providing an important reference for the future control of disease.

Molecular and pathological investigations of Marek's disease outbreaks in vaccinated poultry farms in Plateau State, North Central-Nigeria.

Marek's disease (MD) is a devastating neoplastic disease of poultry caused by MD virus (MDV). MD is one of the several diseases limiting the thriving Nigerian poultry industry. MD is mostly diagnosed in Nigeria based on history and gross lesions without laboratory investigations leading to underreporting of the disease. This study investigated MD outbreaks in poultry farms using polymerase chain reaction (PCR) and histopathology. Tumourous visceral organs were collected from dead chickens presented to veterinary clinics from 110 farms in Plateau State, North Central Nigeria from April 2013 to August 2014. Clinical signs observed in affected chickens were paralysis, stunting and uneven growth. Whilst the gross lesions observed were hepatomegaly, splenomegaly with lymphoma, prominent peripheral nerves and cachexia. The meq gene of MDV­1 was detected by PCR in 55.0% (n = 11/20) of broilers and 71.1% (n = 64/90) of vaccinated layer chicken samples collected. Microscopy revealed severe diffuse lymphocytic infiltrations in the heart, spleen and liver of chickens with tumourous gross lesions. Based on history, gross lesions, detection of meq gene of MDV­1 by PCR and histopathology results, MD was confirmed in the affected farms. Despite vaccination, outbreaks of MD still occurs in poultry farms in Nigeria. This study represents the first confirmatory diagnosis of MD in vaccinated poultry in Nigeria.

Marek's disease in genetically susceptible Cochin chickens in Italy: a case report.

The present study investigates an outbreak of classical Marek's disease (MD) in backyard Cochin chickens reared for hobby in Italy. Examined chickens showed spastic paralysis of the legs and at necropsy, enlargement and discoloration of the peripheral nerves and plexuses that matched microscopic A­ and B­ type MD lesions. Molecular analysis of the meq gene of the detected Gallid alphaherpesvirus 2 (GaHV­2) strain, showed typical markers of low virulence and the strain shared the entire meq gene sequence with strains circulating in Italian backyard chickens. Furthermore, the haplotype B19 of the major histocompatibility complex (MHC) was defined in the affected chickens, showing that the birds possessed a genetic profile of high susceptibility to MD, allowing the appearance of a classical nervous clinical form after infection with an apparently low pathogenicity GaHV­2 strain. Trade of live ornamental purebred chickens occurs frequently between hobby farmers and biosecurity practices, such as quarantine periods, should be applied to avoid the introduction of infected animals. Veterinarians should raise awareness of this issue and promote the use of vaccines against MD.

Genetic characterization of Marek's disease virus in chickens in Thailand reveals a high genetic diversity of circulating strains.

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1). Despite widespread vaccination, MD-related cases have been frequently observed worldwide, including in Thailand. However, no information is available on the genetic characteristics of MDV-1 field strains circulating in chickens in Thailand. This study investigated the geographic distribution and genetic characteristics of MDV-1 field strains circulating in chickens in Thailand between 2013 and 2021 by analysing the Meq and pp38 genes. Out of a total of the 286 clinical samples obtained from 70 chicken farms located in major chicken raising areas of Thailand, 138 samples (48.25%) from 46 chicken farms (65.71%) tested positive for MDV-1 field strains. Results demonstrated that MDV-1 field strains were extensively distributed in major chicken raising areas. Phylogenetic analyses based on the Meq gene revealed that four clusters of MDV-1 circulated in chickens in Thailand between 2013 and 2021. Among these clusters, cluster 1 was the predominant cluster circulating in chickens in Thailand. Additionally, our findings based on molecular characteristics of Meq and pp38 gene/protein suggested that most of the Thai MDV-1 field strains were potentially highly virulent. In conclusion, our data demonstrated the circulation of different clusters of MDV-1 with virulence characteristics in chickens in Thailand, indicating high genetic diversity of MDV-1 in Thailand. This study highlights the importance of more effective vaccine development and routine MDV-1 surveillance for early detection and control of highly virulent MDV-1.

Differential expression profile and in-silico functional analysis of long noncoding RNA and mRNA in duck embryo fibroblasts infected with duck plague virus.

BACKGROUND: Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction. RESULT: RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software. CONCLUSIONS: This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection.

Phylogenetic analyses on Marek's disease virus circulating in Iranian backyard and commercial poultry indicate viruses of different origin.

As neoplastic viruses have been affecting Iranian chicken farms more frequently in recent years, the first step in prevention may therefore be to genetically characterize and systematically identify their source and origin. Recently, we published a phylogenetic analysis based on the meq gene of Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1), that circulated in Iranian backyard and commercial chickens. In the current study, we are reporting for the first time the identification of a 298 aa meq protein containing only two PPPP motifs from an MDV-1-infected unvaccinated backyard turkey. This protein length has never been reported from any turkey species before. According to phylogenetic analysis, a close genetic relationship (0.68%) to several chicken-origin isolates such as the American vv + 648A strain was found. In addition, we identified a standard meq protein from a MDV-1-infected commercial chicken farm. In corroboration with our previous finding from other Iranian provinces, it is likely that the highly identical MDV-1 viruses currently circulating in Iranian chicken farms, which may be indicative of human role in the spread of the virus, have similar Eurasian origin. Our data suggest that regardless of the meq size, MDV-1 circulating in Iran are from different origins. On the other hand, meq sequences from bird species other than chicken have been reported but are very few. Our investigation suggests MDV-1 circulating in turkey do not have species-specific sequences.

First report of Serotype-1 Marek's disease virus (MDV-1) with oncogenic form in backyard turkeys in Turkey: a molecular analysis study.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens. RESULTS: This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64-100% and 99.79-100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies. CONCLUSIONS: These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping.

Molecular detection and phylogenetic analysis of Marek's disease virus virulence-associated genes from vaccinated flocks in southern India reveals circulation of virulent MDV genotype.

Marek's disease (MD) is a re-emerging viral disease of chickens and a serious economic threat to the poultry industry worldwide. Continuous surveillance with molecular investigation is essential to monitor the emergence of virulent Marek's disease virus (MDV) strains and to devise any appropriate vaccination strategy and implement bio-security programmes. In the present study, we investigated the cases of MD outbreaks in vaccinated poultry flocks. The MD outbreak was confirmed through necropsy (mainly visceral tumours), histopathology and viral gene specific PCR. The pathotypes of the field MDV strains were assessed by molecular analysis of three virulence-associated genes, meq, pp38 and vIL-8. The Meq sequence of the field strains analyzed in this study lacked the 59 aa unique to mild strains, indicating that they are potentially virulent strains. Mutation at position 71 and the presence of five proline rich repeats in the transactivation domain, both associated with virulence were observed in these strains; however, the signature sequences specific to very virulent plus strains were absent. Phylogenetic analysis of meq oncogene sequences revealed clustering of the field strains with North Indian strains and with a very virulent plus ATE 2539 strain from Hungary. Analyses of pp38 protein at positions 107 and 109 and vIL-8 protein at positions 4 and 31 showed signatures of virulence. Sequence and phylogenetic analysis of oncogene and virulence-associated genes of field MDVs from vaccinated flock indicated that these strains possessed molecular features of virulent strains.

Report of a new meq gene size: The first study on genetic characterisation of Marek's disease viruses circulating in Iranian commercial layer and backyard chicken.

1. In recent months, several outbreaks with clinical signs of MDV-1 were reported in Iranian parent and laying hen farms, in addition to backyard chickens. Several meq gene sequences from these outbreaks were amplified and molecularly characterised.2. The meq protein sequences revealed three different sizes, namely the standard 339 aa, a shorter form of 338 aa lacking a proline residue at position 191, and a very short (vs) size of 265 aa. Based on sequence and size, the 265 aa meq has never been reported from international research groups before. The protein has only one PPPP repeat motif suggesting it belongs to a highly virulent strain.3. The standard meq sequences showed 100% BLAST identity to the vv+ isolate Polen5. However, the 338 aa form clustered to the clade usually reported from North America.4. This is the first report on genetic analysis of MDV-1 from Iran, but further study is required to obtain a better picture of the diversity and prevalence of different MDV-1 strains circulating in the country's farms, backyard poultry and other bird species.

Quantitative profiling of Marek's Disease Virus in vaccinated layer chicken.

The present study was undertaken to quantify the Marek's Disease Virus (MDV) serotypes in vaccinated commercial layer flocks at 7, 14, 21, 28, 35 and 60-90 days post vaccination (dpv) and to correlate the pathogenic Gallid herpesvirus 2 (GaHV-2, MDV1) load with vaccine viral load of Gallid herpesvirus 3 (GaHV-3, MDV2) and Meleagridis herpesvirus 1 (MeHV-1, MDV3). A total of 25 commercial layer flocks were selected in and around Namakkal district of Tamil nadu, India and the feather pulp (FP) and blood samples were collected. Out of 25 flocks, 14 were revaccinated with bivalent vaccine, six were revaccinated with monovalent vaccine apart from the initial bivalent vaccination done at hatchery and five flocks were not revaccinated. SYBR green based real time PCR was used for absolute quantification of MDV serotypes. The pathogenic MDV1 load had shown an increasing trend until 21 dpv followed by a dip and again had shown a constant uptick between 60 and 90 dpv in the flocks that went on to develop MD outbreak. The flocks which had not encountered any Marek's Disease outbreak had shown increasing trend of MDV2 and 3 load until 21 dpv followed by a slight decrease but maintained a higher load when compared to MDV 1 which had marked a sharp decline between 60 and 90 dpv. Outbreak of MD was observed in seven (28%) out of 25 flocks between 18 and 27 weeks of age. It includes, two out of fourteen farms (14%) revaccinated with bivalent vaccine, two out of six farms (33%) revaccinated with MDV3 vaccine and three out of five farms (60%) without revaccination. The overall mean of vaccine viral load at various stages of dpv was constantly low where as pathogenic MDV 1 load was constantly high between 60 and 90 dpv in the flocks that went on to develop Marek's Disease during later part of life.

Isolation and molecular characterization of a virulent Marek's Disease virus serotype-1from Andhra Pradesh, India.

Marek's disease (MD) is one of the most significant neoplastic diseases of poultry caused by Marek's disease virus (MDV), an oncogenic avian herpesvirus which is responsible for great economic losses to the poultry industry worldwide. MD is being manifested as an acute disease with lymphomas in multiple visceral organs. In the present study, an outbreak of MD was investigated in one of the poultry farms from Andhra Pradesh, India. The gross lesions in the affected birds included lymphomas in different visceral organs like liver, spleen, proventriculus, heart and ovaries. Histopathology revealed presence of uniform lymphoblastoid cell infiltration typical of MD. The isolation of the virus was carried out in duck embryo fibroblast cells. After three blind passages, the cell cultures revealed plaque formation typical of MDV. Further confirmation of the virus was carried out by PCR targeting 132 bp repeats of serotype­1 MDV and the oncogenes Meq and vIL­8 were amplified and sequenced. The nucleotide and phylogenetic analysis of the virus confirmed the virus as virulent serotype­ 1 MDV. The present outbreak suggests the need for change in the vaccination regimen of MD vaccination with appropriate serotype­ 1 MD vaccines in Indian poultry flocks as the HVT and bivalent vaccines are unable to protect the flocks against virulent MDV.

Reemergence of reticuloendotheliosis virus and Marek's disease virus co-infection in Yellow-Chickens in Southern China.

The reticuloendotheliosis virus (REV) and the Marek's disease virus (MDV) cause reticuloendotheliosis (RE) and Marek's disease (MD) in poultry, respectively. According to epidemiological results obtained in our laboratory from 2010 to 2017, the positive rates of REV and MDV co-infection remained at low levels. In the present study, during the period of October 2018 to July 2020, 4 clinical cases with high morbidity (5%-20%) and mortality (2%-10%), caused by the co-infection of REV and vv+ MDV-like strains, were diagnosed and analyzed by histopathological observation, cell cultures and detection with ELISA and IFA, and the PCR and by sequencing of the isolates' genes. Sequencing and the sequence analysis on the complete genomes of the REV strains and the meq genes of the MDV strains were performed. The results, based on the complete genome, LTR, gag, pol, and env genes' nucleotide sequences of the REV strains, showed that the REV isolates and 68.0 % (17/25) of the reference strains were in a same branch, and all had a high sequence similarity (>99.0%). The similarities between the four isolates and a vv+MDV strain GX18NNM4 were very high, up to 99.3-99.8%. Also, the amino acid residuals at locations 71, 77, 80, 115, 139, 176, and 217 were all the same as A, E, Y, A, A, R, and A, respectively, in the meq gene of the four MDV isolates. In addition, the substitutes at P176R and P217A interrupted the stretches of the proline-rich repeat PPPP, indicating that these strains belonged to the vv+ MDV-like category. Our findings indicated that the more recent and frequent reemergence of REV and the subsequent co-infection with vv+ MDV-like strain has become one of the causes of the clinical outbreaks of tumors and is undoubtedly a threat to the poultry industry in southern China.

Molecular characterization of duck plague virus from selected Haor areas of Bangladesh.

Background: Duck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh. Aim: This study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV. Methods: A total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank. Results: Of the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity. Conclusion: Based on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry.

An outbreak in three-yellow chickens with clinical tumors of high mortality caused by the coinfection of reticuloendotheliosis virus and Marek's disease virus: a speculated reticuloendotheliosis virus contamination plays an important role in the case.

Both reticuloendotheliosis and Marek's disease are neoplastic diseases of chickens caused by reticuloendotheliosis virus (REV) and Marek's disease virus (MDV), respectively. The infection of REV or MDV may lead to clinical tumors and also result in immunosuppression and easily allow secondary infection by other pathogens. Here, we investigated a breeder flock of three-yellow chickens in southern China that had been vaccinated with CVI988/Rispens at hatching and had experienced depression, weakness, reduction in weight gain, and an increased death rate after 120 d of age. The morbidity and mortality were 20% and 10%, respectively, at 140 d of age when this infection was diagnosed. The necropsy of the birds revealed significant tumor-like lesions in the heart, liver, spleen, and ceca. Peripheral blood lymphocytes and tumor-like tissues were sampled for PCR detection and for histopathological observation, for virus isolation and the subsequent immunofluorescent assay on the cell cultures and for gene sequencing of the isolated viruses. A REV isolate GX18NNR1 and a MDV isolate GX18NNM5 were both recovered from the sampled bird. Further phylogenetic analysis based on the env gene of REV and the meq gene of MDV demonstrated that GX18NNR1 was closely related to the reference REV strain MD-2, which was isolated from a contaminated commercial turkey herpesvirus vaccine. In addition, the GX18NNM5 was found to belong to the Chinese very virulent MDV strains' cluster. The coinfection of REV and MDV may contribute to tumor outbreaks with high morbidity and mortality in three-yellow chicken flocks.

Molecular characterisation and phylogenetic analysis of Marek's disease virus in Turkish layer chickens.

1. There is no current data about the genotypes of Marek's disease virus (MDV) in Turkish poultry flocks; hence, this study was performed to analyse CVI988/Rispens, turkey herpesvirus (HVT) vaccine viruses and MDV field viruses as well as to perform phylogenetic analysis of MDV in Turkish layer chickens. 2. In 2017 and 2018, a total of 602 spleen samples from 49 layer flocks were collected from the Marmara, West Black Sea and Aegean regions. DNA was extracted from the spleen samples and the samples were analysed by real-time PCR probe assay to detect CVI988/Rispens and HVT vaccine viruses and MDV field strains. Samples found positive for MDV by real-time PCR were subjected to PCR using the Meq gene primers for phylogenetic analysis. 3. Amongst 49 flocks, virulent MDV was detected in nine flocks. CVI988/Rispens and HVT vaccine strains were detected in 47 flocks and HVT in all 49 flocks. Splenomegaly, hepatomegaly and tumours in the oviduct were observed in chickens of affected flocks. Virulent MDV was detected in 120 out of 602 spleen samples. Sequencing and phylogenetic analyses showed that MDVs detected in this study were closely related to MDV strains from Italy, Poland, Saudi Arabia, Iraq, India and China but showed diversity with MDV strains from Egypt and Hungary. Multiple sequence analysis of the Meq protein revealed several point mutations in deduced amino acid sequences. Interestingly, CVI988/Rispens vaccine virus from China (AF493555) showed mutations at position 66 (G66R) and 71 (S66A) along with two other vaccine strains from China (GU354326.1) and Russia (EU032468.1), in comparison with the other vaccine strain CVI988/Rispens (DQ534538). The molecular analyses of the Meq gene suggested that Turkish field strains of MDV are in the class of virulent or very virulent pathotypes. 4. The results have shown that MDV still affects poultry health, and the phylogenetic and amino acid variation data obtained will help in vaccination and control strategies.