Co-infection of vvMDV with multiple subgroups of avian leukosis viruses in indigenous chicken flocks in China
In China, although the ALV eradication program and the MD vaccination strategy greatly reduce the disease burdens caused by the infection of ALV and MDV, the frequent emergence of novel ALV-K or vvMDV in the vaccinated chicken flock challenges the current control strategies for both diseases.
In Guangdong Province, an indigenous chicken flock was infected with neoplastic disease. Hematoxylin–eosin staining of the tissues showed the typical characteristics of MDV and classical ALV infection. The PCR and sequencing data demonstrated that the identified MDV was clustered into a very virulent MDV strain endemic in domestic chickens in China. Moreover, subgroups ALV-A and ALV-K were efficiently recovered from two samples. The full genome sequence revealed that the ALV-K isolate was phylogenetically close to the ALV TW3593 isolate from Taiwan Province.
A co-infection of vvMDV with multiple ALV subgroups emerged in a chicken flock with neoplastic disease in Guangdong Province. The co-infection with different subgroups of ALV with vvMDV in one chicken flock poses the risk for the emergence of novel ALVs and heavily burdens the control strategy for MDV.
KeywordsMDV Multiple ALV Co-infection Molecular analysis
Avian leukosis virus
Avian leukosis virus subgroup A;
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Indirect immunofluorescent assay
Long terminal repeat
Marek’s disease virus
Polymerase chain reaction
Very virulent plus
Very virulent MDV
Avian leukosis virus (ALV) and Marek’s disease virus (MDV) are the most causative agents for neoplastic disease in chickens . ALV is currently classified into seven subgroups (A-E, J and K) in chickens based on the antigenicity of its envelope protein . Infection with avian leukosis virus subgroup A (ALV-A) or ALV-B generally results in classical lymphocytic leukemia, while ALV-J infection mainly causes myeloid leukosis and vascular neoplasms [3, 4, 5]. ALV-C and ALV-D are rare in clinical cases, whereas ALV-K is a novel subtype of ALV recently identified in indigenous Asian chicken flocks [6, 7, 8, 9, 10, 11]. Different from ALV-A, B, C, D, J and K, ALV-E belongs to endogenous ALV. MDV can be clustered into different pathotypes, including mild (m), virulent (v), very virulent (vv) and very virulent plus (vv+) strains. Marek’s disease (MD) caused by MDV is mainly characterized by lymphoproliferative disease in chickens with multiple neuritis or malignant tumors of the internal organs . Except for inducing tumors, the infection of ALV or MDV also causes immunosuppression, which significantly affects the sustaining development of the poultry industry globally . In China, although the ALV eradication program and the MD vaccination strategy greatly reduce the disease burdens caused by the infection of ALV and MDV, the frequent emergence of the novel ALV-K or the very virulent MDV (vvMDV) in the vaccinated chicken flock challenges the current control strategies for both diseases [2, 13, 14]. In this study, an outbreak of vvMDV infection in a vaccinated indigenous chicken flock co-infected with ALV-A, ALV-J and ALV-K was reported in China.
Clinical symptoms and pathological changes
Co-infection of MDV with ALV-A, J and K
Primers for PCR amplification of oncogenic pathogens
Primer sequence 5’→3’
Davidson et al., 1995 
Zhuang et al., 2015 
Zhuang et al., 2015 
Zhuang et al., 2015 
The identified MDVs belonged to vvMDV strains endemic in China
To further investigate whether the MDV detected in the diseased chicken flock is a vaccine strain or vvMDV strain, the meq, gB and pp38 genes were amplified from these samples by PCR and analyzed. The sequence assay for the PCR products revealed that the meq, gB and pp38 genes of the MDV (named GD) were 100% identical to the domestic vvMDV strain GX0101, which has been reportedly circulating on chicken farms in China for a long time (Fig. 1c) (gB and pp38 data not shown). The finding of vvMDV in this vaccinated chicken flock challenges the current MD vaccination strategy in China.
ALV-A and ALV-K were efficiently isolated from the clinical samples
The GD-K ALV-K isolate phylogenetically resembled the TW3593 isolate derived in Taiwan
MDV and ALVs have caused severe economic losses to the poultry industry worldwide . Notably, the frequent co-infection of MDV with ALVs or REV has undoubtedly become a great threat to the healthy development of the poultry industry [17, 18, 19, 20]. In this study, vvMDV and ALV-K were first identified in a chicken flock in southern China. In the field, vvMDV or ALV-K single infection has been reported previously, which could cause immunosuppression in chickens and make them more susceptible to other pathogens. It should also be noted that ALV-K is frequently undetectable due to its poor replication ability [8, 10, 20, 21, 22]. The frequent transportation and communication of the indigenous chickens in southern China and the difficulties in detecting ALV-K were some other possible reasons for co-infection status .
In summary, a co-infection of vvMDV with multiple ALV subgroups was identified in a chicken flock with neoplastic disease in Guangdong Province. Moreover, subgroups ALV-A and ALV-K were efficiently isolated from two samples. To our knowledge, this is the first demonstration of co-infection of vvMDV with the novel ALV subgroup ALV-K. Notably, although the positive rate of ALV-J in these diseased chickens was 80% for PCR, ALV-J could not be efficiently isolated. This interesting finding indicated that co-infections with ALV-A or ALV-K, even vvMDV, might impact the replication of ALV-J. However, how these pathogens interact with each other remains to be further studied. In the case of ALV-A, resistant loci of tvar1, tvar2, tvar3, and tvar4 have been found in some inbred lines of White Leghorn [23, 24]. In addition, single nucleotide polymorphism variants within tva receptor genes in some Chinese chicken breeds have been reported and animals with certain tva alleles are resistant to ALV-A infection [25, 26]. Notably, a recent research has showed that the novel ALV-K shares its tva cell receptor with ALV-A . Thus, the tva receptors in the chicken flock are possibly associated with ALV-A and ALV-K infection status in this study as well. In short, future study should also focus on the polymorphisms of tva receptors in different indigenous chicken breeds in China, test the susceptibility of these indigenous chickens to ALV-A and ALV-K infections, and breed chickens resistant to ALV-A and ALV-K.
The co-infection of vvMDV with different subgroups of ALV identified in a chicken flock poses a risk for the emergence of novel ALVs and burdens the control strategy for MD and highlights the significance of epidemiological monitoring for similar co-infection in indigenous chicken flocks in China.
Layer chickens that were 150 days old and vaccinated with MDV and suffering from neoplastic disease with about 10% neoplastic incidence and 5% mortality were obtained from a large chicken farm in Guangdong Province, China. The owner of the farm gave permission to include ten diseased chickens in this study. Tumor nodules were found on the surface of the organs and skin from these diseased chickens. All experiments complied with institutional animal care guidelines and were approved by the University of Yangzhou Animal Care Committee.
A histopathological assay was conducted as previously described to examine the tissues . Briefly, the livers from the diseased chickens were fixed using 10% formalin buffer, dehydrated in alcohol, and embedded in paraffin. Hematoxylin and eosin staining was then performed, and microscopic changes were observed by light microscopy.
PCR detection for oncogenic pathogens
The homogenates of the PCR-positive tissue samples were filtered through a 0.22 μm filter and inoculated into DF1 cells for 2 h. Then, fresh Dulbecco’s modified Eagle’s medium (DMEM) with 1% fetal bovine serum (FBS) was used for replacement. The supernatant of the cell culture was passed for three serial passages (5–7 days for each passage), and then the infected cells were screened for ALV by PCR and IFA.
Indirect immunofluorescence assay
The infected DF1 cells were fixed with chilled acetone:ethanol solution (3:2) for 5 min and washed once with PBS. Then, they were incubated with the ALV-p27-specific mAb 5D3 for 45 min at 37 °C . After three washes with PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody for another 45 min. After three washes with PBS, the cells were observed under a fluorescence microscope.
For PCR, the gB, pp38 and meq genes were amplified as described . For amplification of ALV and REV genes, 50 μl of reaction volume was used, which consisted of 10 μl of 5 × SF PCR buffer, 1 μl dNTP mixture, 2 μl of each primer, 1 μl of Phanta Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China), 32 μl of ddH2O, and 2 μl of the DNA template. The PCR programs for the pp38 and meq genes were 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s, and then 72 °C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis and then sequenced by Genscript (Nanjing, China). All sequences were aligned with Lasergene 7 and phylogenetically analyzed with MEGA 6.
We thanks for Dr. Jianjun Zhang (Sinopharm Yangzhou VAC Biological Engineering Co.Ltd) for kindly helping us collect the clinical samples.
JY and AQ conceived and designed the experiments. TL, JX, GL, DR and SS performed the experiments. TL, AQ, HS, WG and JY analyzed the data. TL, JX, GL, QX and LL contributed reagents/materials/analysis tools. TL and JY contributed to the writing of the manuscript. TL and JY prepared the figures. All authors read and approved the final manuscript.
This study was supported by the National Key Research & Development (R&D) Plan (2018YFD0500106, 2016YFD0501605), NCFC-RCUK-BBSRC (Grant No. 31761133002 and BB/R012865/1), the Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality (26116120), the Research Foundation for Talented Scholars in Yangzhou University and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The funding bodies did not play direct roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
The diseased chickens with tumor were kindly provided by the farm owner for diagnostics. All experiments complied with institutional animal care guidelines and were approved by the Animal Care Committee of Yangzhou University.
Consent for publication
The authors declare that they have no competing interests.
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