Research note: Molecular epidemiology of Marek's disease virus in China during 2022 and 2023

Abstract

Marek's disease (MD) is a highly contagious and oncogenic viral disease that causes immunosuppression in chickens. A molecular epidemiological investigation of Marek's disease virus (MDV) was conducted in chicken flocks in China during 2022 and 2023. Real-time PCR was used to detect MDV serotype 1 (MDV-1) in 544 clinical tissue samples. MDV-positive samples were subsequently tested for reticuloendotheliosis virus (REV), avian leukosis virus subgroups A and B (ALV-A/B), avian leukosis virus subgroup J (ALV-J), and chicken infectious anemia virus (CIAV). Full-length sequencing of the meq and pp38 genes was performed on 18 selected MDV-positive samples. The results revealed an MDV positive rate of 19.67% (107/544), with 73.83% (79/107) of these samples showing co-infection, predominantly MDV and CIAV (54.21%, 58/107). The MDV positive rates were higher in native chickens (28.43%, 29/102) and Hy-Line layers (27.62%, 29/105), whereas the co-infection rate of MDV with other avian immunosuppressive pathogens was highest in Arbor Acres broilers (91.67%, 22/24). Molecular analysis of the meq gene from circulating field MDV strains revealed typical amino acid substitutions associated with Chinese MDV isolates, including K77E, D80Y, V115A, T139A, P176R, and P217A. Notably, two additional novel substitutions, A88T and Q93R, were identified in 16 of the 18 isolates. Phylogenetic analysis indicated that the 16 isolates clustered with virulent MDV isolates from Guangdong Province (2018), whereas the remaining two grouped with virulent MDV strains circulating one decade earlier. These findings suggest that MDV infection was prevalent in chicken flocks across some regions of China during 2022 and 2023, frequently co-occurring with other avian immunosuppressive pathogens, and that virulent MDV strains with novel molecular characteristics are currently circulating in chicken populations.

Introduction

Marek's disease (MD) is a highly contagious and lymphoproliferative disease of chickens, characterized by immunosuppression, neurological paralysis, and the development of tumors in peripheral nerves, visceral organs, eyes, or skin. The etiological agent, Marek's disease virus (MDV), belongs to the genus Mardivirus within the subfamily Alphaherpesvirinae. MDV strains are classified into three serotypes, among which only serotype 1 exhibits oncogenic potential. Based on pathogenicity, serotype 1 strains are categorized into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+) (Chengula et al., 2025). The MDV genome encodes over 100 genes, among which meq and pp38 are the most critical for viral virulence and oncogenicity (Lachheb et al., 2020). The meq oncogene plays a central role in MDV pathogenesis and contains a basic leucine zipper resembling the Jun/Fos oncogenes at the N-terminus and a transactivation domain of proline-rich repeats at the C-terminus (Motai et al., 2024). The pp38 gene is essential for early cytolytic infection and lymphoid cell transformation (Chengula et al., 2025). The widespread use of the CVI988/Rispens vaccine may have exerted selective pressure leading to progressive increases in MDV virulence, as well as the emergence of novel mutations in the meq gene (Lachheb et al., 2020), thereby enabling the virus to evade vaccine-induced immune protection. Recent epidemiological studies indicate an increasing incidence of MD outbreaks in vaccinated flocks co-infected with other avian immunosuppressive pathogens, including reticuloendotheliosis virus (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) (Nishitha et al., 2021; Adedeji et al., 2024). Such co-infections result in synergistic pathogenic effects in vivo and significantly compromise vaccine efficacy by impairing host immunity (Sun et al., 2017), leading to partial or complete vaccine failure and substantial economic losses in commercial poultry production.

This study investigated MDV infection and co-infection with other avian immunosuppressive pathogens, as well as the genetic characteristics of circulating MDV-1 field strains in chickens flocks in China during 2022 and 2023. The findings highlight the importance of continuous monitoring of MDV evolution and provide valuable insights for the development of effective control strategies.

Materials and methods

Clinical tissue samples

From August 2022 to December 2023, 544 clinical tissue samples (liver and spleen) from diseased or dead chickens were collected from 15 regions of China. All chickens had been vaccinated with the commercial CVI988/Rispens vaccine strain at one day of age.

Detection of MDV, REV, ALV, and CIAV

Genomic DNA and RNA were extracted from homogenized tissues using the Nucleic Acid Extraction Kit (Magnetic beads) (Ascend, Luoyang, China). RNA was reverse-transcribed into cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). According to the Chinese standards GB/T 27644–2011 (Protocol of real-time PCR for detecting Gallid herpesvirus 2) and NY/T 1187–2019 (Diagnostic techniques for chicken infectious anemia), combined with previously reported methods (Li et al., 2012; Dai et al., 2015), quantitative real-time PCR (qPCR) was performed to detect MDV-1 in all samples. Subsequently, MDV-positive samples were further analyzed individually by qPCR to detect REV, ALV subgroups A and B (ALV- A/B), ALV subgroup J (ALV- J), and CIAV.

Sequencing and genetic analysis

Eighteen qPCR-confirmed MDV-positive samples exhibiting gross tumor lesions were selected for full-length sequencing of the meq and pp38 genes; a subset of these specimens underwent histopathological examination, revealing characteristic lesions consistent with MD (data not shown). Specific primers targeting the meq and pp38 gene sequences of MDV-1 were designed for amplification: meq-F: 5′-ATGTCTCAGGAGCCAGAGCC-3′, meq-R: 5′-TCAGGGTCTCCCGTCACCT-3′, pp38-F: 5′-ATGGAATTCGAAGCAGAACAC-3′, and pp38-R: 5′-TTAATTTGATTCAGATTTTGTTTCTCCAG-3′. PCR reactions were performed in a 50-μL volume containing 2 μL of each primer (10 μM), 5 μL of DNA template, and 25 μL of 2 × Rapid Taq Master Mix (Vazyme, Nanjing, China). Thermal cycling conditions: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; final extension at 72°C for 7 min. PCR amplicons were resolved by 1% agarose gel electrophoresis, with expected fragment sizes of 1020 bp (meq) and 873 bp (pp38). Purified products were ligated into the pMD19-T vector (TaKaRa, Dalian, China) and transformed into E. coli DH5α competent cells. Positive recombinant clones were verified by PCR and submitted to Tsingke Biotech (Beijing, China) for Sanger sequencing.

The nucleotide sequences of the meq and pp38 genes from 18 MDV isolates were edited using the EditSeq program in the Lasergene package (DNAStar Inc., Madison, WI, USA). Sequence homology analysis and alignment of the deduced amino acid sequences were performed using the MegAlign program with the Clustal W algorithm. Phylogenetic trees for the meq and pp38 genes were constructed using MEGA version 6.0 with the neighbor-joining (NJ) method and 1,000 bootstrap replicates. Reference viral sequences were retrieved from GenBank for subsequent analysis, including 21 Chinese and 21 international strains. Of these, 11 strains were selected for amino acid sequence alignment of the meq and pp38 genes, 42 for phylogenetic analysis of the meq gene, and 32 for phylogenetic analysis of the pp38 gene.

Statistical analysis

Data analysis was performed using GraphPad Prism v10.4.1 (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics v27.0 (IBM Corporation, Armonk, NY, USA). Categorical data were compared between groups using the chi-square test; statistical significance was defined as P < 0.05.

Results and discussion

Detection of MDV and other avian immunosuppressive pathogens

The qPCR assay was performed to detect MDV-1 in 544 tissue samples collected from clinically diseased chickens. The results showed a positive rate of 19.67% (107/544) for MDV. Subsequent detection of MDV-positive samples for REV, ALV, and CIAV revealed that 73.83% (79/107) were co-infected with MDV and at least one other avian immunosuppressive pathogen, a rate significantly higher than that of MDV single infection (26.17%, 28/107). Among the co-infections, the most prevalent was dual infection with MDV and CIAV (54.21%, 58/107), followed by triple infection with MDV, REV, and CIAV, which accounted for 7.48% (8/107) of the samples (Fig. 1A). These findings indicate that MDV was prevalent in chicken flocks across some regions of China and frequently co-infected with other avian immunosuppressive pathogens, particularly CIAV.

Fig. 1.

Epidemiological analysis of MDV and other avian immunosuppressive pathogens in clinical tissue samples from diseased chickens. (A) MDV single infection and MDV co-infection with other avian immunosuppressive pathogens. Exogenous ALV subgroups A, B, and J were detected in this study. (B) Distribution of MDV infection and co-infection with other avian immunosuppressive pathogens among different chicken breeds. Differences in positive rates between groups were analyzed using the SPSS Chi-Square test. * and ** indicate P < 0.05 and P < 0.01, respectively. (C) MDV positive rates in clinical tissue samples of diseased chickens from 15 regions of China.

Breed distribution of MDV

The MDV positive rates in native chickens (28.43%, 29/102) and Hy-Line layers (27.62%, 29/105) were significantly higher than those in Arbor Acres broilers (15.00%, 24/160), Three-yellow chickens (11.54%, 9/78), and Jing-series layers (Jinghong, Jingfen, and Jingbai; 13.89%, 10/72), whereas the co-infection rate of MDV with other avian immunosuppressive pathogens was highest in Arbor Acres broilers (91.67%, 22/24), significantly exceeding those in Hy-Line layers (62.07%, 18/29) and Jing-series layers (50.00%, 5/10) (Fig. 1B). This discrepancy may be associated with differences in biosecurity practices or breed-specific susceptibility.

Regional distribution of MDV

Of 544 clinical tissue samples collected from 15 regions across China, MDV was detected in samples from 11 regions. MDV positive rates were relatively high in samples from Henan (36.36%, 16/44), Guangdong (46.15%, 12/26), Hunan (62.50%, 5/8), and Guangxi (100%, 2/2) (Fig. 1C). However, all samples were derived from clinical diagnostic submissions rather than systematic, randomized epidemiological sampling, leading to uneven regional sample sizes and potential ascertainment bias in estimates of the regional distribution of MDV.

Sequence analysis of the meq and pp38 genes

Eighteen samples exhibiting typical gross lesions of MD were selected and tested for MDV and other avian immunosuppressive pathogens. Four samples were infected with MDV alone, whereas 14 samples exhibited co-infection, including seven samples positive for MDV+CIAV, three samples positive for MDV+REV, one sample positive for MDV+ALV, two samples positive for MDV+CIAV+REV, and one sample positive for MDV+CIAV+ALV. Full-length meq and pp38 gene sequences were amplified from 18 MDV-positive samples. All sequences were deposited in GenBank under the following accession numbers: meq (PX222275–PX222292), pp38 (PX222293–PX222310). Homology analysis revealed that the nucleotide sequence identities of the meq and pp38 genes among the 18 MDV isolates ranged from 99.3% to 100% and from 99.4% to 100%, respectively.

Alignment analysis of the deduced amino acid sequences of the meq and pp38 genes was conducted using the 18 field MDV isolates and 11 reference MDV strains, including four virulent strains from the USA, two vaccine strains (CVI988 and 814), and five virulent isolates from China. Sequence analysis of the meq gene revealed that substitutions K77E, D80Y, V115A, P176R, and P217A were present in all Chinese field MDV strains; the T139A substitution was observed in most of these Chinese strains; and two novel substitutions (A88T and Q93R) were detected in 16 of the 18 field MDV isolates (Table 1). Notably, the Q93R substitution had not been reported in any of the reference strains. The amino acid substitution A71S and a deletion at position 194 were identified in the attenuated vaccine strains (814 and CVI988), whereas none of the 18 field MDV isolates exhibited these variations. The GXYL20231008 strain shared the same substitution pattern as LS (Accession No. HQ638149), while the JSYC20220829 strain exhibited the same substitution pattern as GX0101 (Accession No. JX844666), LMS (Accession No. JQ314003), and BS/15 (Accession No. MW247181). In contrast, each of the remaining 16 isolates harbored two additional substitutions: either A88T and Q93R relative to GX0101, LMS, and BS/15, or Q93R and T139A relative to SD2012-1 (Accession No. KC511815). We hypothesize that the panel of amino acid substitutions—K77E, D80Y, A88T, Q93R, V115A, T139A, P176R, and P217A—in the meq gene could serve as novel molecular markers for field MDV isolates currently circulating in China. For the pp38 gene, sequence analysis revealed E109G as the only amino acid substitution present in both Chinese MDV isolates and vaccine strains. These findings indicate that the 18 field MDV isolates exhibit molecular characteristics of very virulent strains, distinct from those of vaccine strains. Both the virulence of these isolates and the effects of these Meq amino acid substitutions on viral pathogenicity require in vivo experimental validation.

Table 1.

Sequence alignment of the deduced amino acid sequences of the meq and pp38 genes.

Strain	Type b	meq														pp38
		71 (A)	77 (K)	80 (D)	88 (A)	93 (Q)	115 (V)	119 (C)	139 (T)	153 (P)	176 (P)	180 (T)	194 (P)	217 (P)		109 (E)
GA	v
RB1B	vv
Md5	vv													A
648A	vv+							R		Q	A	A		A
814	m	S	E				A						-			G
CVI988	m	S	E										-			G
GX0101	vv		E	Y			A		A		R			A		G
LMS	vv		E	Y			A		A		R			A		G
LS	vv		E	Y			A				R			A		G
SD2012-1	vv		E	Y	T		A				R			A		G
BS/15	vv		E	Y			A		A		R			A		G
GDMZ20230911 etc.a			E	Y	T	R	A		A		R			A		G
GXYL20231008			E	Y			A				R			A		G
JSYC20220829			E	Y			A		A		R			A		G

^aIndicates the following MDV strains: GDMZ20230911, GDZJ20230323, JSTZ20221209, JSYZ20230224, JSNT20230316, JSNT20230606, JSNT20230609, JSYC20230810, JSTZ20230925, JSTZ20231010, HBHS20221128, HBBD20221215, SDWF20230220, HNLH20230523, HNHY20230531, and HNSY20230626. Strains were designated using provincial and municipal abbreviations along with the sample collection date.

^bIndicates virulence: v, virulent; vv, very virulent; vv+, very virulent plus; m, mild.

"-" indicates an amino acid deletion.

Phylogenetic analysis of the meq and pp38 genes

Phylogenetic analysis of the meq gene revealed that the MDV strains could be classified into two distinct clades: Clade I and Clade II. All 18 field MDV isolates were assigned to Clade I, the predominant genotype currently circulating in China. Of these, 16 isolates clustered with virulent MDV isolates from Guangdong Province in 2018 (Li et al., 2023), while the remaining two grouped with virulent MDV strains circulating one decade earlier, indicating that virulent MDV strains with distinct molecular characteristics were co-circulating in Chinese chicken flocks in recent years. Clade II included vaccine strains, virulent strains from the United States and a limited number of strains from Japan, Poland, Australia, Thailand, and Nigeria. In the phylogenetic tree based on the pp38 gene, the 18 field MDV isolates did not form a unique cluster. Although phylogenetic analysis of the meq gene indicates the co-circulation of distinct MDV lineages in China, a more comprehensive understanding of the evolutionary relationships among MDV strains can be achieved through further whole-genome sequencing.

Funding

This work was supported by the Jiangsu Province University Outstanding Science and Technology Innovation Team Project ([2021] NO.1), 111 Project (D18007), Yangzhou City-University Cooperation Funding Project (YZ2025203), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

CRediT authorship contribution statement

Caihong Yan: Writing – original draft, Validation, Methodology, Conceptualization. Xiaoyu Wei: Investigation, Data curation, Visualization, Formal analysis. Qi Zhong: Investigation, Data curation. Wenjie Jin: Supervision, Resources. Yang Yang: Supervision, Resources. Zhiqiang Wang: Conceptualization, Supervision, Resources. Daxin Peng: Writing – review & editing, Conceptualization, Supervision, Project administration, Funding acquisition.

Disclosures

The authors declare that they have no conflicts of interest.