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. 2023 Nov 14;103(2):103264. doi: 10.1016/j.psj.2023.103264

Research Note: Pathogenetic characteristics of avian encephalomyelitis virus in Guangdong and Jiangxi Provinces, China

Fanfan Zhang *, Yangyang Luo †, Qipeng Wei *, Ligen Xiong *, Quan Xie ‡, Jia Tan *, Chengcheng Wu *, Na Li *, Zhaofeng Kang *,1
PMCID: PMC10697995  PMID: 38035474

Abstract

In recent years, the infection rate of avian encephalomyelitis virus (AEV) infection in chickens has risen significantly, seriously endangering the development of the chicken industry. In order to study the current epidemiological status of AEV in China as well as the genetic and evolutionary patterns of the virus, we conducted a survey and genomic analysis of chicken AEV. The results showed that 46.26% (136/294) of the tissue samples tested (n = 294) were positive for AEV, with the highest positivity rate of 62.24% (61/98) among tissue samples from chickens aged 13 to 18 wk. The complete genomes of 2 representative AEV strains were determined, and the VP1 evolutionary tree results revealed that the 2 representative strains belonged to a novel AEV strain. Multiple alignment analysis showed that the ORF1 genes of the 2 representative strains differed by 82.3 to 99.9% at the amino acid level compared with the reference AEV strain, and the mutations at the key amino acid loci of VP2 and VP3 were the same as those in the chick embryo-adapted strain. The analysis makes up for the molecular epidemiological data and genetic variation of the 2 representative strains. The analysis makes up for the molecular epidemiological data and genetic variation of AEV and provides a basis for further understanding the spread of AEV in China.

KEYWORDS: avian encephalomyelitis virus, surveillance, epidemiological characteristic, phylogenetic analysis

INTRODUCTION

Avian encephalomyelitis (AE) is a highly contagious disease caused by the avian encephalomyelitis virus (AEV) and is characterized by neurological symptoms such as ataxia in young birds (Hauck et al., 2017; Zhang et al., 2023). The virus has a wide range of hosts, with chickens, pheasants, turkeys, quail, and pigeons being susceptible. Young animals show typical neurological symptoms such as depression, rapid head and neck tremors, and ataxia. The prevalence of the disease in chicks is about 40 to 60% and the average mortality rate of sick chickens is about 25% (Zhang et al., 2023). Adult chickens infected with AE are subclinical and do not show neurological signs, but only transient egg-laying decline and reduced hatchability of breeding eggs (Meroz et al., 1990; Calnek, 1998; Zhang et al., 2023). In addition, the virus can also infect offspring chicks through the embryo, which is harmful to the poultry industry. AEV can be transmitted in diseased and normal flocks by horizontal and vertical routes of transmission, with the fecal-oral route being the main mode of horizontal transmission; whereas animals infected by the vertical mode of transmission through embryonic transmission show marked neurological signs after hatching (Toplu and Alcigir, 2004; Welchman et al., 2009).

Avian encephalomyelitis virus, a member of the family Picornaviridae, genus Tremovirus, is a nonenveloped single-stranded positive-sense RNA virus. The genome of AEV is approximately 7 knt in length and only contains 1 open reading frames (ORFs) (Miyamae, 1975). Avian encephalomyelitis virus genome structure is very similar to other small RNA viruses, the genome coding region first synthesizes a polyprotein precursor, which is broken down by intracellular proteases into 4 smaller precursor proteins, L, P1, P2, and P3, of which P1 is the precursor protein of the viral particle coat protein, and the precursor proteins P2 and P3 mainly constitute the viral non-structural proteins. Studies have shown that VP1, the coat protein of AEV, is an important host-protective antigen with good immunogenicity, and it is often used as an immunological diagnostic marker for the clinical detection of AEV (Wei et al., 2008; Hauck et al., 2017).

Since its outbreak in the United States in 1932, AE has now spread widely and become epidemic worldwide (Braune and Gentry, 1971; Itakura and Goto, 1975; Miyamae, 1975; Toplu and Alcigir, 2004; Welchman et al., 2009; Taunde et al., 2017; Lin et al., 2018; Goto et al., 2019; Ali et al., 2021; Wang et al., 2023). In China, since AE was first reported in Guangdong in 1980. Since then, an increasing number of reports on AEV have emerged from various provinces in China, drawing researchers’ attention. As there is no specific treatment for the disease, it can only be prevented and controlled by vaccine (Calnek, 1998). However, in recent years, some commercial laying hens immunized with the AEV vaccine showed paralysis during the breeding period and an unexplained decline in egg production during the peak laying period, with no obvious lesions on autopsy of the diseased chickens, which caused serious losses to the domestic breeding industry.

To address the molecular characteristics and antigenicity of the AEV responsible for the outbreaks in China, we sequenced the VP1 genes of 2 strains of AEV-positive infected chicken from Jiangxi and Guangdong. We analyzed the sequence and systematic development and compared them with 15 reference strains (Table S1) to help understand the genotype and genetic variation of AEV in Jiangxi and Guangdong, providing a reference for the prevention and control of AEV.

MATERIALS AND METHODS

Ethics Statement

All samples were collected on commercial chicken farms by veterinarians during routine diagnostic sampling after permission from the farm owner. No specific permits from an animal ethics committee were required.

Clinical Signs and Diagnostics

In May 2022, in several laying hen farms in Guangdong and Jiangxi, China, flocks around 1 to 4 wk of age continuously showed symptoms of emaciation, paralysis, and head and neck tremors, and wing droop, with no obvious lesions observed after autopsy, and the incidence rate was 7.3%. At the same time, several commercial laying hens around 35 wk of age showed an unexplained decline in egg production during the peak laying period, with egg production returning to normal in about 2 to 3 days. At the onset of the disease, the flocks were clinically stable, with no abnormalities in feeding, drinking, death rate, and respiratory tract. The egg production rate of laying hens can drop up to 23%, and there is no abnormality in the color, hardness, and thickness of eggshells except that the egg weight becomes smaller. Brain, liver, and spleen samples (n = 35) were taken from diseased chickens were sent to our laboratory for pathogen identification (the samples are included in the total sample). Common viral and bacterial pathogens including Newcastle diseases virus (NDV), Marek's disease virus (MDV), egg drop syndrome virus (EDSV), avian reticuloendotheliosis virus (REV), Avian reovirus (ARV), chicken anemia virus (CAV), Gyrovirus galga1 (GyG1), mycoplasma synoviae (MS) and Escherichia coli were firstly examined by standard assays. The results showed that only 2 (5.71%) of the 35 samples tested were positive for MS, 1 (2.86%) was positive for GyG1, and negative for the other pathogens mentioned above. Due to the clinical characteristics of the flocks of the disease, we hypothesized that AEV might be a contributor to the outbreaks in the flocks and attempted to identify and characterize the potential AEVs.

Detection of AEV and Other Related Pathogens

Total RNAs/DNAs were extracted using FastPure Viral DNA/RNA Mini Kit (Vazyme, Nanjing, China) according to the manufacturer's instructions and then were stored at −80°C until used. To investigate whether AEV was associated within these cases, a pair of primers (AEV-F: 5′-GAATTAGCTCCTGGTAAACCTCG-3′, and AEV-R: 5′-CTCTATCGCAACACCCTCAGG-3′ with a predicted product size of 288 bp) for a polymerase chain reaction (RT-PCR) was initially designed based on the VP1 gene of L2Z (GenBank accession no. AY275539), and then a RT-PCR assay was established with the designed primers. The first-strand cDNA synthesis was performed at 25°C for 5 min, 50°C for 45 min, and then 85°C for 2 min to inactivate the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) and followed by 4°C for 5 min.

Fragments were amplified using 2 × Phanta Max Master Mix (Dye Plus) (Vazyme) on the conditions of denaturation at 94°C for 5 min, 35 cycles of 94°C 30 s, 54°C 30 s, 72°C 30 s, and consequently with a final extension at 72°C for 5 min. Expected PCR products were purified, cloned, and sequenced based on the standard procedures. The previously established PCR protocols were used to test other viruses, NDV, MDV, EDSV, REV, ARV, CAV, and GyG1 (primer sequences present in Table S2). In addition, common enteropathogenic germs, including pathogenic E. coli, and Mycoplasma synoviae (MS) were also tested via bacterial isolation and identification with the standard protocols.

Complete Genome Amplification, Sequencing, and Analysis of AEVs

Primers for amplification of the complete genome sequence of AEV field strains were designed based on the AEV L2Z strain (GenBank accession no. AY275539) (primer sequences present in Table S3). Viral RNA was extracted by the method aforementioned. Fragments were amplified using 2 × Phanta Max Master Mix (Dye Plus) (Vazyme) on the conditions of denaturation at 95°C for 3 min, 30 cycles (95°C 15 s, 55°C 15 s, 72°C 1 min), and then with a final extension at 72°C for 5 min. The 5’-and 3’- rapid-amplification of cDNA ends (RACE) for the determination of the terminal sequences of AEV were performed by using a 5’/3’ SMARTer RACE kit (Clontech, Beijing, China) following the manufacturer's instructions. Positive PCR products were subjected to gel purification and afterward cloned into pMD 19-T vectors (TaKaRa, Dalian, China). Three to 5 positive clones of each amplicon were submitted to a commercial sequencing company (Sangon Biotech, Shanghai, China) for sequencing at both directions by Sanger sequencing methodology. Sequences of complete genome were assembled and annotated using the SeqMan in DNAStar Lasergene V 7.10 (DNAStar, Inc., Madison, WI). Phylogenetic analyses of AEV were conducted based on the complete genome and deduced amino acid (aa) sequences of the VP1 protein of AEV, by using the neighbor-joining method (Bootstrap in 1,000 replicates) of molecular evolutionary genetics analysis (MEGA) software (v6.0.2).

RESULTS

AEV Surveillance in Guangdong and Jiangxi Province

Clinical samples collected from chicken farms from 37 farms in Guangdong and Jiangxi province, China, from January 2021 to August 2023 were selected for the AEV prevalence survey. Of 294 chicken brain, liver, and spleen samples examined, 136 (46.26%) were AEV positive. In the context of the sample collected date, The highest detection rate of AEV was found in flock samples collected in June, followed by May (55.56%). Meanwhile, the lowest detection rate of AEV was found in the flock samples collected in February (25.00%), and the detection rate of AEV was also at a low level in January (33.33%), August (33.33%) and September (23.08%) (Table 1). Looking at the different breeds of chickens, laying hens, egg breeders, and broiler breeders showed a high positive rate of AEV. As to the growing stage of chickens, AEVs were detected in flocks of all ages, with the highest prevalence of infection in flocks between 13 to 18 wk, followed by 31 to 36 wk, and >36 wk, respectively (Table 2).

Table 1.

Seasonal distribution of AEV-infected flocks in Guangdong and Jiangxi Provinces, China, 2021–2023.

January February March April May June July August September October November December
2021 25.00% 0.00% 42.86% 33.33% 42.86% 56.25% 33.33% 20.00% 0.00% 33.33% 42.86% 25.00%
2022 40.00% 0.00% 28.57% 33.33% 60.00% 57.89% 41.18% 37.50% 33.33% 50.00% 0.00% 44.44%
2023 0.00% 33.33% 36.36% 50.00% 56.52% 63.64% 50.00% 40.00% / / / /
Total 33.33% 25.00% 36.00% 39.13% 55.56% 60.29% 39.29% 33.33% 23.08% 40.00% 42.86% 38.46%
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Abbreviation: AEV, avian encephalomyelitis virus.

Table 2.

Age distribution of AEV-infected flocks in Guangdong and Jiangxi Provinces, China, 2021–2023.

0–6 wk 7–12 wk 13–18 wk 19–24 wk 25–30 wk 31–36 wk >36 wk
2021 0.00% 0.00% 55.17% 29.41% 22.22% 45.45% 23.08%
2022 14.29% 18.18% 70.73% 36.84% 27.27% 47.06% 44.44%
2023 0.00% 11.11% 57.14% 33.33% 25.00% 39.13% 40.00%
Total 9.09% 13.04% 62.24% 33.33% 25.00% 43.14% 36.59%
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Abbreviation: AEV, avian encephalomyelitis virus.

Complete Genome Sequencing and Genetic Analysis of AEVs

To characterize the genetic features of AEV, the complete genome sequence of 2 representative AEV strain, designated CH/GD202201 and CH/GD202202, was determined and analyzed (Figure 1). The genome length of the 2 AEV strains isolated from chickens was 7,031 bp (excluding the polyA tail), consistent with the genome size of other reported AEV strains (the sequence was deposited into the GenBank under the accession number of OR451211 and OR451212). Like other members of the Picornaviridae family, the 5′ end of the viral genome contains a type IV IRES, which mainly mediates the initiation of viral protein synthesis. This end contains only 1 translation initiation site, encoding a polyprotein precursor, which is hydrolysed and cleaved by the viral protease, leading to the formation of a mature viral protein. A multialignment analysis indicated that CH/GD202201/ CH/GD202202 shared 82.3 to 99.7% at the nt level and 95.3 to 99.9% at the aa level of the polyprotein protein, and 83.2 to 98.0% at the nt level and 97.8 to 99.6% at the aa level of the VP1 protein when compared with reference AEV strains retrieved from GenBank (Table 3). The VP1 protein is the main structural protein of AEV and the main target of the host immune response. Multiple sequence comparison of the VP1 protein revealed that most AEV strain variants share 4 major AA mutations: E3G, H66Y, R120C, and E153K. Multiple sequence comparison of the VP2 and VP3 proteins revealed that our strains (CH/GD202201 and CH/GD202202) share aa mutations such as Q189R (VP2), T63I (VP3), and T63I (VP3) in common with the chick embryo adapted strains (Figure 4), aa mutations such as Q189R (VP2), T63I (VP3) (Figure 2). Based on the phylogenetic analysis of the aa sequences of the CH/GD202201 and CH/GD202202 ORF1 genes determined in this study with the AEV reference strains, CH/GD202201 and CH/GD202202 belonged to the same branch as van_Reokel and GDs29, and were in a different branch from the strains 204C and Pf-CHK1/AEV, and 204C and Pf-CHK1/AEV strains belong to different branches (Figure 3A). According to the aa sequence of VP1 protein, CH/GD202201 and CH/GD202202 belonged to a separate branch, which was a novel AEV strain, and were in different sub-branches from vaccine_A, vaccine_B, vaccine_A_7P), vaccine_B_7P, AEVDL21, L2Z (AY275539), small branches and with Tremovirus_A in a different large branch (Figure 3B).

Figure 1.

Figure 1

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The electropherogram of amplicons of PCR for amplification of the full-length genome sequence of CH/GD202201 and CH/GD202202. M: DL2000 DNA marker; Lane 1–6: 6 overlapping fragments of CH/GD202201; Lane 7–8: 5’ RACE and 3’ RACE products of CH/GD202201; Lane 9–14: 6 overlapping fragments of CH/GD202202; Lane 15–16: 5’ RACE and 3’ RACE products of CH/GD202202; Lane 17: negative control. Abbreviations: PP, polymerase chain reaction; RACE, rapid-amplification of cDNA ends.

Table 3.

Nucleotide (nt) and amino acid (aa) sequence identities in percentage based on a comparison of the whole gene sequence.

% identity to CH/GD202201(nt/aa)
 % identity to CH/GD202202(nt/aa)
Reference strain ORF1 VP1 VP2 VP3 ORF1 VP1 VP2 VP3
AEV(AJ225173) 95.9/98.6 97.4/98.9 96.7/98.6 93.2/96.7 94.6/98.5 95.8/98.9 94.9/98.6 93.2/96.7
van_Reokel(AY517471) 98.1/99.4 95.7/98.9 98.2/100 99.2/98.0 99.5/99.6 97.9/99.6 100/100 99.2/98.0
204C(KF979338) 83.5/96.2 83.2/98.1 81.8/98.2 82.3/95.9 83.5/96.1 83.5/98.1 81.1/98.2 82.3/95.9
L2Z(AY275539) 95.2/96.7 97.2/98.1 97.3/98.6 92.1/94.3 94.2/96.5 95.6/98.1 98.3/98.6 92.1/94.3
UT-Marjanmehr(OP104963) 95.7/98.2 97.4/98.9 96.4/98.2 93.1/96.3 94.4/98.0 95.8/98.9 94.6/98.2 93.1/96.3
AEV(_NC_003990) 95.9/98.6 97.4/98.9 96.7/98.6 93.2/96.7 94.6/98.5 95.8/98.9 94.9/98.6 93.2/96.7
field_A_7P(KY508665) 95.6/98.4 97.3/98.9 96.4/98.2 92.9/96.3 94.3/98.3 95.7/98.9 94.6/98.2 92.9/96.3
vaccine_A(KY508663) 95.8/98.5 97.3/98.9 96.7/98.6 93.1/96.7 94.5/98.4 95.7/98.9 94.9/98.6 93.1/96.7
vaccine_B(KY508661) 95.8/98.6 97.2/98.9 96.7/98.6 93.2/96.7 94.5/98.5 95.6/98.9 94.9/98.6 93.2/96.7
vaccine_A_7P(KY508664) 95.7/98.4 97.3/98.5 96.7/98.6 93.1/96.3 94.4/98.3 95.7/98.5 94.9/98.6 93.1/96.3
vaccine_B_7P(KY508662) 95.7/98.4 97.3/98.5 96.4/98.2 93.1/96.7 94.4/98.3 95.7/98.5 94.6/98.2 93.1/96.7
AEVDL21(OQ749507) 95.8/98.6 97.3/98.9 96.4/98.6 93.2/96.7 94.6/98.5 95.7/98.9 94.6/98.6 93.2/96.7
Tremovirus_A(MF620096) 95.7/98.4 96.8/97.8 96.5/98.2 93.1/96.3 94.5/98.2 95.4/97.8 94.7/98.2 93.1/96.3
GDs29(MF179107) 98.3/99.7 95.8/98.9 98.2/100 100/100 99.7/99.9 98.0/99.6 100/100 100/100
Pf-CHK1/AEV(KT880668) 82.4/95.4 84.1/98.1 82.6/97.3 80.7/95.5 82.3/95.3 84.1/98.1 82.1/97.3 80.7/95.5
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Figure 4.

Figure 4

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Alignment analysis of the amino acid sequences of the VP1 gene associated with embryo-adaption between the identified AEV strain CH/GD202201 and CH/GD202202 and reference AEV strains. Abbreviation: AEV, avian encephalomyelitis virus.

Figure 2.

Figure 2

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Alignment analysis of the nucleotide sequence and amino acid sequences of the VP2 and VP3 gene associated with embryo-adaption between the identified AEV strain CH/GD202201 and CH/GD202202 and reference AEV strains. The incriminated nucleotide (amino acid) positions 566(189) in the VP2 region (A) and 188(63) in the VP3 region (B) are highlighted. Abbreviation: AEV, avian encephalomyelitis virus.

Figure 3.

Figure 3

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Phylogenetic trees of the ORF1 (A), VP1 (B), VP2 (C), and VP3 (D) animo acid sequence of avian encephalomyelitis virus. A bar of 0.1 indicates amino acid substitutions per site. “▲” indicates the strain identified in this study.

DISCUSSION

The prevalence of AEV has caused huge economic losses to the poultry industry (Wang et al., 2023; Zhang et al., 2023). AEV infection in young animals usually leads to neurological symptoms such as ataxia, while in infected adult animals, it does not show clinical symptoms, but only transiently decreases in egg laying and hatchability of breeding eggs. After pathological dissection of the dead chickens, it can be found that the disease has no obvious pathological damage to the internal organs of the animals, while the most serious damage to the organs of the animals is mainly concentrated in the brain, which is manifested as the overall softening of the brain tissue of the diseased chickens, with the blurred demarcation of the cerebral hemispheres, cerebral membrane oedema, and the presence of scattered small hemorrhages, etc., which is the same as the current pathological situation of the AEV outbreaks found in Guangdong and Jiangxi (Miyamae, 1976; Hauck et al., 2017). Up to now, there is no effective treatment for this disease, and it can only be prevented by vaccination. The disease was first reported in China in 1980 and since then AEV infections have been reported in many of the country's major poultry provinces.AE infection is a major concern in large poultry-producing countries because of the threat it poses to the health and productivity of the flocks. In this study, a total of 294 chicken tissue samples suspected to be infected with AEV, covering a period of 3 yr, were found to have a prevalence rate of 46.26% (136/294) and these results are in agreement with other studies (Pohjola et al., 2017; Taunde et al., 2017; Ali et al., 2021; Gethoffer et al., 2021). The positive rate of AEV detected by PCR in Guangdong and Jianxi province from 2021 to 2023 was 37.80%, 46.77%, and 53.41%, respectively. The rising trend in the infection rate of circovirus is apparent. In Finland, Pohjola et al. (2017) reported on the outbreak in that country in 2017; after collecting serum samples from 457 chickens from 51 chicken farms, they found that these sera were 86% positive for AEV antibodies. These data suggest that AEV infections are common in chickens worldwide. The disease can occur throughout the year but is more likely to occur during the rainy summer months. Because AEV is stable, can survive in the environment for long periods of time, and is not easily removed, rain contaminates feed and water sources, etc., and this environment facilitates the spread of the virus from flock to flock. Therefore, appropriate biosecurity measures (including regular cleaning and disinfection of facilities and equipment) on poultry farms, regardless of the season, can help reduce the risk of disease transmission. In addition, we found that AEV was detected significantly more frequently in flocks 13 to 18 wk of age than in flocks of other ages, which could be attributed to the occurrence of infection in flocks due to a decline in maternal antibody levels. In addition, an increase in the infection rate of the virus was also observed during the peak laying period of the flock, with a positive rate of 43.14%. After the chickens lay eggs into the egg-laying period, the stress of egg-laying and nutritional depletion makes the hens' physical condition easy to deteriorate, affecting their own immunity, which will lead to outbreaks of pathogenic bacteria lurking inside the hens' bodies and an increase in the rate of infection of diseases in vitro (Ali et al., 2023). Especially like influenza, and egg reduction syndrome, these viruses, long-term latent in the body of the hen, young chickens do not show clinical symptoms, and once the peak egg-laying period, hen immunity will be reduced when the outbreak, which is why many laying hens are prone to peak morbidity and lead to egg production on the rate of not go one of the reasons (Zhuang et al., 2015; Parent et al., 2020; Najimudeen et al., 2021; Xu et al., 2022).

Currently, the serotypes of all AEV isolates remain essentially the same. However, the pathogenicity of AEV in susceptible animals varies greatly, and it can be classified into enterophilic and neurophilic types according to its histophilicity in susceptible animals. The wild strains prevalent in nature are enterophilic, which can infect animals through the oral route and stimulate the body to produce specific immune responses; the wild strains can produce highly neurophilic strains, i.e., Van Roekel strains, after successive passages in the brain tissue of chick embryos; the subcutaneous and intramuscular routes of inoculation can infect the chicks and produce obvious neurological symptoms, but rarely stimulate the body to produce effective humoral immune responses (e.g., the Van Roekel strains, which are highly neuronal). However, it rarely stimulates an effective humoral immune response (Shafren and Tannock, 1991). Currently, only the full gene sequences of GDs29, VanRoekel, L2Z, Pf-CHK1/AEV, and 204C strains have been determined from all AEV isolates, and some gene sequences of some local isolates of SD, SX, and YL strains have been sequenced by researchers. Therefore, the AEV gene sequence library needs to be replenished urgently.

The total length of the genome sequences of the 2 AEV strains isolated in this study was 7,031 bp, which was the same size as the domestic isolates GDs29 and AEVDL21, and belonged to the same clade as GDs29 and VanRoekel. According to the analysis of the VP1 region, VP2, and VP3 regions, the variability of VP2 and VP3 proteins was higher, while the VP1 protein was more conserved. The results indicate that this change of organ tropism is caused by 2-point mutations in the VP2 and VP3, which is similarly to Coxsackievirus, in which 2-point mutations in the VP2 and VP3 change the binding behavior of the virus (Hauck et al., 2017). Mutations at both sites were also found in the 2 strains of AEV virus that we sequenced and may be responsible for the outbreaks that occurred in chickens that were immunized with the AEV vaccine. Furthermore, it remains to be determined whether both mutations are necessary to alter the biological tropism.

In recent years, there have been more and more reports about AEV and the economic losses caused to the chicken industry. At present, prevention is mainly based on the AEV vaccine, and drug treatment is ineffective (Lin et al., 2018; Sarma et al., 2019). Therefore, it is necessary to strengthen the daily feeding management and biosecurity measures of chickens, improve the breeding environment, enhance the resistance of chickens to AEV, and prevent secondary infection or secondary infection. From the results of the current analyses, the degree of variation of the 2 strains of AEV is small and the epidemic is relatively stable. In this study, amplification and whole genome sequencing of 2 strains of AEV-infected chickens in Guangdong were performed to understand the genotypes and genetic variation of AEV in these areas and to provide a reference for the prevention and control of AEV.

In conclusion, the present study demonstrated that AEV is widely prevalent in laying hens and can cause morbidity in 1 to 4 wk-old and 35 wk-old commercial laying hen flocks. In this study, we determined the complete genome sequences of a representative AEV strain from a 1 to 4 wk-old morbid chicken (CH/GD202201) and a 35-wk-old commercial laying hen (CH/GD202202) from Guangdong Province, China. This study bridges the gap in the understanding of the molecular epidemiology, viral genetics, pathogenicity, and pathogenesis of AEV in Jiangxi and Guangdong provinces, and provides a basis for further understanding of the spread of AEV in China.

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ACKNOWLEDGMENTS

This research was supported by Jiangxi Special Fund for Agro-scientific Research in the Collaborative Innovation Project (JXXTCXQN202101), the earmarked fund for Jiangxi Agriculture Research System (JXARS-09), and the earmarked fund for Jiangxi Provincial Livestock and Poultry Breeding Industry Joint Tackling of Challenges Project (2022JXCQZY04).

DISCLOSURES

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2023.103264.

Appendix. Supplementary materials

mmc1.docx (24KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (24KB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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