Prevalence and Molecular Characteristics of FAdV-4 from Indigenous Chicken Breeds in Yunnan Province, Southwestern China

Fowl adenovirus-induced hepatitis–pericardial effusion syndrome outbreaks have been increasingly reported in China since 2015, resulting in substantial economic losses to the poultry industry. The genetic diversity of indigenous chicken results in different immune traits, affecting the evolution of these viruses. Although the molecular epidemiology of fowl adenovirus serotype 4 (FAdV-4) has been well studied in commercial broiler and layer chickens, the prevalence and genetic characteristics of FAdV-4 in indigenous chickens remain largely unknown. In this study, samples were collected from six indigenous chicken breeds in Yunnan province, China. FAdV-positive samples were identified in five of the six indigenous chicken populations via PCR and 10 isolates were obtained. All FAdVs belonged to serotype FAdV-4 and species FAdV-C. The hexon, fiber, and penton gene sequence comparison analysis demonstrated that the prevalence of FAdV-4 isolates in these chickens might have originated from other provinces that exported chicks and poultry products to Yunnan province. Moreover, several distinct amino acid mutations were firstly identified in the major structural proteins. Our findings highlighted the need to decrease inter-regional movements of live poultry to protect indigenous chicken genetic resources and that the immune traits of these indigenous chickens might result in new mutations of FAdV-4 strains.


Introduction
Fowl adenovirus (FAdV) is a DNA virus belonging to the genus Aviadenovirus in the family Adenoviridae [1].Based on variations in the antigenic structure, FAdVs can be categorized into three groups: Group I fowl adenoviruses, which are derived from the respiratory tracts and livers of chickens, turkeys, geese, and ducks [2], Group II fowl adenoviruses, primarily isolated from the lymphocytes and livers of turkeys and known as hemorrhagic enteritis viruses (HEV) that primarily induce hemorrhagic enteritis in turkeys and marble spleen disease in pheasants [3], and Group III fowl adenoviruses, referred to as egg drop syndrome viruses (EDSV) [4].Group I FAdV is a prevalent pathogen in fowl species and can be further divided into five subgroups: A, B, C, D, and E [5].Additionally, it consists of 12 serotypes, ranging from FAdV-1 to FAdV-11, with FAdV-8 further divided into two types: 8a and 8b [6].Group I FAdVs causes fowl inclusion body hepatitis (IBH), hydropericardium hepatitis syndrome (HHS), and gizzard erosion (GE), posing significant risks to the poultry sector and impeding its development [7].Notably, FAdV-4 is the causative agent of HHS, which is distinguished by the presence of pericardial effusion and liver necrosis [8].
The coat proteins of fowl adenoviruses, namely, fiber, penton, and hexon, exhibit a significant degree of homology across all adenoviruses and play a crucial role in determining the efficacy of viral infection [9].In the case of the group I fowl adenoviruses, the majority possess two fiber proteins, whereas FAdV-4 possesses two fiber proteins of varying lengths [10].The interactions between viruses and hosts are mediated by fiber proteins [11], whereas viral internalization during the infection cycle is facilitated by penton proteins [12].Hexon genes, owing to their conserved nature and the antigenicity of the proteins they encode, are utilized as efficient tools for rapid type inference [6].
Chickens of various age groups are susceptible to FAdVs, with broilers aged 3-5 weeks exhibiting the highest vulnerability.Since 2015, there has been a notable increase in reports concerning FAdVs across several provinces in China, particularly broiler-producing provinces, such as Anhui, Shandong, Guangdong, and Guangxi, as evidenced by the substantial number of clinical HHS samples isolated from these regions [13].Although commercial vaccination against FAdV-4 has been implemented, FAdV-4 infection still occurred sporadically.In most of the previous studies on the isolation and sequencing of FAdVs from commercial broiler and layer chickens [13][14][15], no systematic epidemiological investigation and study of the molecular characteristics of FAdVs from indigenous chickens has been performed.
Indigenous chickens are an important source of genetic and phenotypic diversity.However, infectious diseases threaten the health of chickens.Previous studies have revealed that the immune responses of different kinds of indigenous chicken breeds after being infected with pathogens such as Newcastle disease virus showed significant differences [16,17].The diversity of the MHC, which regulates specific immune responses, was confirmed in Chinese indigenous chickens [18].Thus, these viruses might have some mutations under the different selective pressures from these indigenous chickens.Yunnan province, situated in the southwestern region of China, is renowned for its abundant animal resources and is often referred to as the "Kingdom of Animals".The province has plenty of unique indigenous chicken breeds including the Wuliangshan black-bone chickens, La-bai high leg Chickens, Yanjin black-bone chickens, Xichou black-bone chickens, Yunlong shortfoot chickens, Wuding strong chickens, Qinhua chickens, and so on.These indigenous chicken breeds have significant value as precious species resources within China.However, there is a lack of reports on the prevalence of FAdVs in indigenous chickens in this region and the molecular characteristics of these viruses have remained largely unknown.Within the scope of this study, we successfully isolated and identified FAdVs from indigenous chicken populations, thereby elucidating the epidemiological attributes of FAdV-4 in specific chicken populations in Southwest China.These findings suggest that FAdV-4 exists among indigenous chicken breeds in Yunnan province and has some new mutations.

Sample Collection and Processing
To investigate the prevalence of FAdVs in the indigenous chickens in Yunnan province, a total of 478 swab samples including oropharyngeal and cloacal swabs of the same chicken from clinically healthy indigenous chickens and 28 tissue samples from suspected FAdVs infections were collected to detect FAdVs from January 2022 to June 2023.The tissue and swab samples were collected and treated as previously described [19,20].The samples were obtained by adding tissue to a PBS buffer containing penicillin and streptomycin, followed by grinding in liquid nitrogen, subjecting to three freeze-thaw cycles, and subsequently centrifuging at 4 • C and 8000× g to obtain the supernatant.Similarly, the collected swabs underwent three freeze-thaw cycles and were then centrifuged to obtain the supernatant.The supernatant derived from the samples was further filtered through a 0.22 µm filter and stored at a temperature of −80 • C. The specific details of the sample collection are presented in Table 1.

Virus Isolation
The presence of FAdVs was confirmed via polymerase chain reaction (PCR), as described previously [21].All positive samples were inoculated into LHM cells, according to a previous method [22].The LMH cells were cultivated in F12 (Gibco, Grand Island, NE, USA) medium supplemented with 10% FBS (Gibco, Grand Island, NE, USA) for the purpose of passaging.Upon reaching 80% confluence, the filtered supernatant derived from the diseased material was diluted by a factor of 5 and subsequently introduced into the LMH cells.Following a 2 hours incubation period, the adsorption process was substituted with F12 medium containing 2% FBS to sustain cell proliferation.The cells were then cultured in 5% CO 2 , 37 • C incubators for a duration of 3 to 5 days.The cells were be monitored daily for signs of swelling, rounding, and aggregation into grape-bunch-like structures.If these characteristics were observed in the majority of cells, the cultured cells and supernatant were collected.After three cycles of freeze-thawing, these samples were filtered using a 0.22 µm filter and then inoculated into LMH cells following the previously described method.After three passages, the cultured cells and supernatant were collected and stored at −80 • C.

DNA Extraction and PCR
Genomic DNA was isolated from the culture supernatants using an Accurate Biology Viral Genomic DNA/RNA Extraction Kit (Accurate Biology, Changsha, China).The samples were analyzed by PCR using specific primers (Supplementary Table S1).Analyses were conducted using 1% agarose gel electrophoresis to determine the presence of positive samples.Amplification of the hexon, fiber1, fiber2, and penton genes was performed on these positive samples using the specific primers listed in Supplementary Table S1.The target amplification products were purified by 1% agarose gel electrophoresis, and the desired bands were excised.A gel extraction kit (Omega, Norcross, GA, USA) was used to purify the positive products, which were then cloned into a Blunt-zero plasmid vector (Vazyme, Nanjing, China).Subsequently, these clones were transformed into E. coli DH5α receptor cells (AlpalifeBio, Shenzhen, China), and the positive clones were selected and sent to Sango Biotech (Shanghai, China) for sequencing.

Sequence Comparison and Phylogenetic Analysis
The nucleotide sequences of hexon, penton, fiber1, and fiber2 were aligned with publicly available reference sequences obtained from the National Library of Medicine (NCBI) using the MegAlign program, which is part of the DNAStar software suite version 5.01 (DNAStar, Madison, WI, USA).Supplementary Table S2 provides detailed information on the reference sequences.Phylogenetic trees for the hexon, penton, fiber1, and fiber2 genes were constructed using the neighbor-joining method implemented in MEGA version 11.0.

Phylogenetic Analysis of FAdVs Isolated from Indigenous Chickens in Yunnan Province
To investigate the phylogenetic characteristics of FAdVs circulating in indigenous chickens in Yunnan province, the hexon, fiber, and penton genes of the 10 FAdVs were sequenced and submitted to GenBank.Phylogenetic analysis of the hexon, fiber1, fiber2, and penton genes of these 10 FAdVs clustered them into the same branch, which belonged to the FAdV-4/C group (Figures 1-3).
To investigate the phylogenetic characteristics of FAdVs circulating in indigenous chickens in Yunnan province, the hexon, fiber, and penton genes of the 10 FAdVs were sequenced and submitted to GenBank.Phylogenetic analysis of the hexon, fiber1, fiber2, and penton genes of these 10 FAdVs clustered them into the same branch, which belonged to the FAdV-4/C group (Figures 1-3).Figure 2. Phylogenetic analysis of the fiber genes.(A) Phylogenetic tree for the fiber1 genes of 10 isolated strains and 27 reference strains created using MEGA 11.0 software using the neighbor joining method.(B) Phylogenetic tree for fiber2 genes of 10 isolated strains and 18 reference strains created using MEGA 11.0 software using the neighbor-joining method.The black triangle indicates isolated strains in this study.
Figure 2. Phylogenetic analysis of the fiber genes.(A) Phylogenetic tree for the fiber1 genes of 10 isolated strains and 27 reference strains created using MEGA 11.0 software using the neighbor joining method.(B) Phylogenetic tree for fiber2 genes of 10 isolated strains and 18 reference strains created using MEGA 11.0 software using the neighbor-joining method.The black triangle indicates isolated strains in this study.
The fiber1 genes of YNYL/2022, YNXJ/2022, and YNBL-8/2023 were closely related to the isolates from Shandong, Henan, Heilongjiang, and Guangxi.The other strains shared genetic similarities with isolates from the Shandong, Anhui, and Guangdong provinces.The hexon genes of the 10 FAdV-4 strains in this study were compared with the sequences published in the NCBI, and a table of homology between the Yunnan isolates and other isolates was compiled (Supplementary Table S3).The hexon genes of all 10 Yunnan isolates were highly homologous to the GDMZ virus isolated from Guangdong province and the SCDY virus isolated from Sichuan province.YNBL-7/2023, YNFD/2022, YNLP/2022, YNNC-1/2022, YNNJ/2022, YNXJ/2022, and YNYL/2022 were also most closely related to AH712 isolated from Anhui province.YNBJ-5/2023, YNBL-7/2023, Figure 3. Phylogenetic analysis of the entire penton gene.Phylogenetic tree for penton genes of 10 isolated strains and 28 reference strains created using MEGA 11.0 software using the neighborjoining method.The black triangle indicates isolated strains in this study.
For the fiber 2 gene, YNLP/2022 shared the highest genetic relationship with the Guangxi isolate.The other strains shared genetic similarities with isolates from Shandong, Anhui, and Guangdong provinces in China.

Molecular Characteristics of FAdV-4 Isolated from Southwest China
A thorough examination was undertaken to analyze the amino acid mutations in the sequencing data of the hexon, penton, fiber1, and fiber2 proteins.The investigation revealed the existence of multilocus amino acid mutations, particularly in the hexon protein of the FAdV-4 strain examined in this study.Furthermore, additional amino acid mutations were observed in the penton, fiber1, and fiber2 proteins.
The fiber1 protein has been implicated in viral invasion.Prior research has demonstrated that chick embryo lethal orphan (CELO) viruses, specifically FAdV-1, can interact with coxsackieviruses and adenovirus receptors (CARs) through fiber1 when initiating para-infection through fiber 2 proteins [26].However, the key amino acid sites in fiber1 have not been identified.Compared with ON1 from Canada and MX-SHP95 from Mexico, the isolate from southwest China had a deletion of 428 amino acid sites in fiber1, which was consistent with FAdV-4 isolated from other regions of China.The mutation sites in the fiber1 protein included S25P, T46A, N63S, K165R, T217A, and L255F (Table 5).Among these, aa46 was identical to the strains ON1 from Canada and MX-SHP95 from Mexico.
Previous studies have identified many conserved amino acid mutations in fiber2 proteins, such as G219D, P307A, V319I, and A380T, which are found in all highly virulent FAdV-4 strains, some of which may be associated with virulence [27].These mutations were also found in isolates from the indigenous chicken breeds of Yunnan province.Five amino acid insertions at aa11-15 was observed in these isolated FAdV-4 strains when compared with the ON1 and MX-SHP95 strains, consistent with the other FAdV-4 strains isolated in China.The fiber2 protein of these viruses also showed some mutations, including E136G, D142G, P384S, S413R, and A475T (Table 6).It is worth noting that the sites D219, Q232, T261, T300, A305, A307, I319, and T380 of the fiber2 protein of theses FAdV-4 strains were identical to the highly virulent MX-SHP95 strain but differed from the ON1 strain.
a Bold-type letters indicate the mutations when compared with the other FAdV-4 strains isolated in this study or ON1 and MX-AHP5.
a Bold-type letters indicate the mutations when compared with the other FAdV-4 strains isolated in this study or ON1 and MX-AHP5.
a Bold-type letters indicate the mutations when compared with the other FAdV-4 strains isolated in this study or ON1 and MX-AHP5.
a Bold-type letters indicate the mutations when compared with the other FAdV-4 strains isolated in this study or ON1 and MX-AHP5.

Discussion
FAdVs, especially FAdV-4, can induce high mortality rates in chickens and can be transmitted via horizontal and vertical transmission, posing a great threat to the safety of indigenous chickens.Before 2015, IBH and HHS associated with FAdVs infections, including FAdV-4, FAdV-8a, FAdV-8b, and FAdV-11, were sporadically reported in poultry in China, with low mortality rates [28].However, since 2015, outbreaks of HHS, mainly associated with FAdV-4, have occurred in broiler-producing provinces such as Shandong, Henan, Jiangsu, Guangdong, Guangxi, and Anhui [29].Subsequently, the FAdV-4 strain has spread rapidly throughout the country and among layer chickens, resulting in huge economic losses to the poultry industry.FAdV-4 is the dominant serotype, but FAdV-8a, FAdV-8b, and FAdV-11 also are noted in China [22,30].Indigenous chicken breeds are valuable genetic resources for breeding.Yunnan province, located in southwest China, hosts multiple types of indigenous chickens.In this study, we showed that five of six indigenous chicken populations tested positive for FAdVs.Moreover, all FAdVs detected in this study were identified as FAdV-4.Thus, our results confirmed the prevalence of FAdV-4 but not FAdV-8a, FAdV-8b, or FAdV-11 in indigenous chicken breeds in Yunnan province.
Zhang et al. demonstrated that FAdV-4 strains isolated from central China exhibit clustering with viruses isolated from India, as determined by the hexon sequence, suggesting the potential origin of these viruses as India [31].However, upon analyzing the genome sequence of three FAdV-4 strains isolated in 2015, it was observed that these strains displayed the highest nucleotide similarity with early Chinese strains.This finding suggests the potential origin of these viruses as early FAdV-4 strains in China [32].A prior investigation similarly discovered that FAdV-N22 obtained from a live Newcastle disease vaccine, exhibited a substantial degree of sequence similarity with JSJ13, which was isolated from birds affected by infectious bursal disease in China during the period 2012-2013 [33].This suggests that the contamination of live vaccines may have a significant impact on the occurrence of HHS outbreaks in Chinese poultry.Numerous studies have documented that the viral excretion of FAdV-4 through the respiratory and digestive systems can persist for a duration of two weeks [34,35].Furthermore, it has been observed that FAdV-4 can be effectively disseminated among fowl populations through aerosol transmission [33].Recent investigations have identified the presence of FAdV-4 in Taizhou geese [36], Cherry Valley ducks [37], mandarin ducks [38], and Muscovy ducks [38], with a high degree of genetic similarity to viruses isolated from chickens.These findings suggest the potential occurrence of the cross-species transmission of FAdV-4.
The hexon, fiber, and penton gene sequence comparison analysis in this study demonstrated that all 10 FAdV-4 strains isolated from indigenous chickens in Yunnan province had the highest nucleotide similarity with viruses previously reported in other provinces, such as Guangdong, Sichuan, Anhui, Heilongjiang, Shandong, Guangxi, and Henan, indicating that the prevalence of FAdV-4 isolates in these indigenous chickens might have originated from other provinces in China.Although Yunnan province hosts multiple types of indigenous chickens, the productive performance of these indigenous chickens is relatively poorer than that of hybrids of commercial broiler chickens.Thus, most of the chicks were imported into Yunnan province from other provinces, such as Guangdong, Guangxi, Henan, and Shandong.Moreover, poultry products from these developed poultry provinces were brought into Yunnan province to satisfy the consumption needs of the people.This might have resulted in the introduction of FAdV-4 into Yunnan province.It suggests that the scale of broiler breeding in Yunnan province should be expanded to meet the demand of meat and eggs in Yunnan province as much as possible, so as to reduce the demand for live poultry transported from other provinces.
The identification of four essential FAdV-4 genes, specifically hexon, penton, fiber1, and fiber2, has been documented [39][40][41].Of these genes, hexon and fiber2 are of particular significance in determining the virulence of FAdV-4 and are strongly related to the emergence of highly pathogenic FAdVs [42,43].The discovery of FAdV-4 in Southwest China implies that FAdV-4 is transmitted between different chicken breeds.The different adaptations of FAdV-4 in indigenous flocks of different breeds have resulted in numerous amino acid mutations in the isolates from Southwest China.Many of the mutations were previously unreported and were concentrated in the hexon and fiber genes.Previous research suggests that the hexon protein is crucial to determining the virulence of FAdV-4.The substitution of the amino acid isoleucine (I) with arginine (R) at position 188 of the hexon protein is anticipated to greatly enhance the virulence of FAdV-4 [44].In the hexon protein, several mutations have been reported, including I188R, Q193R, and E195Q [45].However, some mutations, including Y17H, W97R, N147D, T199I, T254S, R290G, N389D, T414I, S429N, T465A, D599G, N601D, W637R, M707T, T735I, G747D, V816A, and V914A, have not been previously reported.Furthermore, the hexon protein from the indigenous chicken isolate displayed mutations at aa193 and aa195, which were identical to those observed in the avirulent strain ON1 from Canada and the virulent strain MX-SHP95 from Mexico.Amino acid mutations also occurred in the fiber1 protein, including the previously unreported mutations S25P, A46T, N63S, K165R, and L255F.In the fiber2 protein, the mutations G219D, P307A, V319I, and A380T were observed in these isolates, which were consistent with previously reported amino acid mutations that may be closely related to virulence [27].However, the mutations E136G, D142G, E232Q, P384S, S413R, and A475T, which have not been previously reported, were also identified.Notably, the aa46 and aa255 mutations in the fiber1 protein were consistent with strains isolated from other countries and the aa430 mutation was consistent with the virulent Mexican strain MX-SHP95.Previous studies have demonstrated that the MHC system, which plays important roles in immune response, shows high genetic diversity in Chinese indigenous chickens, indicating that the immune traits of these indigenous chickens might have large differences [46].Recent studies have confirmed that FAdV-4 can induce cellular pathways in chickens to produce interferon and antigen presenting molecules (MHCI/II) [47].Thus, the different immune traits of these indigenous chickens might have resulted in the mutations in the FAdV-4 strains isolated in our study.However, further research is required to determine the biological significance of these mutations in recombinant chimeric viruses.
In summary, our results reveal the existence of FAdV-4 in indigenous chickens in Yunnan province.Moreover, several mutations in the major structural proteins of FAdV-4 were identified, indicating that FAdV-4 may have undergone evolutionary adaptation to suit the indigenous chicken breeds.Thus, it is imperative to conduct further investigations to ascertain the potential impact of these mutations on viral virulence.More importantly, to protect indigenous chicken breeds in Yunnan province the monitoring and vaccination against FAdVs should be strengthened.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms11112631/s1,Table S1: Primers used in study; Table S2: Details of fowl adenovirus isolates for sequence alignment and phylogenetic analysis; Table S3: Information on strains with the highest homology to the FAdV-4 strains isolated in our study.Reference [48] is cited in the supplementary materials.
Author Contributions: J.L. was responsible for experimental design, data analysis, and manuscript writing.B.X. designed the experiments and revised the manuscript.F.C., R.Z., Y.Z., G.G., X.C., S.L. and W.M. collected samples.X.H. was responsible for data processing.L.Y. and T.R. reviewed and edited the manuscript.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.Phylogenetic analysis of the entire hexon gene.Phylogenetic tree for the hexon genes of 10 isolated strains and 41 reference strains created using MEGA 11.0 software using the neighbor-joining method and 1000 bootstrap replicates.The black triangle indicates the isolated strains in this study.

Figure 1 .
Figure 1.Phylogenetic analysis of the entire hexon gene.Phylogenetic tree for the hexon genes of 10 isolated strains and 41 reference strains created using MEGA 11.0 software using the neighborjoining method and 1000 bootstrap replicates.The black triangle indicates the isolated strains in this study.

Figure 3 .
Figure 3. Phylogenetic analysis of the entire penton gene.Phylogenetic tree for penton genes of 10 isolated strains and 28 reference strains created using MEGA 11.0 software using the neighbor-joining method.The black triangle indicates isolated strains in this study.

Funding:
This research was funded by the Academician (Expert) Workstation of Yunnan Province Program (202305AF150127), Yunnan Ten Thousand Talents Plan Leading Talents of Industrial Technology Project of China (YNWR-CYJS-2019-020), National Key Research and Development Program of China (2022YFD1601900), and Yunnan Fundamental Research Projects (202301AT070488).Institutional Review Board Statement:This study was performed in accordance with the regulations and recommendations of the Ministry of Science and Technology of the People's Republic of China, "The Care and Use of Laboratory Animals", and was approved by the Animal Welfare Ethics Committee of Yunnan Agricultural University (permit number: 202203056).

Table 1 .
Surveillance statistics for FAdVs in indigenous chickens in Yunnan province, Southwest China from 2022 to 2023.

Table 2 .
Accession numbers of all FAdV-4 isolates in this study.

Table 3 .
Amino acid variations in the hexon proteins of the FAdV-4 strains identified in this study.

Table 4 .
Amino acid variation in the penton proteins of the FAdV-4 strains identified in this study.

Table 5 .
Amino acid variation in the fiber1 proteins of the FAdV-4 strains identified in this study.

Table 6 .
Amino acid variation in the fiber2 proteins of the FAdV-4 strains identified in this study.