Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China

In several parts of China, there have been a large number of hydropericardium syndrome (HPS) outbreaks caused by serotype 4 fowl adenovirus (FAdV4) in broiler chickens since 2015. These outbreak-associated FAdV-4 strains were distinct from previous circulating strains which did not lead to severe HPS outbreaks. To better understand the molecular epidemiology of the currently circulating FAdV strains for effective diagnosis and treatment of HPS, we isolated 12 HPS outbreak-associated FAdV-4 strains from different regions in central China and investigated their molecular characteristics by performing phylogenetic analyses based on the hexon genes. Our results indicated the FAdV-4 strains in this study all belonged to serotype FAdV-4, species FAdV-C. And in comparison with ON1, KR5, MX-SHP95, PK-01, PJ-06 strains within the cluster where outbreak-associated FAdV-4 strains were located, the nucleotide sequence divergence were 1.31, 1.10, 1.42, 2.77 and 2.84%, respectively. Phylogenetic analyses revealed the hexon genes of the 12 outbreak-associated strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor and we suggested that these outbreak-associated FAdV-4 strains originate from earlier strains in India.

Fowl adenoviruses (FAdVs) are non-enveloped double stranded DNA-viruses and belong to the genus Aviadenovirus, family Adenovirida together with other four genera: Mastadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus [11]. Based on restriction enzyme digest pattern and serum cross-neutralization test, FAdVs have been grouped into 5 species (FAdV-A to FAdV-E) with 12 serotypes (FAdV-1 to 8a and 8b to 11) [12]. Serotype 4 fowl adenovirus (FAdV-4), the causative agent of HPS, is a member of the species Fowl Adenovirus C [4,13]. The genome of FAdV-4 encodes a number of non-structural proteins and three structural proteins: hexon, penton and the fiber protein. The hexon gene of FAdVs is the longest and consists of hypervariable Loop L1 (HVR1-4) regions, making it a hotspot for research on taxonomy and antigenic shift of FAdVs [10,[14][15][16]. Hexon protein is the predominant target for induction of serotype-specific neutralizing antibodies [13].
Since 2015, clinical cases of HPS have been increasing in many regions of China, including Shandong, Hubei, Jiangsu, Anhui, Jiangxi, and Henan (Fig. 1a). These outbreaks were characterized by high mortality and no seasonal characteristics and mainly concentrated in small and medium broiler farms, rearing chickens and ducks [17]. However, to date, little is known regarding the molecular and genetic evolution characteristics of these potentially devastating FAdV strains that remain circulating in China. Hence, in this study, we aimed to investigate the molecular epidemiology of these currently circulating strains in chicken flocks in central China. Phylogenetic trees were constructed based on the hexon genes to establish the origin and genetic relationships of FAdV strains. It was found that FAdV field strains circulating after 2015 were closely related to the Indian strains PK-01 and PJ-06.

Materials and methods
Origin of the strains  Table 2.

Genomic DNA extraction
The presence of FAdV in each sample was confirmed by virus isolation followed by PCR. Initially, 5 mg of liver was cut into tiny pieces and fully ground in sterilized PBS (weight/volume = 1:3) under aseptic conditions. Following complete grinding, the suspension of liver tissue in PBS was frozen at −20°C and thawed at 37°C three times before centrifugation at 8000 rpm at 4°C for 10 min. The supernatant was then collected, filtered through a 0.22 μm filter, and inoculated onto a confluent monolayer of CEF (chicken embryo fibroblast) cells prepared from 9day SPF embryonated chicken eggs. After incubation for 1 h at 37°C, 1.5 ml of DMEM media containing 2% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) were added to the 6-well cell culture plate. 72 h later, supernatants were collected and filtered through a 0.22 μm filter for DNA extraction. Genomic DNA from these field samples was extracted using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, Dalian, China), and stored at −20°C before use. Quantification and quality of DNA extracts were determined with a Nanodrop spectrophotometer (Thermo Scientific, USA).

PCR
The hexon gene was divided into two fragments (Named fragment A and B) because of its long length in this study.

Sequencing and phylogenetic analysis
The respective bacterial clones harboring recombinant plasmids of fragment A and fragment B were sequenced by Sangon Biotech. Each sample was sequenced twice (one with the primers designed in this study and the other with the sequencing primers of the PEASY-Blunt Cloning Vector). The obtained sequences were edited using DNAStar software, and deposited into GenBank. Homology analyses to identify gene homologs were performed with the BLAST program (NCBI). Phylogenetic trees were constructed using MEGA software by the neighbor-joining analysis with 1000 bootstrap replicates, Maximum Composite Likelihood method. The phylogenetic datasets for analyses included 12 FAdV-4 isolates from this study and 24 reference strains ( Table 2).

Results
Before 2014, FAdV-4 circulation also existed in China, such as ON1 (2004), JSJ13 (2013), but there were no significant HPS outbreaks. However, since July 2015, HPS outbreaks have been reported in Henan, Shandong, Anhui, Jiangxi, Jiangsu, and Hubei provinces of China (Fig. 1a), which causes huge losses and continues to threaten the poultry industry. In this study, 12 FAdV field strains from different regions in central China (Fig. 1b) were isolated in HPSoutbreak chicken flocks and propagated in CEF cells. After PCR amplification and sequencing of the hexon genes, the obtained complete hexon gene sequences were deposited

(partial cds) Japan
Twelve strains were isolated and confirmed to be FAdV-4 positive by PCR. Sequenced Hexon genes from these strains were edited using DNAStar software, and deposited into GenBank under the accession number KX640901-KX640912. 24 strains from GenBank were used as reference strains. Serotypes, species and origins of these strains were provided. "-" indicated unknown into GenBank under the following accession numbers: KX640901-KX640912 (Table 2). Hexon genes were used to analyze whether changes in hexon protein could explain the sudden outbreak. A phylogenetic tree based on the hexon protein included 12 outbreak-associated FAdV-4 strains from this study and 24 strains from GenBank (Table 2) (Fig. 2b). These mutations may help to explain the sudden outbreaks.
However, compared with Indian strains PK-01 and PJ-06, the hexon proteins only contain 2, 5 amino acid substitutions respectively, despite of 25 amino acid deletions at the N end, which do not lead to decline of the virulence. And the genetic relationship between them is very close. Thus, we postulate that ancestors of the strains is not the local strains but earlier strains in India.

Discussion
FAdVs are commonly present in chicken farms worldwide [2]. HPS is the most severe disease associated with FAdV infection, which has been attributed exclusively to serotype 4. Before 2014, FAdV-4 circulation had been reported in China, but there were no severe HPS outbreaks. However, since July 2015, outbreaks of HPS occurred with sudden high mortality rates in broilers in many small and middle chicken forms in central China, even the duck farms, leading to tremendous economic losses. Recent years, the frequency of HPS have also been increasing in many countries, such as India (2014) [18], Canada (2011) [19], Hungary (2013) [20], Korea (2012) [21], Japan (2012) [22], and Poland (2016) [10]. The ON1 and JSJ13 strains in local regions in China didn't lead to severe clinical symptoms. The phylogenetic tree revealed that the strains circulated in China before 2014 and after July 2015 had different ancestors, which lead to this phenomenon. And the strains circulated in China now derived from earlier strains in India, with some mutations and 25 amino acid deletions; however, the exact role of the deletion in these FAdVs still needs to be elucidated in future studies.
Recently, it has been demonstrated that FAdV-4 strains circulating in China carried deletion within ORF19, which might lead to higher virulence [3]. And in comparison with JSJ13 and JH13 (isolated from China in 2013), the strains HB1510 (isolated from China in 2015) and MX-SHP95 (isolated from Mexico), KR-5 (isolated from Australia) carried a deletion of 33 nt in ORF29 [3]. This indicated that FAdV strains also carried changes in other genes and the exact of these changes in relation to antigenic variation and pathogenicity should be further investigated.
Currently, commercial vaccines against HPS have not yet been developed in China due to the lack of knowledge and understanding of FAdVs. Shengwang Liu and Huixin Li also confirmed that HPS outbreak is attributed to the emergence of FAdv strains in chickens in China, and what's worse, FAdV species C, D, and E were cocirculating in chicken flocks in China, which made the infection more complex [23]. The molecular and phylogenetic analysis based on hexon genes in this study may further the understanding of FAdV evolution and provide relevant information for developing vaccines against the disease.

Conclusions
FAdV-4 strins is continuously affecting the poultry industry of China all over the world. In this study, we investigated the molecular epidemiology and characteristics of HPS outbreak-associated FAdV-4 strains based on the hexon genes. It showed that FAdV strains isolated in central China in this study all belonged to serotype FAdV-4, species FAdV-C. Besides, FAdV-4 strains (represented by FAdV ON1) which did not cause many HPS outbreaks before 2015 cluster together with the Austria strain KR5, Mexico strain MX-SHP95. FAdV strains now circulating in central China since 2015 are closely related to the Indian strains PK-01 and PJ-06, which revealed that the strains now originated from the earlier strains in India. This study also provides new information about the prevalence of those FAdV-4 strains currently circulating in central China, which contributes to the prevention and contral, even elimination of the strains.