Introduction

Fowl adenoviruses (FAdVs) infect many avian species [1] and five species named Fowl aviadenovirus A to E (FAdV-A to -E) are known; these are members of the genus Aviadenovirus of the family Adenoviridae. Among the 12 serotypes that are grouped into five species of FAdV, serotypes FAdV-4 and FAdV-10 belong to FAdV-C [1, 2]. FAdV-4 is known as the major causative agent of hydropericardium syndrome (HPS), which is a highly pathogenic disease in poultry [1, 3].

The virions of FAdVs possess 240 hexons, 12 penton bases at vertices, and one or two fibers projecting from each penton base [1, 4]. The genes for these major proteins have attracted considerable attention because they are important for a comprehensive understanding of viral pathogenesis and immunogenicity. Genetic studies of FAdV-4 have reported phylogenetic analysis of partial hexon gene sequences of various isolates [5–12]. Whole genome or full-length hexon genes were phylogenetically analyzed for the FAdV-4 reference isolate, KR5 and a Canadian FAdV-4 isolate, ON1, both of which are non-virulent [13, 14]. In addition, comparison of fiber-2 genes of HPS isolates and nonpathogenic isolates has been attempted [13, 15]. Recently, the whole genome sequences of HPS-inducing FAdV-4 isolates were determined in China [16, 17] and Mexico [18]. Since the initial reports on HPS outbreaks in Korea [19, 20], the disease has occurred around the country [21–24]. Therefore, further molecular investigation of FAdV-4 isolates from HPS cases is required. To our knowledge, only the nucleotide sequences of partial hexon genes were analyzed in previous studies of Korean FAdVs. These include a few reports [21, 22, 24] comparing HPS-associated FAdV-4 with other isolates.

In this study, the entire sequences of three structural genes from FAdV-4 isolates associated with HPS outbreaks in Korea were analyzed along with those of foreign FAdV-4 and other representative FAdV serotypes. In addition, comparison of amino acids for these genes was performed, which focused on variations in fiber-2 amino acids between HPS and non-HPS isolates of Korean and foreign origin.

Results and discussion

Four Korean FAdV-4 isolates previously isolated from HPS-affected chickens and designated as Kr-Changnyeong, Kr-Andong, Kr-Yeoju, and Kr-Gunwi [23], were used in this study. From the chickens’ liver tissues, DNAs for hexon, penton base, and fiber-2 genes were acquired by polymerase chain reaction (PCR) and their nucleotide sequences were obtained as described in Fig. 1 and Table S1. Nucleotide sequences and their deduced amino acids for these genes were analyzed for the Korean and foreign isolates for which full-length nucleotide sequences for each gene were available in GenBank using BioEdit [25] and MEGA [26].

Fig. 1
figure 1

Phylogenetic trees based on amino acid sequences from hexon, penton base and fiber-2 genes of Korean FAdV-4 isolates and foreign FAdV isolates, including FAdV-4. For the Korean FAdV-4 isolates, the full-length DNA sequences for each gene were acquired by PCR amplification for liver tissues of HPS-affected chickens in the four different farms, from which the four Korean FAdV-4 were isolated, and cloning into pUC19 vector for DNA sequencing by an automated DNA analyzer (ABI 3730XL; Applied Biosystems, USA) and a universal primer set. For each gene, deduced amino acid sequences from Korean FAdV-4 isolates were aligned with those from foreign FAdV-4 isolates and other representative FAdV isolates of different serotypes for which full-length sequences were available in GenBank using BioEdit (version 7.1.3) [25]. The trees were created by the neighbor-joining method with MEGA (version 5.05) [26]. The GenBank accession numbers for each isolate are shown in the parentheses. The numbers at the branch points show the bootstrap values calculated from 1000 bootstrap replicates, and the scale bars indicate the numbers of amino acid substitutions per site. *Kr-Changnyeong/Korea represents three Korean FAdV-4 isolates (Kr-Changnyeong/Korea, Kr-Andong/Korea, and Kr-Yeoju/Korea) sharing identical amino acid sequences for hexon and penton base genes, and Kr-Changnyeong/Korea represents all four Korean FAdV-4 isolates with identical amino acid sequences for the fiber-2 gene. a hexon; b penton base; c fiber-2

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The entire hexon gene in all four Korean FAdV-4 isolates was 2811 bases in length. The nucleotide sequence identity of the hexon gene among the four Korean FAdV-4 isolates was 99.8–100 %, and these isolates showed identity of 97.5–99.0 % to ten Indian, two Russian, and two Chinese FAdV-4 isolates, and 99.2–99.3 % to ON1/Canada, KR5/Japan, and MX-SHP95/Mexico. Whereas 936 amino acids were deduced from the hexon gene in all four Korean isolates, 937 amino acids were deduced from that of other closely related isolates, including MX-SHP95/Mexico and ON1/Canada. A missing amino acid in the four Korean isolates corresponded to E195 or Q195 of the foreign isolates (Fig. S1a). This deletion might be dispensable for viral pathogenicity, considering that the Korean isolates were from HPS cases [23].

The entire penton base gene in all four Korean FAdV-4 isolates was 1578 bases in length. Nucleotide sequence identity of penton base genes among the four Korean FAdV-4 isolates was 99.9–100 %. These isolates showed identity of 99.1–99.4 % to 14 foreign FAdV-4 isolates of Indian, Canadian, Japanese, Pakistani, or Chinese origin, and 99.6–99.7 % to MX-SHP95/Mexico. Five hundred twenty-five amino acids encoded by the penton base gene were deduced from the four Korean FAdV-4 isolates as well as FAdV-4 isolates of foreign origin, except for NIAB-NIBGE 01/Pakistan, which contained three additional amino acids (D493 to R495). The four Korean isolates shared a unique amino acid, S127, which was identified as N127 in foreign FAdV-4 isolates (Fig. S1b).

Fiber proteins have received significant attention because of their roles in tissue tropism and virulence [1, 27, 28]. Among the FAdVs, FAdV-A and -C have two fiber genes (fiber-1 and fiber-2), whereas FAdV-B, -D, and -E have a single fiber gene [13]. In terms of antigenicity, the fiber-2 protein subunit of FAdV-4 induced greater protection against HPS than fiber-1, thus demonstrating its important role during viral infection [29]. In the present study, the entire fiber-2 gene in all four Korean FAdV-4 isolates was 1425 bases in length and the nucleotide sequences among the four Korean isolates were identical. The fiber-2 gene of these isolates was 95.5–96.8 % identical to that of 11 foreign FAdV-4 isolates of Indian, Canadian, Japanese, Pakistani, Russian, and Chinese origin; and was 99.0 % to that of AG234/Mexico and MX-SHP95/Mexico. For the fiber-2 gene for all four Korean FAdV-4 isolates, 474 amino acids were deduced. Five amino acids (E11 to P15) were missing for the four Korean isolates, compared to those of other FAdV-4 isolates, except for ON1/Canada, AG234/Mexico, and MX-SHP95/Mexico, which shared this deletion at the same position (Fig. S1c). Since both the Korean and the two Mexican isolates caused HPS [18, 23, 30], these residues appear to be dispensable for the pathogenicity of FAdV-4. The four Korean isolates shared five unique amino acids (I279, D294, A295, W343, and L372) that were not identified in the other FAdV-4 isolates. Two motifs have been recognized in the N-terminal tail region of fiber proteins [31]. The RKRP motif, predicted to be the nuclear localization signal, is conserved among FAdV-8 and -11 isolates [31]. A similar sequence, KRP/A, was in FAdV-4 and -10 isolates [13]. In the present study, KRP was found in the four Korean isolates as well as ON1/Canada, AG234/Mexico, and MX-SHP95/Mexico, whereas KRA was found in other isolates (Fig. S1c). When the sequence of this motif was KRP, the amino acids E11 to P15 were missing. These five amino acids might be related to the function of nuclear localization. The VYPY motif is known to be involved in the interaction with penton base proteins [31]; the VYPY/F sequence is found in FAdVs [13, 31, 32]. VYPF was conserved in all FAdV-4 isolates compared in this study (Fig. S1c), which is consistent with the finding that VYPF is conserved in all FAdV fiber-1 and fiber-2 sequences [13].

Fiber-2 protein is known to play a role in virulence [27]; therefore, amino acid variations between HPS and non-HPS isolates were analyzed to identify amino acids relevant for FAdV-4 virulence. Among FAdV-4 isolates analyzed based on their fiber-2 genes (Fig. S1c), only four foreign isolates were confirmed to be from HPS cases, whereas two were non-HPS isolates [13, 14, 16–18, 30]. For the other isolates, it was not clear whether they were from HPS cases. Hence, we included two additional HPS isolates, JP/LVP-1/96 from Japan and H-1 from India, whose amino acid sequences are available in a report [15]. In this analysis (Table 1), D219 and T300 were conserved among ten HPS isolates but were not present in the non-virulent ON1/Canada and KR5/Japan isolates, which had G219 and I300, respectively. However, D219 and T300 (A300 in some isolates) were also found in other FAdV-4 isolates; it was not clear if they were from HPS cases (Fig. S1c). According to a previous study [13], D219 and T380 are conserved among HPS isolates compared to non-virulent isolates. Another study confirmed the presence of D219 in HPS isolates, compared to G219 in a non-virulent isolate, KR5/Japan [15]. A recent study [18] showed that D219, T300, and T380 are found in pathogenic isolates including MX-SHP95/Mexico, as compared to different amino acids in the same positions of KR5/Japan. We identified D219 and T300 as amino acids with possible relevance to virulence, which is consistent with previous reports. Although T380 was identified in foreign HPS isolates, the four Korean isolates had A380, which was also identified in the non-HPS isolates ON1/Canada and KR5/Japan. These data suggest that T380 may not be responsible for virulence or is less significant than other defined amino acids.

Table 1 Comparison of variable amino acid sequences from fiber-2 genes among HPS and non-HPS isolates of Korean and foreign origin
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Based on previous reports comparing the partial hexon gene, Korean FAdV-4 isolates from HPS cases were phylogenetically close to KR5/Japan [21, 22, 24]. The phylogenetic analysis based on the hexon, penton base, and fiber-2 genes in the present study showed that the four Korean FAdV-4 isolates were closer to MX-SHP95/Mexico than other Asian isolates, whereas FAdV-4 isolates from India and China were closely related (Fig. 1a–c). This indicates that the genetic relationship among the FAdV-4 isolates is independent of geographic distribution. This is consistent with a recent report that found FAdV-4 isolates are not phylogenetically grouped according to geographic regions [12].

In the present study, the hexon, penton base, and fiber-2 genes of Korean FAdV-4 isolates were analyzed with those of foreign FAdV-4 isolates. Importantly, the amino acid sequences of fiber-2 genes were compared for HPS and non-HPS isolates to identify potential amino acids relevant for the virulence of FAdV-4. These data provide information for the molecular properties of FAdV-4 and will help to better understand the pathogenicity and immunogenicity of FAdV-4.