Species Fowl aviadenovirus B Consists of a Single Serotype despite Genetic Distance of FAdV-5 Isolates

Abstract

Fowl adenoviruses (FAdVs) are infectious agents, mainly of chickens, which cause economic losses to the poultry industry. Only a single serotype, namely FAdV-5, constitutes the species Fowl aviadenovirus B (FAdV-B); however, recently, phylogenetic analyses have identified divergent strains of the species, implicating a more complex scenario and possibly a novel serotype. Therefore, field isolates of the species were collected to investigate the contemporary diversification within FAdV-B, including traditional serotyping. Full genomes of fourteen FAdV-B strains were sequenced and four strains, possessing discriminatory mutations in the antigenic domains, were compared using virus cross-neutralization. Essentially, strains with identical antigenic signatures to that of the first described divergent strain were found in the complete new dataset. While chicken antiserum against FAdV-5 reference strain 340 could not neutralize any of the newly isolated viruses, low homologous/heterologous titer ratios were measured reciprocally. Although they argue against a new serotype, our results indicate the emergence of escape variants in FAdV-B. Charge-influencing amino acid substitutions accounted for only a few mutations between the strains; still, these enabled one-way cross-neutralization only. These findings underline the continued merit of the cross-neutralization test as the gold standard for serotyping, complementary to advancing sequence data, and provide a snapshot of the actual diversity and evolution of species FAdV-B.

Keywords: fowl adenovirus; fowl adenovirus 5; molecular typing; serotyping.

Figure 1

Amino acid alignment of hexon loop-1 sequences originating from two fowl adenovirus 5 strains: the reference strain 340 and another one (40440). The newly sequenced divergent strains are identical on the amino acid level to strain 40440 on this stretch. Differences are highlighted; furthermore, amino acid substitutions affecting the electrostatic charge of the protein are denoted using red squares.

Figure 2

Phylogenetic analysis of Fowl aviadenovirus B strains based on the (A) complete genome sequence, (B) the hexon amino acid sequence and the (C) hexon loop-1 (L1) nucleotide sequence. Trees (A,B) were rooted on fowl adenovirus 8a, whereas tree (C) was midpoint rooted. (A) Branch lengths were short within the species Fowl aviadenovirus B; thus, the red highlighted branch of the species is shown separately. From this tree, the green highlighted branch of the newly sequenced strains was separated again to reveal branch lengths among these strains too. (C) The 26 identical strains: 160, 177, 2255, 5626, 8844, 45871, 08-21472, 08-8669, 09-7470-2, 09-7473-2, 14/24408, 15/1401, 15/3466, 15/368, 15/4616, 15/6270, 15/6541, 17/25702, 18/11753, 15/4225, 18/6238, 18/6239, 18/907, 19/7209, GB 1643, K318/09. See Table 2 for further details.

Figure 3

Three-dimensional major capsid protein models of strains 340 and 40440 (both serotype fowl adenovirus 5 [FAdV-5]) and FAdV-8a: (A) complete hexon; (B) fiber knob (amino acid stretches for strain 340: 349–553, 40440: 349–554, FAdV-8a: 314–524); and (C) penton base (amino acid stretches for strain 340: 76–542, 40440: 77–543, FAdV-8a: 81–552). The upper strips of each subset show the monomer subunits in cartoon representation; here, amino acid substitutions affecting the electrostatic charge of the protein are highlighted. For strains 340 and 40440, substitutions compared to each other, and for fowl adenovirus 8a, substitutions compared to strain 340, are highlighted. Red and blue are charged (negatively and positively, respectively), green are polar, and white are nonpolar amino acids. The lower strips of each subset represent the electrostatic surface potentials of the trimeric (hexon and fiber knob) or pentameric (penton base) proteins, where red color represents regions with a potential value below −2.0 kT, white represents 0.0 kT, and blue represents regions above +2.0 kT.