Complete genome sequences of avian paramyxovirus serotype 6 prototype strain Hong Kong and a recent novel strain from Italy: evidence for the existence of subgroups within the serotype

Abstract

Complete genome sequences were determined for two strains of avian paramyxovirus serotype 6 (APMV-6): the prototype Hong Kong (HK) strain and a more recent isolate from Italy (IT4524-2). The genome length of strain HK is 16236 nucleotide (nt), which is the same as for the other two APMV-6 strains (FE and TW) that have been reported to date, whereas that of strain IT4524-2 is 16230 nt. The length difference in strain IT4524-2 is due to a 6-nt deletion in the downstream untranslated region of the F gene. All of these viruses follow the "rule of six". Each genome consists of seven genes in the order of 3'N-P-M-F-SH-HN-L5', which differs from other APMV serotypes in containing an additional gene encoding the small hydrophobic (SH) protein. Sequence comparisons revealed that strain IT4524-2 shares an unexpectedly low level of genome nt sequence identity (70%) and aggregate predicted amino acid (aa) sequence identity (79%) with other three strains, which in contrast are more closely related to each other with nt sequence 94-98% nt identity and 90-100% aggregate aa identity. Sequence analysis of the F-SH-HN genome region of two other recent Italian isolates showed that they fall in the HK/FE/TW group. The predicted signal peptide of IT4524-2 F protein lacks the N-terminal first 10 aa that are present in the other five strains. Also, the F protein cleavage site of strain IT4524-2, REPR downward arrow L, has two dibasic aa (arginine, R) compared to the monobasic F protein cleavage site of PEPR downward arrow L in the other strains. Reciprocal cross-hemagglutination inhibition (HI) assays using post-infection chicken sera indicated that strain IT4524-2 is antigenically related to the other APMV-6 strains, but with 4- to 8-fold lower HI tiers for the test sera between strain IT4524-2 and the other APMV-6 strains. Taken together, our results indicated that the APMV-6 strains represents a single serotype with two subgroups that differ substantially based on nt and aa sequences and can be distinguished by HI assay.

Fig. 1

Antigenic analysis of the indicated APMV-6 strains by a hemagglutination-inhibition (HI) assay using antisera specific to strains HK or IT4524-2. The anti-HK and IT4524-2 sera were collected 14 days following a single infection in 2-week old chickens.

Fig. 2

Nucleotide sequence alignment of the F mRNA 3’ UTRs of the six APMV-6 strains for which sequences are available. The sequences for strains IT4524-2 and HK are provided in full because they have little sequence identity. The other strain sequences are listed below, with nt identity to strain HK is indicated by dots. The unfilled rectangle on the left represents the stop codon of the F gene ORF, and the solid rectangle on the right represents the GE transcription signal of the F gene. The sequences are positive-sense, and the sequence numbers refer to the position in the UTR.

Fig. 3

Global pairwise comparison of the complete sequence of the genome of strain IT4524-2 with complete (HK, TW, and FE) and partial (IT4526 and IT6895-1) sequences of other APMV-6 strains. Percent sequence identity (y-axis) is plotted versus position in the genome (x-axis). The comparisons with strains IT4526 and IT6895-1 involve only the F-SH-HN gene. The analysis was performed with the software mVISTA Limited Area Global Alignment of Nucleotides (LAGAN) (http://genome.lbl.gov/vista/index.shtml) (Brudno et al., 2003; Robinson et al., 2009).

Fig. 4

Nucleotide sequence alignment of the leader (A) and trailer (B) regions of the indicated APMV-6 strains. Dots indicate identity with strain IT4524-2. Sequences are negative-sense.

Fig. 5

Amino acid sequence alignment of the C-terminal domain of the V proteins (A) and W proteins (B) of the indicated APMV-6 strains. Conserved cysteine (C) residues are underlined, dots indicate identity with strain IT4524-2, and gaps are indicated by dashes. The sequences are numbered according to the complete V and W proteins.

Fig. 6

Sequence alignments of the N-terminal region of the F protein (A) and 5’ end of the F mRNA (B) of the six APMV-6 strains. This illustrates an N-terminal truncation in the signal peptide of strain IT4524-2 compared to the others due to a difference in the position of the translational start site. (A) Amino acid sequence alignment of the N-terminal region of the F proteins of the indicated APMV-6 strains. The polar N-terminus (n region), hydrophobic core (h region, underlined) and C-terminus (c-region) of the signal peptide are indicated. The arrow indicates the predicted cleavage site of signal peptidase. Amino acid identity relative to strain IT4524-2 (top sequence) is indicated by dots; gaps in the IT4524-2 sequence compared to the others are indicated with dashes. (B) Nucleotide sequence alignment of the upstream end of the F mRNA of the indicated APMV-6 strains. The sequence is positive-sense, the GS signal is italicized, the remainder of the 5’ UTR is capitalized, and the ATG translational start sites are underlined. Nucleotide identity relative to strain IT4524-2 (top sequence) is indicated by dots.

Fig. 7

Alignment of the F protein cleavage site sequence of APMV-6 strain IT4524-2 with those of other APMVs. Basic aa were underlined and the cleavage position was indicated.

Fig. 8

Phylogenetic trees based on the aa sequences of the F (A) and HN (B) proteins of the six strains of APMV-6 and representative strains of APMV serotypes 1–4 and 7–9. The trees were constructed by bootstrap analysis (1,000 replicates) using the neighbor-joining of the Poisson-corrected values for aa differences in the MEGA 4.0 phylogenetic analysis program (Tamura et al., 2007). All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons. The scale bar shows the number of substitutions per site. Bootstrap values are shown at the nodes.

Fig. 9

Phylogenetic tree based on the nt sequence of the F genes of the indicated APMV-6 strains, and proposed scheme for classification of APMV-6 strains. The unrooted tree was constructed by bootstrap analysis (1,000 replications) using the neighbor-joining of the Kimura-2-parameter method for nt differences in the MEGA 4.0 phylogenetic analysis program. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons. Scale bar shows number of base substitutions per site. Bootstrap values are shown at the nodes. The percent genetic distances of complete F genes were computed by same program. The matrix of genetic distance among APMV-6 strains is not shown.