Complete genome sequence of avian paramyxovirus (APMV) serotype 5 completes the analysis of nine APMV serotypes and reveals the longest APMV genome

Abstract

Background: Avian paramyxoviruses (APMV) consist of nine known serotypes. The genomes of representatives of all APMV serotypes except APMV type 5 have recently been fully sequenced. Here, we report the complete genome sequence of the APMV-5 prototype strain budgerigar/Kunitachi/74.

Methodology/principal findings: APMV-5 Kunitachi virus is unusual in that it lacks a virion hemagglutinin and does not grow in the allantoic cavity of embryonated chicken eggs. However, the virus grew in the amniotic cavity of embryonated chicken eggs and in twelve different established cell lines and two primary cell cultures. The genome is 17,262 nucleotides (nt) long, which is the longest among members of genus Avulavirus, and encodes six non-overlapping genes in the order of 3'N-P/V/W-M-F-HN-L-5' with intergenic regions of 4-57 nt. The genome length follows the 'rule of six' and contains a 55-nt leader sequence at the 3'end and a 552 nt trailer sequence at the 5' end. The phosphoprotein (P) gene contains a conserved RNA editing site and is predicted to encode P, V, and W proteins. The cleavage site of the F protein (G-K-R-K-K-R downward arrowF) conforms to the cleavage site motif of the ubiquitous cellular protease furin. Consistent with this, exogenous protease was not required for virus replication in vitro. However, the intracerebral pathogenicity index of APMV-5 strain Kunitachi in one-day-old chicks was found to be zero, indicating that the virus is avirulent for chickens despite the presence of a polybasic F cleavage site.

Conclusions/significance: Phylogenetic analysis of the sequences of the APVM-5 genome and proteins versus those of the other APMV serotypes showed that APMV-5 is more closely related to APMV-6 than to the other APMVs. Furthermore, these comparisons provided evidence of extensive genome-wide divergence that supports the classification of the APMVs into nine separate serotypes. The structure of the F cleavage site does not appear to be a reliable indicator of virulence among APMV serotypes 2-9. The availability of sequence information for all known APMV serotypes will facilitate studies in epidemiology and vaccinology.

Figure 1. Gene map of APMV-5 and comparison with prototype strains for the other eight APMV serotypes (maps not to scale).

Individual genes are indicated by rectangles, with the gene names given at the top of the Figure. The amino acid length(s) of the encoded protein(s) is shown in each box. The nt length of the gene is shown over each box. The lengths of the non-translated upstream and downstream regions (which also are represented in the total gene length) are indicated by underlined values immediately over each box. The nt lengths of the non-coding leader, intergenic, and trailer regions are shown under each map, and the total genome nt length is shown in parentheses to the right. The SH gene is present in APMV-6 but not in the other APMVs. Upstream UTRs include the GS sequence and the downstream UTRs include the GE sequence. The prototypes strains used here and in the other Figures and Tables are as follows: APMV-5, APMV-5/budgerigar/Kunitachi/74 (present study); APMV-1, Newcastle disease virus (strain LaSota/46); APMV-2, APMV-2/Chicken/California/Yucaipa/56; APMV-3, APMV-3/PKT/Netherland/449/75; APMV-4, APMV-4/duck/Hongkong/D3/75; APMV-6, APMV-6/duck/HongKong/18/199/77; APMV-7, APMV-7/dove/Tennessee/4/75; APMV-8, APMV-8/goose/Delaware/1053/76; APMV-9, APMV-9/duck/New York/22/1978.

Figure 2. Alignment and comparison of Leader and trailer sequences of APMV-5 with other APMV prototype strains.

Sequence alignments (negative-sense) of the (a) 3′-leader (b) 5′-trailer regions of APMV-5 with those of prototypes strains of the other eight APMV serotypes, and (c) terminal complementarity between the leader and trailer of APMV-5. Dots in (a) and (b) indicate identical nucleotides to APMV-5; crosses in (c) indicate complementarity. Gaps are indicated by dashes. The numbers in the parenthesis indicate total number of nucleotides.

Figure 3. Sequence alignments of P gene editing site and C-terminal region of V protein of all APMV prototype strains.

Sequence alignments of (a) the RNA editing site in the P gene (shown in negative-sense) and (b) the C-terminal domain of the V protein of APMV-5 versus prototype viruses of the other eight APMV serotypes. The editing site is underlined. Dots indicate nucleotides identical to those of APMV-5. The conserved cysteine residues in V protein are bolded and marked by stars. The numbers in the parenthesis indicate the corresponding nucleotide position in the complete genome (a) and amino acid position of V gene (b).

Figure 4. Phylogenetic tree of representative members of family Paramyxoviridae.

Phylogenic analysis of the complete genome (a) and respective, F (b) and L (c) proteins of members of family Paramyxoviridae. The numbers represents the bootstrap values among different viruses. The phylogenetic trees were analyzed by average distance using MEGA 4.1, Molecular Evolutionary Genetics Analysis software.