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

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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↓L, has two dibasic aa (arginine, R) compared to the monobasic F protein cleavage site of PEPR↓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.

Introduction

Paramyxoviruses are pleomorphic, enveloped viruses containing a negative-sense, single-stranded RNA genome. These viruses have been isolated from a great variety of mammalian and avian species around the world, and in some cases from fish and reptiles (Lamb and Parks, 2007). Paramyxoviruses are classified under the family Paramyxoviridae, which includes two subfamilies, Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae is further divided into five genera: Morbillivirus (including measles [MeV] and canine distemper [CDV] viruses), Rubulavirus (including simian virus 5 [SV5, now also known as parainfluenza virus 5], mumps virus [MuV], and human parainfluenza virus [HPIV-2]), Respirovirus (including Sendai virus [SeV] and HPIV-1), Henipavirus (comprising Hendra virus [HeV] and Nipah virus [NiV]) and Avulavirus (comprising avian paramyxovirus [APMV] serotype 1, also known as Newcastle disease virus [NDV], and APMV serotypes 2–9). The subfamily Pneumovirinae comprises two genera: Pneumovirus (including human and bovine respiratory syncytial viruses [HRSV and BRSV]), and Metapneumovirus (comprising human and avian metapneumoviruses [HMPV and AMPV]) (Lamb et al., 2005).

The genomes of paramyxoviruses range from 13 to 19 kb and contain 6–10 genes that code for up to 12 different proteins. For the members of subfamily Paramyxovirinae, efficient genome replication depends on the total genome nucleotide (nt) length being an even multiple of six, known as ‘rule of six’ (Kolakofsky et al., 1998). The genome termini consist of extragenic regions, called the 3′-leader and 5′-trailer: the 3′-leader region contains the genome promoter, and the trailer encodes the 3′ end of the antigenome containing the antigenome promoter. Each gene starts with a conserved gene-start (GS) sequence and ends with a conserved gene end (GE) sequence. Transcription begins at the 3′-leader region and proceeds in a sequential manner by a start–stop mechanism that is guided by short, conserved gene-start (GS) and gene-end (GE) signals that flank each gene (Lamb and Parks, 2007). The genes are separated by non-coding intergenic sequences (IGS) that are conserved in length and sequence among the different gene junctions for some genera (Respirovirus, Morbillivirus, and Henipavirus) and are non-conserved in sequence or length for others (Rubulavirus, Avulavirus, Pneumovirus, and Metapneumovirus). All members of family Paramyxoviridae encode a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutinin-neuraminidase (HN) or glycoprotein (G), and a large polymerase protein (L). Most members of subfamily Paramyxovirinae encode two additional proteins, V and W (or I, in case of genus Rubulavirus), from alternative open reading frames (ORFs) in the P gene that are accessed by RNA editing. In addition, members of the family Pneumovirinae and genus Rubulavirus contain a small gene designated SH, which encodes a small hydrophobic protein (SH).

Paramyxoviruses isolated from avian species fall into two distinct groups based on gene map and antigenic and sequence relationships: the APMVs of genus Avulavirus, and the avian metapneumoviruses of genus Metapneumovirus. The APMVs have been divided into nine different serotypes based on hemagglutination inhibition (HI) and neuraminadase inhibition (NI) assays (Alexander, 2003). NDV is an economically important disease of poultry and is the most studied member of the genus Avulavirus. Very little information is available about the molecular and biological characteristic and pathogenicity of APMV-2 through 9. APMV-2, -3, -6 and -7 have been associated with disease in domestic poultry (Zhang et al., 2007, Alexander and Collins, 1982, Warke et al., 2008a, Saif et al., 1997). APMV-3 and APMV-5 has been implicated in a severe pulmonary disease of birds (Jung et al., 2009, Nerome et al., 1978). The pathogenicity of the remaining APMV serotypes is not known. The APMV-6 virus causes mild respiratory disease and is associated with a drop in egg production in turkeys (Alexander, 1997). Experimental pathogenesis studies showed that APMV-6 was avirulent in chickens (Chang et al., 2001, Warke et al., 2008b). Recently, complete genome sequences have been determined for representative strains of all of the APMV serotypes except APMV-5: APMV-2 (Subbiah et al., 2008), APMV-3 (Kumar et al., 2008), APMV-4 (Nayak et al., 2008, Jeon et al., 2008), APMV-6 (Chang et al., 2001), APMV-7 (Xiao et al., 2009), APMV-8 (Paldurai et al., 2009) and APMV-9 (Samuel et al., 2009). This has substantially increased our understanding of the members of genus Avulavirus, but further studies are necessary to characterize the extent of diversity within the serotypes.

APMV-6 strain duck/HongKong/18/199/77 was first isolated from a domestic duck in Hong Kong (HK) in 1977 and is considered the prototype for the entire serotype (Shortridge et al., 1980). APMV-6 strain duck/Taiwan/Y1/99 (TW) was isolated from a domestic duck in Taiwan in 1999 (Chang et al., 2001). APMV-6 strain goose/FarEast/4440/2003 (FE) was isolated from a goose in the Far East of Russia in 2003 (GenBank accession no. EF569970). APMV-6 strains continue to be isolated from wild birds around the world (Warke et al., 2008a, Stanislawek et al., 2002). To date, the complete genome sequences of APMV-6 strains TW and FE have been determined.

APMV-6 is an atypical member of the genus Avulavirus in having a genome organization, 3′-leader-NP-P/V-M-F-SH-HN-L-trailer-5′, that includes an SH gene between F and HN genes (Chang et al., 2001), which is not found in the other APMV serotypes sequenced to date. The biological function of the APMV-6 SH protein is not known. However, the SH proteins of SV5 and MuV appears to play an essential role in blocking the TNF-alpha-mediated apoptosis pathway (Wilson et al., 2006). The SH protein of HRSV has ion channel activity in planar lipid bilayers (Gan et al., 2008). HRSV from which the SH gene was deleted was fully viable in cell culture, but was slightly attenuated in mice and chimpanzees (Bukreyev et al., 1997 and Whitehead et al., 1999).

As noted, APMV-6 strains have been isolated from a wide range of avian species from different parts of the world. But little is known about the serological and genetic relationships among these strains. This information is important for understanding virus evolution and epidemiology and for the development of vaccines against these viruses. As a first step towards understanding the serological and genetic relationships among APMV-6 strains, we report the complete genome sequences of the APMV-6 prototype strain HK as well as a strain, duck/Italy/4524-2/07 (IT4524-2), that was isolated in Italy in 2007. We also determined sequences for the F, SH and HN genes of two more Italian strains, IT4526 and IT6895-1, that also were isolated in 2007, and compared the phylogenetic relatedness of all known APMV-6 strains to representative strains of the other APMV serotypes as well as members of other genera of family Paramyxoviridae. The results suggested that APMV-6 contains two subgroups that can be distinguished by sequence and antigenic comparisons.

Section snippets

Virus and cells

APMV-6/duck/Italy/4524-2/07 (IT4524-2), APMV-6 /duck/Italy/4526/07 (IT4526), and APMV-6/teal/Italy/6895-1/07 (IT6895-1) were kindly provided by Dr. Ilaria Capua (Istituto Zooprofilattico Sperimentale delle Venezie, Padova, Italy). APMV-6 /duck/HongKong/18/199/77 (HK) was received from the National Veterinary Services Laboratory, Ames, Iowa, USA. The viruses were propagated in 9-day-old embryonated specific pathogen free (SPF) chicken eggs. Infected allantoic fluid was harvested 3 days

Growth characteristic of APMV-6 strains

APMV-6 prototype strain HK and the more-recently isolated strains IT4524-2, IT4526, and IT6895-1 each replicated to a titer of 258 HAU/ml in 9-day-old embryonated chicken eggs. Since the growth characteristics and in vitro host spectrum of these strains were not known, we evaluated their replication, with and without added trypsin (1 μg/ml), in six established cell lines that each represent a different species of origin: chicken embryo fibroblasts (DF-1), African green monkey kidney (Vero) cells,

Discussion

APMVs constitute the genus Avulavirus and are divided into nine serotypes based on HI and NI assays. Among these serotypes, APMV-1 (NDV) is well characterized because it causes severe disease in poultry worldwide. A great deal of information is available on the antigenic and genetic relationships among APMV-1 strains isolated from different parts of the world. Recently, we and others have reported complete genome sequences for representative strains of APMV-2, 3, 4, 6, 7, 8 and 9. However, very

Acknowledgements

We thank Drs. Ilaria Capua and Isabella Monne at IZSV, Italy for helpful support and advice. We also thank Anandan Paldurai, Flavia Dias and Dianel Rockemann for their excellent technical assistance and help. “This research was supported by NIAID contract no. N01A060009 (85% support) and NIAID, NIH Intramural Research Program (15% support). The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention of trade names,

References (47)

  • M. Subbiah et al.

    Complete sequence of the genome of avian paramyxovirus type 2 (strain Yucaipa) and comparison with other paramyxoviruses

    Virus Res.

    (2008)
  • S. Xiao et al.

    Complete genome sequence of avian paramyxovirus type 7 (strain Tennessee) and comparison with other paramyxoviruses

    Virus Res.

    (2009)
  • D.J. Alexander

    Newcastle disease and other avian paramyxoviridae infections

  • D.J. Alexander

    Avian paramyxoviruses 2–9

  • D.J. Alexander et al.

    Pathogenecity of PMV-3/Parakeet/Netherland/449 /75 for chickens

    Avian Pathol.

    (1982)
  • J.D. Bendtsen et al.

    Improved prediction of signal peptides: signal 3.0

    J. Mol. Biol.

    (2004)
  • M. Brudno et al.

    LAGAN and multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA

    Genome Res.

    (2003)
  • A. Bukreyev et al.

    Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse

    J. Virol.

    (1997)
  • P.C. Chang et al.

    Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks

    J. Gen. Virol.

    (2001)
  • S.W. Gan et al.

    Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain

    Protein Sci.

    (2008)
  • W.J. Jeon et al.

    Full-length genome sequence of avain paramyxovirus type 4 isolated from a mallard duck

    Virus Genes

    (2008)
  • P.R. Johnson et al.

    Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity

    J. Virol.

    (1987)
  • A. Jung et al.

    Avian paramyxovirus serotype 3 infection in Neopsephotus, Cyanoramphus, and Neophema species

    J. Avian Med. Surg.

    (2009)
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