key: cord-0004881-uutppde3
authors: Meng, X. -J.; Paul, P. S.; Halbur, P. G.; Lum, M. A.
title: Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe
date: 1995
journal: Arch Virol
DOI: 10.1007/bf01309962
sha: 045ca16ba40806eadbfe0b8f420cb81bea7c10b5
doc_id: 4881
cord_uid: uutppde3

The putative membrane (M) protein (ORF 6) and nucleocapsid (N) protein (ORF 7) genes of five U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) with differing virulence were cloned and sequenced. To determine the genetic variation and the phylogenetic relationship of PRRSV, the deduced amino acid sequences of the putative M and N proteins from these isolates were aligned, to the extent known, with other PRRSV isolates, and also other members of the proposed arterivirus group including lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). There was 96–100% amino acid sequence identity in the putative M and N genes among U.S. and Canadian PRRSV isolates with differing virulence. However, their amino acid sequences varied extensively from those of European PRRSV isolates, and displayed only 57–59% and 78–81% identity, respectively. The phylogenetic trees constructed on the basis of the putative M and N genes of the proposed arterivirus group were similar and indicated that both U.S. and European PRRSV isolates were related to LDV and were distantly related to EAV. The U.S. and European PRRSV isolates fell into two distinct groups, suggesting that U.S. and European PRRSV isolates represent two distinct genotypes.

3'; DP966: 5'AATGGGGCTTCTCCGG 3'). The sequences were combined and analyzed by MacVector (International Biotechnologies, Inc) and GeneWorks (IntetliGenetics, Inc.) computer programs. Phylogenetic analyses were conducted with the aid of the PAUP software package version 3.1.1 (David L. Swofford, Illinois Natural History Survey, Champaign, IL). PAUP employs the maximum parsimony algorithm to construct phytogenetic trees. The nucleotide sequence data reported in this paper have been deposited with GenBank database under the accession numbers U18748 (ISU-1894), U18749 (ISU-22), U18750 (ISU-3927), U18751 (ISU-55) and U18752 (ISU-79).

Analysis of the nucleotide sequences encoding the putative M and N proteins of the five U.S. PRRSV isolates indicated that, like LV [20] and VR 2385 [18] , the M and N genes of all the five U.S. isolates overlapped by 8 base pairs (bp) (Fig. 1) . The AACC motif located 13 nucleotides upstream of the N gene is believed to be the leader-body junction site for subgenomic RNA 7 during PRRSV transcription [18, 21] . Numerous substitutions in the nucleotide sequence were distributed randomly throughout the M and N genes in all the five isolates when compared to VR 2385 ( Fig. 1 ). There is no correlation between the virus virulence and the genetic variation in the M and N genes among these U.S. isolates analyzed. Most of the substitutions are third base silent mutation when converted to amino acid sequences (Fig. 2) . Insertions and deletions were found in the nucleotide sequences of the M and N genes between the U.S. isolates and LV, but not among the U.S. isolates (Fig. 1) . The deduced amino acid sequences of the M and N genes from the five U.S. PRRSV isolates were then aligned with the corresponding sequences of two other U.S. isolates, Minnesota isolate VR 2332 [-22] , only N gene) and Iowa isolate VR 2385 [18] , one Canadian PRRSV isolate IAF-exp91 [17] , only N gene), two European PRRSV isolates, LV E20J and PRRSV isolate 10 (PRRSV-10) [5] , two LDV strains, LDV-C [7] and LDV-P [14] , and EAV [6] (Fig. 2) . The amino acid sequence of the N gene among the eight North American PRRSV isolates were highly conserved (Fig. 2b) , and displayed 96-100~ amino acid sequence identity (Table 1) . However, the N protein of all North American PRRSV isolates shared only 57-59~o amino acid sequence identity with that of the two European isolates when the inserted amino acids of the European isolates were included in the alignment ( Table 1 ), suggesting that the North American and the European isolates may represent two different genotypes. The M protein of all the U.S. isolates with varying virulence was also highly conserved, and displayed higher sequence similarity with the M proteins of the two European isolates (Fig. 2a) , ranging from 78 to 81~ amino acid identity ( Table 1 ). The N gene of all the The leader-body junction site motif, AACC, upstream of N gene is underlined TACAAATAAGGT~GC~CTCACTATGc``/3AGCA~TAGTTGcACTCCTTPGGGGGGTGTACTCAGC-- North American PRRSV isolates shared 49-50~o amino acid sequence identity with that of the two LDV strains, whereas the two European PRRSV isolates shared only 40-41~o amino acid identity with that of the LDV strains when the inserted amino acids of the European PRRSV isolates were included (Table 1) . Two regions of amino acid sequence insertions, "KKSTAPM" and "ASQG", were found in the N protein of the two European PRRSV isolates when compared to the eight North American PRRSV isolates as well as two LDV strains and EAV (Fig. 2b) . These results indicated that the U.S. PRRSV isolates were more closely related to LDV than were the European PRRSV isolates, and that PRRSV may have undergone divergent evolution in the U.S. and in Europe before their association with PRRS was recognized in swine [22] . The M and N genes of the North American and European PRRSV isolates shared only t5-17~ and 22-24~ amino acid sequence identity with those of EAV, respectively. The sequence homology of PRRSV with LDV and EAV suggests that these viruses are closely related and may have evolved from a common ancestor [22, 24] . The evolutionary relationships of PRRSV with other members of the proposed arterivirus group were determined on the basis of the amino acid sequence of the M and N genes. Bootstrapped parsimony (1000 replicates) with branch-and-bound search option were performed to find the tree with shortest length (most parsimonious). The trees were rooted by assuming EAV as the outgroup for the N genes and LDV as the outgroup for the M genes. The phylogenetic tree drawn for the N gene is essentially the same as that drawn for the M gene (Fig. 3) . The PRRSV isolates fall into two distinct groups. All the North American PRRSV isolates thus far sequenced are closely related and formed one group. The two European PRRSV isolates are closely related and comprised another group. Recently, portions of the N gene from seven Spanish PRRSV isolates have been determined, and displayed 96-97~o sequence homology with the corresponding region of LV [29] . This again indicates that the European PRRSV isolates are highly conserved. Both the U.S. and European PRRSV isolates are related to LDV strains and distantly related to EAV (Fig. 3) . The North American and European PRRSV isolates represent two distinct genotypes (Fig. 3) .

A striking feature of RNA viruses is their rapid evolution resulting in extensive sequence variation [12] . Direct evidence for recombination between different positive-strand RNA viruses has been obtained [16] . Western equine encephalitis virus appears to be an evolutionarily recent hybrid between Eastern equine encephalitis virus and another alphavirus closely related to Sindbis virus [10] . Thus, the emergence of PRRSV and its close relatedness to LDV and EAV is not surprising. Although the capsid or nucleocapsid protein has been used for construction of evolutionary trees of many positive-strand RNA viruses, proteins with conserved sequence motifs such as a RNA-dependent RNA polymerase or a RNA replicase, would be more suitable for phylogenetic studies [12] . Further sequence information of both North American and European PRRSV isolates would facilitate the evolutionary studies of PRRSV. a) 1 7 Phylogenetic analyses of PRRSV 

Comparison of porcine alveolar macrophages and CL 2621 for the detection of porcine reproductive and respiratory syndrome (PRRS) virus and anti-PRRS antibody

Serologic survey for Lelystad and VR-2332 strains of porcine respiratory and reproductive syndrome (PRRS) virus in US swine herds

Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332)

Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North American and experimental reproduction of the disease in gnotobiotic pigs

Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the Arterivirus group

Equine arteritis virus is not a togavirus but belongs to the coronavirus-like superfamily

Map location of lactate dehydrogenaseelevating virus (LDV) capsid protein (Vpl) gene

Complete genomic sequence and phylogenetic analysis of the lactate dehydrogenaseelevating virus (LDV)

Porcine reproductive and respiratory syndrome

Western equine encephalitis virus is a recombinant virus

Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line

Evolution and taxonomy of positive-strand RNA viruses: implication of comparative analysis of amino acid sequence

A nested set of eight RNAs is formed in macrophages infected with lactate dehydrogenase-elevating virus

Lactate dehydrogenase-elevating virus (LDV): subgenomic mRNAs, mRNA leader and comparison of 3'-terminal sequences of two LDV isolates

Coronavirus: organization, replication and expression of genome

Phylogenetic analyses of PRRSV 755

RNA recombination in animal and plant viruses

Identification of major differences in the nucleocapsid protein genes of a Quebec strain and European strains of porcine reproductive and respiratory syndrome virus

Molecular cloning and nucleotide sequencing of the T-terminal genomic RNA of the porcine reproductive and respiratory syndrome virus

Development of a radiolabeled nucleic acid probe for the detection of encephalomyocarditis virus of swine

Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV

Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence

The PRRS virus

Porcine reproductive and respiratory syndrome: an overview

Lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus, a new group of positive strand RNA virus

The carboxyl-terminal part of putative Berne virus polymerase is expressed by ribosomal frame shifting and contains sequence motifs which indicate that toro and coronaviruses are evolutionarily related

Coronaviruses: structure and genome expression

Endemic porcine reproductive and respiratory syndrome virus infection of nursery pigs in two swine herds without current reproductive failure

Evolution of RNA virus

Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR)

Mystery swine disease in the Netherlands: The isolation of Lelystad virus

Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome virus

Lelystad virus and the porcine epidemic abortion and respiratory syndrome

The authors would like to thank W. 

Received November 2, 1994