Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].     

Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates belonging to the European genotype 1 have recently emerged in South Korea, suggesting potential problems for disease control. In the present study, we attempted to determine the complete nucleotide sequence of the first Korean type 1 PRRSV isolate, designated KNU-07. The full-length genome of KNU-07 was found to be 15,038 nucleotides in length, which was 60 nucleotides shorter than the type 1 prototype strain Lelystad due to a notable 60-bp deletion within the nonstructural protein 2 (NSP2). The KNU-07 genome was shown to consist of a 221-nucleotide (nt) 5′ untranslated region (UTR), a 14,703-nt protein-coding region, and a 114-nt 3′ UTR, followed by a 42-73-bp poly(A) tail. A nucleotide sequence comparison of the KNU-07 genome with 20 complete PRRSV genomes revealed a 10.5–13.3% and 39.5–40.3% divergence from type 1 and type 2 strains, respectively, at the genome level, indicating a high similarity to the virus strains commonly identified as the European genotype. In order to investigate genetic variation and to understand the molecular evolution of the type 1 isolate in Korea, extensive phylogenetic analyses were performed using the ORF5 and ORF7 nucleotide sequences of published type 1 PRRSV isolates. The data further indicated that the newly emerging type 1 isolate KNU-07 belongs to the recently proposed pan-European subtype 1. Taken together, the results of this study describe the genomic characterization of the type 1 PRRSV isolated in South Korea, suggesting a recent introduction of the virus typical for this genotype that has commonly appeared worldwide.     

Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 44 nucleotide changes, 13 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.     

Genetic characterization of the Korean porcine reproductive and respiratory syndrome viruses based on the nucleocapsid protein gene (ORF7) sequences

To investigate the genetic characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), we determined the complete sequence of the nucleocapsid protein gene (ORF7) from 105 PRRSV isolates from all nine Korean prefectures during the years 2003 through 2006. These sequences were then analyzed along with the published ORF7 sequences for two Korean PRRS viruses (PL97-1/1997 and LMY/2002) and 36 non-Korean viruses. The ORF7 nucleotide sequence identities among the 107 Korean PRRS viruses ranged from 86.2 to 100%, corresponding to 85.4 to 100% identity at the amino acid level. All of the Korean isolates examined belonged to the North American genotype. The ORF7 gene sequence from the North American prototype virus (VR-2332) and its derived vaccine virus (Ingelvac PRRS MLV) was 90.0–100% identical to the various ORF7 sequences of the Korean isolates, with corresponding amino acid identities from 91.0 to 100%. In the phylogenetic tree obtained by neighbor-joining analysis, all of the Korean PRRSVs were divided into four groups. Our ORF7 sequence data also revealed no correlations between the date or place of collection and the distribution of PRRSV in Korea. North American genotype PRRSVs may have been introduced into Korean swine herds some time ago; these viruses apparently radiated nationwide within a relatively short period of time. Within the North American genotype PRRSVs from around the world, the Korean PRRSVs did not emerge as a single independent clade overall, and their immediate relationships with the PRRSVs from other countries could not be determined.     

The primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain VR-2332 is located in the middle of the GP5 ectodomain

Summary.  Pigs infected with porcine respiratory and reproductive syndrome virus (PRRSV) strain VR-2332 were found to generate high levels of antibodies (Abs) that bound in an indirect ELISA to synthetic peptides representing segments of the primary envelope glycoprotein (GP5) ectodomain of this virus. Use of overlapping GP5 ectodomain peptides of various length indicated that the epitope recognized by the Abs was located in the middle of the ectodomain (amino acids 36-52), in the same relative segment that contains the single linear neutralization epitope of the closely related mouse arterivirus, lactate dehydrogenase-elevating virus (LDV). The VR-2332 GP5 segment exhibits 77% amino acid homology with the corresponding GP5 ectodomain segments of both the European PRRSV strain Lelystad virus (LV) and LDV. This explains some observed crossreaction between the pig Abs and neutralizing anti-LDV monoclonal Abs with peptides representing the GP5 ectodomains of VR-2332, LV and LDV. The GP5 binding Abs of pigs seem to be the primary PRRSV neutralizing Abs, since the well timed appearance in sera of all VR-2332 infected pigs of GP5 peptide binding Abs correlated 100% with the appearance of neutralizing Abs and earlier studies indicated that GP5 of PRRSV, like that of other arteriviruses, contains the main neutralization epitope of PRRSV. In addition, one neutralizing anti-LDV monoclonal Ab that is specific for the GP5 ectodomain epitope of LDV also strongly neutralized both PRRSV strains, VR-2332 and LV. The PRRSV GP5 epitope is associated with an N-glycan that is conserved in both PRRSV genotypes and all LDV isolates. This N-glycan may impede the humoral immune control of PRRSV in infected pigs and might be responsible for the low immunogenicity of PRRSV when injected into mice. Received April 2, 2002; accepted July 9, 2002     

Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus

Summary The 3-portion of the genome of a U.S. isolate of the porcine reproductive and respiratory syndrome (PRRS) virus, ATCC VR-2332, was cloned and sequenced. The resultant 3358 nucleotides contain 6 open reading frames (ORFs) with homologies to ORFs 2 through 7 of the European strain of the PRRS virus and other members of the free-standing genus of arteriviruses. Both VR-2332 and the European isolate (called the Lelystad virus) have been identified as infectious agents responsible for the swine disease called PRRS. Comparative sequence analysis indicates that there are degrees of amino acid identity to the Lelystad virus open reading frames ranging from 55% in ORF 5 to 79% in ORF 6. Hydropathy profiles indicate that the ORFs of VR-2332 and Lelystad virus correspond structurally despite significant sequence differences. These results are consistent with the biological similarities but distinct serological properties of North American and European isolates of the virus.     

Discriminating between serological responses to European-genotype live vaccine and European-genotype field strains of porcine reproductive and respiratory syndrome virus (PRRSV) by peptide ELISA

A peptide ELISA was developed based on an immunodominant and hypervariable epitope in the ORF4 envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). The peptide sequence was derived from the Porcilis live-attenuated PRRSV vaccine strain (genotype 1, European). Antibodies induced by the field PRRSVs currently circulating in Poland were not detected by the Porcilis ORF4 peptide ELISA. In contrast, Porcilis-vaccinated animals seroconverted in the ORF4 peptide ELISA at 21 days post-vaccination. Maximal titers were seen 30-92 days post-vaccination; most sera had endpoint titers between 1:1000 and 1:100,000. In a paired format, where sera were assayed in two separate ELISAs using ORF4 peptides derived from the genetically very closely related Porcilis and Lelystad PRRSV strains, it was possible to differentiate between antibodies induced by these two viruses. The Porcilis and Lelystad ORF4 peptide ELISAs had sensitivities of 89 and 100%, respectively. Thus, ORF4 peptide ELISA afforded specific detection of antibodies induced by an European-genotype live-attenuated vaccine PRRSV strain (Porcilis). The results suggest that specific ORF4 peptide ELISAs can be custom-made for European-genotype PRRSV strains, using general peptide design criteria described in this work. Thus, ORF4 ELISAs may be generally useful, to monitor safety and operational aspects of European-genotype live-attenuated PRRSV vaccine virus use in populations with circulating field European-genotype PRRSVs.     

Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field

The objective of this study was to characterize a porcine reproductive and respiratory syndrome virus (PRRSV) isolated from United States pigs experiencing high morbidity (50%) and mortality (20%). The PRRSV isolate, designated NC16845b, was characterized through phenotypic analysis and genomic sequencing and compared to Type 2 PRRSV isolates VR-2332, MN184 and VR-2385. NC16845b demonstrated slower replication in vitro compared to the three other isolates and grew to a peak titer of 5.4 × 10⁵ plaque forming units (PFU) per ml at 60 h post inoculation, which was 4- to 13-fold less than the peak titer of the other three viruses. NC16845b plaques were intermediate size averaging 3.3 mm in diameter that was larger than MN184 plaques and smaller than VR-2385 and VR-2332. Using Northern blot analysis, viral and subgenomic RNA were detected that demonstrated variable levels of hybridization in some open reading frames (ORF) compared to the other viruses. NC16845b is 15,389 nucleotides in length and ORF 5 restriction fragment length polymorphism (RFLP) analysis demonstrated a 1-18-2 pattern. Among all available Type 2 complete genome sequences, NC16845b showed the highest nucleotide homology (91.2%) to atypical PRRSV strain JA142. Compared to prototype VR-2332, NC16845b demonstrated marked nucleotide variability within non-structural protein (nsp) 1β and nsp2, and a nucleotide deletion of 24 bases in nsp2. Sequence homology with VR-2332 and MN184 was 88.4% and 82.9%, respectively; homology with the ORF2-7 of VR-2385 was 90.4%. Collectively, these data indicate that, compared to prototype Type 2 PRRSV isolates, NC16845b exhibited slower in vitro growth properties, had regions of heterogeneity within ORF1a that corresponded to at least two individual virus quasispecies, and also contained a continuous 8 amino acid deletion in the nsp2 protein.     

Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype

Summary.  Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1β, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype. Received August 28, 1999 Accepted December 16, 1999     

Genomic characterization of two Chinese isolates of <Emphasis Type="Italic">Porcine respiratory and reproductive syndrome virus</Emphasis>

Summary. The genomes of two isolates of Porcine respiratory and reproductive syndrome virus (PRRSV) from China, designated HB-1(sh)/2002 and HB-2(sh)/2002, were sequenced and analyzed. The size of the genomes of HB-1(sh)/2002 and HB-2(sh)/2002 were 15,411 and 15,373 nucleotides respectively, excluding the poly(A) tails. Comparative analysis with the genomic sequences of another Chinese isolate (BJ-4) and North American (VR2332) and European (Lelystad virus, LV) viruses revealed that HB-1(sh)/2002 shared 89.8% identity with BJ-4 and VR2332, but only 54.7% with LV; while HB-2(sh)/2002 shared 89.4% and 89.5% identity with BJ-4 and VR2332 respectively and 54.3% with LV, indicating that the two new Chinese isolates were related to the North American PRRSV genotype. Phylogenetic analysis based on the nucleotide sequence of the structural protein ORFs showed that the two new Chinese isolates belong to same genetic subgroup. HB-2(sh)/2002 additionally exhibited variations in the NSP2 nonstructural protein encoded by ORF1 and the structural protein GP3 encoded by ORF3 in comparison with other North American PRRSV isolates, namely a 12 amino acids deletion in Nsp2 and one amino acid deletion in GP3 were found in HB-2(sh)/2002. Therefore, HB-2(sh)/2002 was a novel strain with unique deletions.     

Molecular characterization and recombination analysis of porcine reproductive and respiratory syndrome virus emerged in southwestern China during 2012–2016

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen causing tremendous economic losses to the swine industry. To investigate the prevalence of PRRSV of genotype 2 (North American type, NA-type) in southwestern China, the Nsp2 hypervariable region (Nsp2 HV) and ORF5 of 61 PRRS viruses collected during 2012–2016 were sequenced and analyzed. All the virus detected clustered into the JXA1-like (52/61), VR-2332-like (7/61), and NADC30-like (2/61) sub-genotypes. Five deletions in Nsp2 HV were detected in addition to the typical 30aa discontinuous deletion in HP-PRRSV, and two of these five were not reported previously. Strikingly, two PRRS virus (SCnj16 and SCcd16) isolated in 2016 contained the classic HP-PRRSV molecular marker in the Nsp2-coding region, but belonged to the NADC30-like sub-genotype on the ORF5 gene. Further recombination and phylogenetic analysis on the two complete genomic sequences revealed that they may have originated from recombination events between the NADC30 and Chinese HP-PRRSV strains. The present study suggests that the endemic PRRSVs in the region have continuously evolved and new vaccine strategies are necessary for more efficient control of the virus.     

Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine

Fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolate genomes were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200, and 403 amino acid deletions in the second hypervariable region of nsp2, were found to be stable in the nsp2 coding region after in vivo infection (Faaberg et al., 2010). Furthermore, 3 of 4 mutants achieved replication kinetics similar to wt virus by study end. We hypothesized that other mutations elsewhere in the virus may have contributed to their replication fitness in swine. To further assess the stability of the engineered viruses, all sequenced genomes were compared and contrasted. No specific mutations occurred in all nsp2 deletion mutant genomes that were not also seen in the parent genome of Type 2 PRRSV strain VR-2332. Second site (non-nsp2) deletions and/or insertions were not evident after replication in swine. The number of point mutations seen increased slightly with deletion size, but even the largest deletion (403 aa) had very few consensus mutations. Thus, our findings provide further substantiation that the nsp2 deletion mutant genomes were genetically stable after in vivo passage.     

Molecular epidemiology of porcine epidemic diarrhea virus in China

Since early 2006, porcine epidemic diarrhea virus (PEDV) has been reemerging in immunized swine herds. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. The entire ORF3 genes of 12 PEDV field strains and one vaccine strain were sequenced. The ORF3 genes of Chinese PEDV field strains (excluding CH/GSJIII/07) contain a single 672- or 675-nucleotide (nt) ORF, which encodes a 223- or 224-aa-long peptide. However, the CV777 vaccine strain and CH/GSJIII/07 contain a 276-nt ORF because of a 49-nt deletion at nt 245–293. The Chinese PEDV field strains and PEDV reference strains are divided into three groups based on the phylogenetic relationship of their ORF3 genes. Chinese PEDV field strains (excluding CH/GSJIII/07) have a close phylogenetic relationship to Korean strains and are genetically different from the PEDV vaccine strains. However, CH/GSJIII/07 has a close phylogenetic relationship to two vaccine strains, suggesting that it might have evolved from a live vaccine strain. Chinese PEDV field strains (excluding CH/GSJIII/07) can be differentiated from PEDV vaccine strains by a nested RT-PCR method.     

The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.

GP5, the principal envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV), contains a hypervariable region within the ectodomain which is responsible for generating diversity in field isolates. The purpose of this study was to gain insight into the possible origin of this diversity by following GP5 sequence changes in pigs exposed to PRRSV strain VR-2332 in utero. A region of the PRRS virus genome containing portions of ORF4 and ORF5 was amplified directly from tissues of infected pigs from birth to 132 days of age. We observed the emergence of a new PRRSV population, identified by a single nucleotide change in the ectodomain. The Asp to Asn change at amino acid 34 was also found as a minor component in pigs that expressed the wild-type sequence. The results from this study suggest that the variability in the ectodomain of ORF5 is the result of positive or negative selection, of which the mechanism remains to be determined.     

Complete genome analysis of a novel norovirus GII.4 variant identified in China

The complete genome sequence of a novel norovirus strain GZ2010-L87 identified in Guangzhou was analyzed phylogenetically in this study. The RNA genome of the GZ2010-L87 strain is composed of 7,559 nucleotides. The phylogenetic analysis based on open reading frame (ORF) 2 revealed that the strain belongs to the GII.4 genotype, forming the new cluster GII.4-2009 which was also identified in Asia and the USA since 2009. Furthermore, phylogenetic analyses of the full genome and the different open reading frame sequences of GZ2010-L87 and other representative strains suggested that the novel strain did not undergo recombination. Comparative analysis with the consensus sequence of 31 completely sequenced norovirus GII.4-2009 genomes showed 86 mismatched nucleotides (56 in ORF1, 16 in ORF2, and 14 in ORF3), resulting in 19 amino acid changes (9 in ORF1, 3 in ORF2, and 7 in ORF3). Furthermore, 12 variable sites were found on the capsid protein of norovirus GII.4-2009, and most were located at the P2 domain. Meanwhile, based on comparison with other GII.4 clusters, 14 sites were shown specific to the novel cluster. In summary, the genome of the new GII.4-2009 variant GZ2010-L87, which was first identified in China, was extensively characterized with a large panel of genetically diverse noroviruses. The genomic information obtained from the novel variant can be used not only as a full-length norovirus sequence standard in China but also as reference data for future evolution research.     

Neutralizing antibody responses of pigs infected with natural GP5 N-glycan mutants of porcine reproductive and respiratory syndrome virus

In order to assess the effect of the N-glycans associated with the GP5 neutralization epitope of porcine reproductive and respiratory syndrome virus (PRRSV) on the neutralizing antibody (Ab) response of swine, groups of young pigs were infected with PRRSV strains differing in N-glycosylation pattern. The humoral immune response to strain VR-2332, harboring four potential N-glycan sites, was compared to that of two natural field isolates carrying mutations either abolishing the N-glycosylation site at position 44 (N44) or the two N-glycosylation sites in the hypervariable region upstream of the neutralization epitope (HV-1). The pigs were bled at intervals and their sera were assayed for neutralizing Abs by indirect and competition ELISAs using peptides containing the GP5 neutralization epitope, and selectively for infectivity neutralization of a number of PRRSV strains. In addition, viremia was monitored by quantitative RT-PCR, and anti-N-protein Ab formation was measured by HerdChek ELISA. The neutralizing Ab responses as measured by peptide ELISA varied greatly between individual pigs infected with each PRRSV strain. Some pigs generated high titers of peptide binding Abs between 7 and 28 days post infection (p.i.), whereas other pigs had not generated a response by 90 days p.i. However, the HV-1-infected pigs generated Abs to the neutralization epitope more rapidly and to a 5-10 times higher level than VR-2332-infected pigs, and the Abs neutralized the homologous HV-1 virus 10-20 times more efficiently than PRRSV strains VR-2332, N44, MN184, or SDSU73. In contrast, most N44-infected pigs generated neutralizing Abs only after 42 days p.i. and only to low levels. The results suggest that the deletions of the N-glycans or other amino acid substitutions in the GP5 ectodomains of the mutants affect the immunogenicity of the neutralization epitope and the specificity of the Abs raised to it but not the sensitivity of the virions to Ab neutralization.     

Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages

Restriction fragment length polymorphism (RFLP) analysis is one of the tools commonly used to study the molecular epidemiology of porcine reproductive and respiratory syndrome viruses (PRRSVs). As PRRSVs are genetically variable, the stability of the RFLP pattern of a PRRSV during in vivo replication was evaluated by carrying out 13 sequential pig-to-pig passages (P1 to P13) of PRRSV ATCC VR-2332 in three independent pig lines for a total of 727 days. During P1 the pigs were inoculated with a homogeneous inoculum (CC-01) prepared through a series of plaque purifications, and during P2 to P13 the pigs were inoculated with a tissue filtrate from the corresponding pig in the previous passage. Fifteen viral plaque clones were directly isolated from CC-01 and the day 7 serum of each pig of each passage, open reading frame 5 of the clones was sequenced, and the clones were compared to CC-01 to assess the mutation rates and RFLP patterns (obtained by digestion with MluI, HincII, and SacII) over time. Among the 495 viral clones recovered during the passages, 398 clones, including CC-01, had pattern 2-5-2 (MluI-HincII-SacII); however, the remaining 97 viral clones showed different patterns (2-6-2 [P2], 1-5-2 [P3], 2-5-4 [P7], and 2-1-2 [P10]). Importantly, the MluI site that was reported to be present in only one of the PRRS modified live virus vaccine strains (Ingelvac) and its parental strain (ATCC VR-2332) can disappear during in vivo replication. Furthermore, sequence homology between CC-01 and clones with pattern 2-5-2 or clones with other patterns differed by 0.05 to 1.58% and 0.5 to 1.45%, respectively, suggesting that RFLP analysis cannot accurately predict genetic relatedness between PRRSVs. Collectively, precaution should be taken when the molecular epidemiology of PRRSVs is evaluated by RFLP analysis.     

Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate

Summary.  Presently, one of the most economically important pathogens affecting swine is the porcine reproductive and respiratory syndrome virus (PRRSV). This virus is prevalent in herds throughout the world and continues to pose a significant threat as newer and more virulent disease phenotypes emerge. In this report we describe the full-length nucleotide sequence of a Canadian PRRSV isolate, designated PA8. A consecutive sequence of 15,411 nucleotides was obtained from a set of overlapping cDNA clones. In order to determine the extent of genetic variation among isolates recovered from swine in Canada and the US, as well as to understand the molecular mechanisms governing the evolution of PRRSV, the full-length sequence of PA8 was compared with that of two US isolates, VR2332 and 16244B. The genomic sequence of PA8 shared 98.2% and 99.2% identity with 16244B and VR2332, respectively. The untranslated regions (UTR) at the 5′ and 3′ ends of the genome were very well conserved. Notable exceptions include an eight nucleotide difference at the 5′ end of the 5′ UTR of VR2332 relative to PA8 and 16244B and a two nucleotide difference in the 3′ UTR of PA8 relative to VR2332 and 16244B. In contrast to PA8 and VR2332, 16244B possessed two nucleotide differences within the RNA pseudoknot structure of the ribosomal frameshift region between open reading frame (ORF)1a and ORF1b. Amino acid differences were distributed throughout the genome, however they appeared to be most extensive in Nsp1β and ORF5 of the nonstructural and structural coding regions, respectively, suggesting that the evolutionary pressure to conserve these viral genes is somewhat lower. Received January 10, 2000/Accepted May 22, 2000