Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus,Québec reference strain

Summary The cDNA sequence of the 3-terminal genomic region of the Québec IAF-exp91 strain of porcine reproductive and respiratory syndrome virus (PRRSV) was determined and compared to those of other reference strains from Europe (Lelystad virus) and US (ATCC VR2385, MN-1b). The sequence (2834 nucleotides) which encompassed ORFs 3 to 7 revealed extensive genomic variations between the Québec strain and Lelystad virus (LV), resulting from high number of base substitutions, additions and deletions. The ORFs 5, 3, and 7 seemed to be relatively the most variable; the predicted encoding products of the Québec and LV strains displayed only 52%, 54%, and 59% amino acid identities, respectively. Nevertheless, in vitro translation experiments of the structural genes (ORFs 5, 6, and 7) and radio-immunoprecipitation assays with extracellular virions gave results similar to those previously reported for LV. In contrast, close genomic relationships were demonstrated between Québec and US strains. Taking together, these results indicate that, although structurally similar, North American PRRSV strains belong to a genotype distinct from that of the LV, thus supporting previous findings that allowed to divide PRRSV isolates into two antigenic subgroups (U.S. and European).The nucleotide sequence data reported in this paper will appear in the EMBL and GenBank nucleotide sequence databases under accession number L40898.     

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.     

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.     

Sequence and genomic organization of a rabbit hemorrhagic disease virus isolated from a wild rabbit

Rabbit hemorrhagic disease virus (RHDV) is a member of the caliciviridae family. The nucleotidic sequence of a full-length cDNA of one RHDV isolate (RHDV-SD) is reported. The genome is 7437 bases long and includes two ORFs, ORF1 (7034 b) and ORF2 (353 b), coding for the polyprotein and the Vp12, respectively. The coding sequence for the second structural protein (the capsid protein, Vp60) is located at the 3 end of ORF1. Comparison of RHDV-SD with the German RHDV isolate revealed 470 nucleotide substitutions (96% homology). The deduced amino acid sequences of the two isolates are closely related (98% identity), and no hypervariable region could be identified either in the structural or nonstructural proteins.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z29514.     

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

Summary 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.     

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.     

Molecular cloning and sequencing of the coat protein gene of a Nebraskan isolate of tobacco necrosis virus: the deduced coat protein sequence has only moderate homology with those of strain A and strain D

Summary A partial nucleotide sequence spanning the coat protein (CP) gene of a Nebraskan isolate of tobacco necrosis virus (TNV-NE) has been determined. The sequence contains at least four open reading frames (ORFs). The 5-terminal ORF encodes a protein that has 86% and 38% homology with the polymerases of strains A (TNV-A) and D (TNV-D), respectively. The second and third ORFs probably encode 10.7 kDa and 6.2 kDa proteins (p 10.7 and p 6.2). These are respectively 90% and 96% amino acid homologous encoded by similar ORFs in TNV-A but only 26% and 20% homologous with those in TNV-D. The fourth 3-proximal ORF encodes the 30.3 kDa CP. The amino acid sequence of TNV-NE CP is only 51% and 44% homologous to those of TNV-A and TNV-D, respectively. Thus, the CP genes of TNV-NE, TNV-A, and TNV-D are quite different. Like the sequences to the 5 side of the CP gene, that of TNV-NE is more closely related to TNV-A than to TNV-D.This study was a cooperative investigation of the ARS, USDA, and the Nebraska Agricultural Experiment Station, under Project 21-12. It is published as paper no. 10147, Journal Series, Nebraska Agricultural Experiment Station. The nucleotide sequence data reported in this paper have been entered in the GenBank databases under the accession number L 04261.     

Complete cDNA sequence of a South American isolate of potato virus X

The complete cDNA sequence corresponding to the genomic RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3, 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476-615 of ORF 1 and 64-100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.     

Nucleotide sequence of the 3′-terminal region of citrus tatter leaf virus RNA

The 3-terminal sequence of citrus tatter leaf virus lily isolate (CTLV-L) was determined from cloned cDNA. The sequence contains two open reading frames (ORFs). ORF1 encodes a protein that contains consensus sequences associated with the RNA-dependent RNA polymerase. ORF2, which is in a different reading frame within ORF1, can encode a 36 kD protein, putatively identified as a movement protein. CTLV-L coat protein (CP) was found to be located in the C-terminal region of the polyprotein encoded by ORF1. Evolutionary relationships and classification of capilloviruses is discussed.The nucleotide sequence data reported in this paper have been submitted to GenBank nucleotide sequence database and have been assigned the accession number D14455.     

Recombination and phylogeographical analysis of Lily symptomless virus

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.     

Concurrent porcine circovirus type 2a (PCV2a) or PCV2b infection increases the rate of amino acid mutations of porcine reproductive and respiratory syndrome virus (PRRSV) during serial passages in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic and antigenic variability. The purpose of this study was to determine if porcine circovirus type 2 (PCV2) infection increases genetic variability of PRRSV during serial passages in pigs and to determine if there is a difference in the PRRSV mutation rate between pigs concurrently infected with PCV2a or PCV2b. After 8 consecutive passages of PRRSV alone (group 1), PRRSV with PCV2a (group 2), or PCV2b (group 3) in pigs, the sequences of PRRSV structural genes for open reading frame (ORF) 5, ORF6, ORF7 and the partial non-structural protein gene (Nsp) 2 were determined. The total number of identified amino acid mutations in ORF5, ORF6, ORF7 and Nsp2 sequences was 30 for PRRSV infection only, 63 for PRRSV/PCV2a concurrent infection, and 77 for PRRSV/PCV2b concurrent infection when compared with the original VR2385 virus used to infect the passage 1 pigs. Compared to what occurred in pigs infected with PRRSV only, the mutation rates in ORF5 and ORF6 were significantly higher for concurrent PRRSV/PCV2b infected pigs. The PRRSV/PCV2a pigs had a significantly higher mutation rate in ORF7. The results from this study indicated that, besides ORF5 and Nsp2, the PRRSV structural genes ORF6 and ORF7 were shown to mutate at various degrees when the PRRSV was passaged over time in vivo. Furthermore, a significantly higher mutation rate of PRRSV was observed when pigs were co-infected with PCV2 highlighting the importance of concurrent infections on PRRSV evolution and control.     

Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate

Summary.  The complete sequence of a North American tobacco rattle virus (TRV) isolate, ‘Oregon yellow’ (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 of TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than -SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses, ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses vis also discussed. Accepted March 21, 1998     

Nucleotide sequence analysis of an infectious laryngotracheitis virus gene corresponding to the US3 of HSV-1 and a unique gene encoding a 67 kDa protein

Summary The DNA sequence of 4005 nucleotides from the Kpnl O and part of Kpnl K fragments in the short unique region of infectious laryngotracheitis virus (ILTV) was determined. The sequence contained two complete and one partial open reading frames (ORFs). The partial ORF was open at the 5 end of the sequence and represented the NH₂-terminal 118 amino acids (aa) of a polypeptide. Its partial predicted protein product exhibited significant homology to the US2 gene product of HSV-1 (herpes simplex virus type 1) and its homologs in other herpesviruses. ORF 2 is 471 aa long and could encode a protein of 53.8 kDa which shared aa homology with the protein kinases encoded by HSV-1 US3 and its gene homologs. Analysis of the ORF 2 aa sequence revealed domains characteristic of protein-serine/threonine (S/T) kinases of cellular and viral origin. The ORF 3 encoded a predicted protein of 601 aa (Mr 67.5 kDa) which exhibited limited homology (18% overall identity) with the UL47 protein (major tegument protein) of HSV-1. Northern (RNA) blot hybridization and metabolic inhibitors were used to characterize the ILTV protein kinase and the 67K mRNAs. The data revealed that protein kinase is a gamma-1 gene encoding a 1.6 kb mRNa, while the 67K ORF is a gamma-2 gene encoding a 2 kb mRNA.     

Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States

Recently, isolates of porcine reproductive and respiratory syndrome virus (PRRSV) that possess nucleotide sequences similar to European isolates have been reported in United States herds. The origin, diversity and prevalence of European-like North American PRRSV isolates in the U.S. remain unknown. Nucleotide sequence analysis of the 12 kb ORF1 of a North American isolate, SDPRRS 01-08 (01-08), showed 93.7% identity with Lelystad virus (LV), the prototypic European isolate, but only 58% identity with VR-2332, the prototypic North American isolate. Comparisons between LV and 01-08 at the peptide sequence level of the predicted non-structural proteins (Nsp) showed that Nsp9 (98.9% amino acid identity) was the most conserved and the least conserved was Nsp2 at 90.6% identity. For the purpose of comparison, GP5, the principal envelope structural protein, showed a 93.5% identity between 01-08 and LV. The most dramatic differences between the Nsp2 proteins of LV and 01-08 were a single 17 amino acid deletion between residues 734 and 750, as well as several amino acid differences. The same deletion was identified in the Nsp2 in five of seven other EuroPRRSV isolates submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. The remaining two isolates contained small deletions, but in other regions of Nsp2. Peptide sequence diversity in the form of hypervariability and deletions in Nsp2 demonstrate that European-like PRRSV isolates in the USA represent a heterogeneous group. Furthermore, areas in Nsp2 with deletions and amino acid hypervariability localize to regions that are predicted to be immunologically important.     

Baculovirus Expression of Proteins of Porcine Reproductive and Respiratory Syndrome Virus Strain Olot/91. Involvement of ORF3 and ORF5 Proteins in Protection

Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tu¨bingen isolates and between 61–64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.     

Sequence analysis of the 3'-end of feline calicivirus genome.

The nucleotide sequence of the 3'-end of the Japanese F4 strain of feline calicivirus (FCV) RNA was determined from a cloned cDNA of 3.5 kbp. We found three open reading frames (ORFs). The largest ORF encoded a 668-amino acid protein of 73,588 Da, which was presumably the capsid precursor protein of FCV and had significant amino acid sequence homology with the VP3 of picornaviruses. A small ORF at the extreme 3'-end was compared with that of the F9 strain of FCV, a vaccine strain originally from the U.S. Highly conserved amino acid sequences were shown, suggesting that this ORF might be functional and encode a putative 106-amino acid protein of 12,153 Da. The other ORF in the 5'-flanking region of the cDNA had consensus amino acid sequences conserved among the RNA-dependent RNA polymerases.     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

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     

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA

Summary.  The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3 803 nucleotides and has five open reading frames (ORFs). The 5′ ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria. Accepted August 25, 1997 Received June 18, 1997     

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.