[Cell therapy of critical lower limb ischemia (problems and prospects)].

Critical limb ischemia is a syndrome that combines several peripheral artery diseases with different ethiology and pathogenesis but with similar prognosis, high morbidity and mortality. Possibility of surgical and conservative treatment of critical limb ischemia almost completely exhausted. Some hopes have arisen due to progress in cell technology. The article provides a critical analysis of pathogenic prerequisites of stem/progenitor cells for the treatment of patients with a critical limb ischemia in detail the basic results of preclinical and clinical studies on the safety and efficacy of cell technology. Unsolved problems and prospects of practical application are also discussed.

First Report of Recombinant Potato virus Y Strains in Potato in Jalisco, Mexico.

Potato virus Y (PVY) is a serious problem for potato production worldwide. The virus reduces both tuber yield and quality, and recent spread of recombinant strains of PVY in potato production areas is largely credited with the spread of potato tuber necrotic ringspot disease (PTNRD) (1). In Mexico, recombinant strains of PVY were reported in at least two states, Chihuahua (4) and the State of Mexico (3); however, no surveys have been conducted in other potato-producing areas, and the spectrum of PVY isolates circulating in the country has remained uncharacterized. In October 2011, a small-scale survey of seed potato was conducted in the state of Jalisco, Mexico, to identify PVY isolates present in fields. Twelve seed potato fields were inspected visually. These represented various generations of seed potato, from nuclear to G2. Leaf samples were collected from plants displaying mosaic, crinkling, and yellowing symptoms, and were tested for PVY. Fifty samples were collected from cultivars Fabula, Mondial, Fianna, Gigant, Caesar, and Adora. Of the 50 leaf samples collected, seven were PVY-positive using the Immuno-strip Kit (Agdia, Elkhart, IN), and six of these were determined to have a N-serotype according to the typing by the Pocket Diagnostics lateral flow kit (Forsite Diagnostics, Ltd., York, UK). PVY-positive samples came from cultivars Fabula (2 with N serotype), Mondial (4 with N serotype), and Fianna (1 with O serotype). Extracts of the seven PVY-positive leaf samples were applied to Whatman FTA cards (Sigma, St. Louis, MO), dried, and transported to the Plant Virology Laboratory at the University of Idaho for further characterization. All samples immobilized on FTA cards were subjected to RNA extraction and standard reverse transcriptase (RT)-PCR typing using a set of PVY-specific primers (2) to determine the strain type. All PVY isolates were recombinant. The six N-serotype samples were found to contain recombinant PVYNTN isolates and produced characteristic bands of 181 and 452 bp in RT-PCR, which indicated the presence of two recombination junctions in the HC-Pro/P3 and VPg regions typical of European PVYNTN isolates. The one O-serotype sample was identified as a recombinant PVYN-Wi/N:O isolate, and produced 181 and 689 bp bands in RT-PCR, which indicated the presence of one recombination junction in the HC-Pro/P3 region. Sequence analysis of RT-PCR products amplified from five samples with N serotype identified them as PVYNTN isolates, and from the one with O serotype identified it as PVYN-Wi/N:O isolate. Sequence comparisons confirmed that N serotype samples contained PVY isolates most closely related to typical PVYNTN sequences (Accession No. EF026075), while the O serotype sample contained the PVY isolate most closely related to PVYN-Wi from Europe (HE608963). The data obtained suggest the presence of two different types of PVY recombinants, PVYNTN and PVYN-Wi, in seed potato in Jalisco. Additional surveillance for these recombinant isolates may be needed, as well as a survey of their effects on tuber quality in production areas. This is the first report of recombinant isolates of PVY often associated with PTNRD circulating in seed potato in Jalisco, Mexico. References: (1) S. M. Gray et al. Plant Dis. 94:1384, 2010. (2) J. H. Lorenzen et al. Plant Dis. 90:935, 2006. (3) V. R. Ramirez-Rodriguez et al. Virol. J. 6:48, 2009. (4) L. Robles-Hernandez et al. Plant Dis. 94:1262, 2010.

First Report of Grapevine fleck virus in Idaho Grapevines.

Idaho has a growing viticulture industry, with nearly 1,600 acres of wine grapes (Vitis vinifera L.). Production is largely concentrated in two locations, the Snake River valley, which includes Canyon County in the southwest, and the Clearwater River valley, primarily Nez Perce County in the northwest. Grapevine fleck virus (GFkV) belongs to the genus Maculavirus, family Tymoviridae, comprising positive-sense, single-stranded RNA viruses with ca. 7.6-kb genome (3). It is one of five non-mechanically transmitted viruses associated with the fleck disease complex and has been previously documented to occur in the neighboring state of Washington (2). Main sources of wine grape nursery material imported to Idaho reside in Washington or in California, and it is important to monitor virus status of the planting material brought to the state. However, no information was available on the occurrence and prevalence of GFkV in wine grapes in Idaho. During three growing seasons in 2009 through 2011, random grapevine samples were collected in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce counties. A total of 434 samples were tested by one step RT-PCR using GFkV-specific primers, GFkVf: 5'-TGACCAGCCTGCTGTCTCTA-3' and GFkVr: 5'-TGGACAGGGAGGTGTAGGAG-3' designed to amplify a fragment of the GFkV capsid protein gene (1). Twenty-four samples tested positive for GFkV by RT-PCR and produced the expected 179-bp DNA fragment. These samples came from five vineyards sampled across all surveyed counties, and represented seven wine grape cultivars, including Pinot Noir, Cabernet Sauvignon, Syrah, Lemberger, Riesling, Chardonnay, Pinot Gris, and one unknown table grape cultivar. Twelve PCR products were cloned into the pGEM-T Easy plasmid vector (Promega), sequenced (numbered ID1 to 12, available upon request), and confirmed to represent fragments of the GFkV CP gene between positions 6,453 and 6,631 in the genome of GFkV isolate MT48 (GenBank Accession No. AJ309022.1). Eight of the Idaho GFkV sequences (ID2, ID3, ID7 to 11, and ID12) matched closely with other GFkV sequences from Washington State, Italy, India, and South America, showing 97 to 99% identity at the nucleotide level in pair-wise comparisons. Four GFkV sequences from Idaho (ID1 and ID4 to 6) showed only modest (90 to 92%) identity in pair-wise comparisons with GFkV sequences available in GenBank. Consequently, in phylogenetic reconstructions eight Idaho GFkV sequences clustered in the same lineage with the six GFkV sequences deposited in GenBank, and four other GFkV sequences were placed outside of this main clade. It is possible that this phylogeny of the Idaho GFkV reflects different sources of the virus-infected planting material brought to the state. In the absence of symptoms expressed in wine grape cultivars infected with GFkV, laboratory methods remain the only tool to detect the virus. To our knowledge, this is the first report of GFkV found in wine grapes in Idaho demonstrating its substantial presence in production areas. References: (1) G. Gambino and I. Gribaudo. Phytopathology 96:1223, 2006. (2) R. A. Naidu et al. Plant Dis. 94:784, 2010. (3) S. Sabanadzovic et al. J. Gen. Virol. 82:2009, 2001.

First Report of Beet severe curly top virus in Jalapeño Pepper in Chihuahua, Mexico.

Curly top is a serious problem in many irrigated crops in the semiarid areas in the western United States. The disease is caused by a complex of leafhopper-transmitted curtoviruses, one of which, Beet mild curly top virus (BMCTV), was previously found in chili pepper in Zacatecas and Aguascalientes, Mexico (3). During the past few years, sporadic symptoms similar to curly top disease were observed in jalapeño pepper in the south-central area of Chihuahua State. Symptomatic plants were scattered in otherwise healthy looking pepper stands and displayed stunting and yellowing. Affected leaves were brittle, showed upward curling, and a distinct green vein pattern with interveinal yellowing. In June and August of 2010, field surveys were conducted in Cordillera-Escuadra, Meoqui-Estacion Consuelo, Meoqui-Lomas del Consuelo, and Delicias-Presa Francisco I Madero. Ninety-four leaf samples were collected from symptomatic jalapeño pepper plants and subjected to ELISA and PCR testing for curly top. Of the 94 samples, 11 were found to be positive by triple-antibody sandwich-ELISA with polyclonal antibodies against curly top (2). To confirm the identification of curly top and type the specific curtovirus identified, four ELISA-positive samples were subjected to a PCR analysis using a virus-specific primer set for curtovirus typing designed by Chen et al. (1). All four samples tested produced a single 720-bp band with primers BSCTVv2688 and BGc396 (1) characteristic of the Beet severe curly top virus (BSCTV). These curly top-specific PCR amplicons were sequenced and found to be 99% similar to the BSCTV nucleotide sequence in the C1 gene region (GenBank Accession No. X97203); corresponding sequences were deposited in GenBank under Accession Nos. JF437870 to JF437873. To our knowledge, this is the first report of the curly top virus in the State of Chihuahua, demonstrating that curly top is established and common in jalapeño pepper here and will need surveillance in other vegetable crops under irrigation. References: (1) L. F. Chen et al. Plant Dis. 94:99, 2010. (2) J. Durrin et al. Plant Dis. 94:972, 2010. (3) R. Velásquez-Valle et al. Plant Dis. 92:650, 2008.

Monitoring of motor disorders in 7-day-old rats with severe hypoxic-ischemic injury of the brain.

Seven-day old rats were subjected to unilateral ligation of the common carotid artery followed by 2-h exposure to a gas mixture consisting of 8% oxygen and 92% nitrogen. Locomotor function of rats was monitored weekly. Functional deficit in these animals persisted for at least 3 months. The exercise tests of rotarod, hanging, and narrowing track were most informative. Our results can be used in preclinical studies of new drugs for the therapy of perinatal brain injury.

Study of the efficiency of transplantation of human neural stem cells to rats with spinal trauma: the use of functional load tests and BBB test.

Human ensheating neural stem cells of the olfactory epithelium were transplanted to adult male rats immediately after contusion trauma of the spinal cord at T9 level rostrally and caudally to the injury. Voluntary movements (by a 21-point BBB scale), rota-rod performance, and walking along a narrowing beam were monitored weekly over 60 days. In rats receiving cell transplantation, the mean BBB score significantly increased by 11% by the end of the experiment. The mean parameters of load tests also regularly surpassed the corresponding parameters in controls. The efficiency of transplantation (percent of animals with motor function recovery parameters surpassing the corresponding mean values in the control groups) was 62% by the state of voluntary motions, 37% by the rota-rod test, and 32% by the narrowing beam test. Morphometry revealed considerable shrinking of the zone of traumatic damage in the spinal cord and activation of posttraumatic remyelination in animals receiving transplantation of human neural stem cells.

First Identification of an Unusual Recombinant Potato virus Y Strain in Potato in Mexico.

Potato virus Y (PVY) has been reported in potato crops in Mexico (3), with tobacco necrotic variants found in the central State of Mexico (4). Nevertheless, many individual states are currently declared PVY free and distribution of individual strains of PVY in potato in different states of Mexico and in different solanaceous crops had not yet been studied. A limited field PVY survey was conducted on potato in the State of Chihuahua in August 2009. More than 900 random potato leaf samples were collected from cvs. Snowden, Atlantic, FL1867, Felsina, Fianna, Gigant, and Alpha. Seven were found to be PVY-positive and had been collected from cvs. Fianna, Snowden, and FL1867. The PVY status of the collected samples was initially determined with the PVY-specific Immunostrips (Bioreba, Reinach, Switzerland) and by double-antibody sandwich-ELISA using the polyclonal PVY detection kit (Agdia, Elkhart, IN). To determine the strain specificity of these PVY isolates following ELISA tests, the infected original samples were inoculated onto tobacco plants at the four-leaf stage and symptom appearance and development were observed for 8 weeks side-by-side with control isolates PB-Oz (PVYO), N4 (PVYNTN), and Mont (PVYN) (1), followed by the standard PVY strain typing by reverse transcription (RT)-PCR (2). Only one of the PVY-positive samples, originally from symptomless potato cv. Fianna, induced systemic PVY infection in tobacco by producing stunting, mosaic, and vein clearing. No systemic vein necrosis, characteristic of isolates Mont and N4, was observed in Nicotiana tabacum cvs. Burley, Xanthi, or Samsun after inoculation with this isolate during all 8 weeks of observation. This isolate, PVY-M3, was typed as a PVY recombinant by RT-PCR, with two recombinant junctions characteristic of European PVYNTN strains (2). It was further analyzed by triple-antibody sandwich-ELISA using four PVYO and PVYN strain-specific monoclonal antibodies. Monoclonals 1F5 (Agdia) and SASA-N (Scottish Agriculture Science Agency [SASA], Edinburgh) reacted to this isolate and identified PVY-M3 serologically as PVYN serotype, characteristic of other PVYNTN recombinants. Monoclonals MAb2 (Agdia) and SASA-O (SASA), specific to PVYO and PVYC strains, did not react to PVY-M3. Taken together, the combination of biological, serological, and molecular characteristics define this recombinant isolate from Mexico as belonging to the same PVY strain group represented by the isolate PVY-L26 (1). To our knowledge, this is the first report of such an unusual PVYNTN recombinant strain from Mexico. Presence of this isolate, with no vein necrotic symptoms induced on tobacco and with PVYNTN genome, will necessitate development of new detection methods for the seed potato industry in Mexico. References: (1) X. Hu et al. Virus Res. 143:68, 2009. (2) J. L. Lorenzen et al. Plant Dis. 90:935, 2006. (3) L. P. Moreno et al. Rev. Mex. Fitopatol. 22:187, 2004. (4) V. R. Ramirez-Rodriguez et al. Virol. J. 6:48, 2009.

[Problems and prospects of experimental modeling of hypoxy-ischemic lesions in the central nervous system].

Perinatal hypoxy-ischemic brain lesions are one of the main causes of mortality and dysfunction of the central nervous system in the neonatal period accounting for high disability rate among survivors. Numerous animal models were proposed to study this problem in ante-, intra-, and neonatal periods of ontogenesis. This paper is devoted to the analysis of the adequacy of these models. The processes of brain development in laboratory animals are considered along with etiopathogenetic factors that can be reproduced on the models of perinatal hypoxy-ischemic lesions in CNS. The available data on such models and their correspondence to known clinical syndromes are summarized. Current trends in the development of new models of hypoxy-ischemic brain lesions are discussed.

First Report of Grapevine leafroll-associated virus-3 in Six Wine Grape Cultivars in Idaho.

In recent years, wine grape (Vitis vinifera) acreage in Idaho has expanded because of favorable climatic conditions for premium wine production. Nearly 95% of the 491.7 ha (1,215 acres) of wine grapes are in the Snake River Valley with Canyon County accounting for 81% of the vines. Previous studies have shown that grapevine leafroll disease (GLD) is the most widespread and economically significant virus disease in wine grapes in Washington and Oregon (1,2). However, little is known about the incidence and economic impact of GLD on wine grapes in Idaho. During the 2008 growing season, leaf samples were collected from approximately 25 individual grapevines of red-berried cultivars (Cabernet Sauvignon, Merlot, Syrah, and Petit Syrah) showing GLD symptoms and white-berried (Chardonnay) cultivars with suspected GLD symptoms growing in 10 geographically separate vineyards in Canyon County. An additional five samples were collected from a Lemberger block in Elmore County. Petiole extracts from these samples were tested by single-tube reverse transcription (RT)-PCR with primers LC 1 (5'-CGC TAG GGC TGT GGA AGT ATT-3') and LC 2 (5'-GTT GTC CCG GGT ACC AGA TAT-3') specific for the heat shock protein 70 homologue (HSP-70 gene) of Grapevine leafroll-associated virus-3 (GLRaV-3) (3). All samples, except the Petit Syrah, produced a single band of the expected size of 546 bp. ELISA with GLRaV-3-specific antibodies (BIOREBA AG, Reinach, Switzerland) confirmed the presence of the virus in samples that were positive in RT-PCR. GLRaV-3-specific amplicons were cloned in pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA) and 2 to 3 independent clones per isolate were sequenced in both orientations. A pairwise comparison of 22 sequences, six from Chardonnay (GenBank Accessions GQ344810, GQ344811, GQ344823, GQ344824, GQ344825, and GQ344826), five from Cabernet Sauvignon (GQ344807, GQ344808, GQ344809, GQ344827, and GQ344828), four each from Merlot (GQ344815, GQ344816, GQ344817, and GQ344818) and Syrah (GQ344819, GQ344820, GQ344821, and GQ344822), and three from Lemberger (GQ344812, GQ344813, and GQ344814) showed 87 to 100% identity at the nucleotide level and 92 to 100% identity at the amino acid level. A pairwise comparison of HSP-70 sequences of GLRaV-3 isolates from Idaho with corresponding sequences of GLRaV-3 isolates from GenBank showed nucleotide sequence identities between 88% (AJ748519) and 100% (DQ780885). Phylogenetic analysis of HSP-70 sequences from Idaho and GenBank showed clustering of Idaho sequences into five groups, with 12 sequences clustering with a Washington isolate (DQ780885), six sequences in a second group clustering with an isolate from Tunisia (AJ748522), two sequences in a third group clustering with an isolate from Austria (AJ748513), and one sequence each in groups four and five clustering with isolates from Italy (AJ748520) and Washington (DQ780889), respectively. The clustering was not cultivar- or vineyard-specific, suggesting separate introductions of different GLRaV-3 isolates in planting materials. To our knowledge, this is the first report of GLRaV-3 in grapevines grown in Idaho. These and previous results (1,2), indicate the wide distribution of GLRaV-3 in several grapevine cultivars in the Pacific Northwest Region. References: (1) R. R. Martin et al. Plant Dis. 89:763, 2005. (2) R. A. Naidu et al. (Abstr.) Phytopathology 96(suppl.):S83, 2006. (3) M. J. Soule et al. Plant Dis. 90:1461, 2006.

[Combined treatment including ozonotherapy of patients with viral hepatitis ].

Patients with viral hepatitis have disturbances of biliary tract motor function with the tendency to hypertonus of Oddi's sphincter, changes of physic-colloid properties of bile with increase in density of gall and hepatic bile, pH shift to acid side, microlites formation, disorders in biochemical composition of bile. More than 80% patients have biliar insufficiency. According to our data, with the purpose to correct of disturbances of hepatic exocrine function in patients with viral hepatitis and to prevent stone formation, it is reasonable to use together with antiviral therapy also intravenous injection of ozonated physiological solution and preparations of ursodeoxycholic acid.

Complete nucleotide sequence and genome organization of pineapple mealybug wilt-associated virus-1.

Pineapple mealybug wilt-associated virus-1 (PMWaV-1; family Closteroviridae, genus Ampelovirus) belongs to a complex of mealybug-transmissible viruses found in pineapple worldwide. In this study, the complete genome of PMWaV-1 was sequenced and found to be 13.1 kb in length, making it the smallest in the family. The genome encoded seven open reading frames (ORFs) and was unusual for an ampelovirus due to the lack of an intergenic region between the RdRp and p6 ORFs, an ORF encoding a relatively small coat protein (CP), and the absence of an ORF encoding a coat protein duplicate (CPd). Phylogenetic analyses placed PMWaV-1, plum bark necrosis stem pitting-associated virus and some grapevine leafroll-associated viruses in a distinct clade within the genus Ampelovirus.

Identification of Potato virus Y Strains Associated with Tuber Damage During a Recent Virus Outbreak in Potato in Idaho.

Potato virus Y (PVY) causes substantial losses in potato production by decreasing yields and affecting the quality of potato tubers. Management of PVY in potato is dependent primarily on potato seed certification programs to prevent or limit initial levels of virus inoculum. Prior to 1990, the ordinary strain of PVY (PVYO) was the predominant virus in North America. PVYO induces clear foliar symptoms in many potato cultivars, allowing successful management in seed potato through a combination of visual inspections and limited laboratory testing. In recent years, necrotic strains of PVY (PVYN, PVYNTN, and PVYN:O) have begun to spread in the United States, many of which induce mild symptoms in potato, making them more difficult to manage through visual inspections. In addition to reducing yield, necrotic isolates may also cause external and internal damage in tubers of susceptible cultivars, which is known as potato tuber necrotic ringspot disease (PTNRD). Tuber necrotic strains of PVY have been reported across the northern United States (1,2,4), although limited information is available on their incidence and spread in commercial potato production. During June and July of 2007, 38 random samples were collected from three different commercial fields displaying disease problems (cvs. Russet Ranger, Alturas, and Russet Burbank) in the vicinity of Idaho Falls, ID. Plants collected showed various degrees of mosaic and leaf yellowing. By using double-antibody sandwich (DAS)-ELISA and reverse transcription (RT)-PCR, 25 of these plants were identified as PVY positive. The mutiplex RT-PCR assay (3) confirmed that nine plants were infected with PVYNTN and 11 with PVYN:O. No RT-PCR products were amplified from five samples. During September and October of 2007, 25 tuber samples (cv. Russet Burbank) showing various degrees of unusual internal symptoms (e.g., brown spots) were collected near Idaho Falls, ID. Twenty-two tubers were found PVY positive by DAS-ELISA, and multiplex RT-PCR determined 13 of those were PVYNTN, three were PVYO, one was a PVYNTN/N:O mixture, and one was a PVYO/N:O mixture. No RT-PCR products were amplified from four samples. In October 2007, six tubers showing distinct external tuber damage characteristic of PTNRD (cv. Highland Russet) were collected near Twin Falls, ID. All six tubers were determined to be PVY positive by DAS-ELISA, and RT-PCR identified five as infected with PVYNTN and one with PVYN:O. All the mixtures were easily separated by inoculating tobacco plants followed by subsequent testing of individual plants. Asymptomatic tubers from the same lot not showing PTNRD damage were found PVY negative by DAS-ELISA and RT-PCR. All PVYNTN isolates collected during 2007 were inoculated into tobacco plants (Nicotiana tabacum L. cv. Xanthi) and confirmed to induce systemic vein necrosis. Limited sequencing of four of the PVYNTN isolates determined that they contained recombinant junctions 2 and 3, identifying them as being related to the European strain of PVYNTN (3). The data suggest an increase in distribution and incidence of necrotic strains of PVY in commercial, potato-production areas in Idaho during an outbreak in 2007 and the potential for an increase in PTNRD. References: (1) P. M. Baldauf et al. Plant Dis. 90:559, 2006. (2) J. M. Crosslin et al. Plant Dis. 90:1102, 2006. (3) J. H. Lorenzen et al. Plant Dis. 90:935, 2006. (4) L. M. Piche et al. Phytopathology 94:1368, 2004.

The complete nucleotide sequence and genome organization of tomato chlorosis virus.

The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the "rattlesnake tail" of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.

Nucleotide sequence and taxonomy of Cycas necrotic stunt virus. Brief report.

Cycas necrotic stunt virus (CNSV) is the only well-characterized virus from gymnosperm. cDNA segments corresponding to the bipartite genome RNAs (RNA1, RNA2) were synthesized and sequenced. Each RNA encoded a single polyprotein, flanked by the 5' and 3' non-coding regions (NCR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs, which were characteristic of viruses in the genus Nepovirus. The polyproteins showed higher sequence identities to Artichoke Italian latent virus, Grapevine chrome mosaic virus and Tomato black ring virus, all of which belong to subgroup b of the genus Nepovirus, than to other nepoviruses. Phylogenetic analysis of RNA dependent RNA polymerase and coat protein also showed closer relationships with these viruses than other viruses. The data obtained supported the taxonomical status of CNSV as a definitive member of the genus Nepovirus, subgroup b.

Nucleotide sequence of a Japanese isolate of Squash mosaic virus. Brief report.

The complete nucleotide sequence of the RNA-1 of Squash mosaic virus (SqMV) was determined using a Japanese isolate (Y-SqMV). The sequence consisted of 5865 nucleotides excluding the poly (A) at the 3' terminus and contained a single long open reading frame with a coding capacity for a protein of Mr209971. Analysis of the deduced amino acid sequence suggested a genomic organization typical of comoviruses. The nucleotide sequence of the RNA-2 of Y-SqMV was also determined and compared with the SqMV isolates from the United States. The larger and smaller capsid protein (CP) coding region was compared to those of K-SqMV and Z-SqMV, which represent two subgroups of SqMV. The larger CP gene of Y-SqMV showed 93.0% and 88.0% identities with those of K-SqMV and Z-SqMV, respectively at the nucleotide level. The smaller CP gene of Y-SqMV was 94.1% and 88.4% identical with those of K-SqMV and Z-SqMV. The results suggested that the Japanese SqMV isolate (Y-SqMV) is distinct from those in the United States, and might represent a third subgroup.

Satsuma dwarf and related viruses belong to a new lineage of plant picorna-like viruses.

Satsuma dwarf virus (SDV) and two closely related viruses, Citrus mosaic (CiMV), and Naval orange infectious mottling (NIMV), seriously affect citrus varieties grown in Japan and East Asia. All three viruses have icosahedral particles built of two proteins encapsidating two single-stranded genomic RNAs. The natural mode of transmission of these SDV-like viruses is unknown, and they were previously placed among tentative members of the family Comoviridae. Recently, a complete genome of SDV was sequenced, and its replication-related proteins were found only distantly related to those of viruses from the family Comoviridae (Iwanami T., Kondo Y., and Karasev A.V. J Gen Virol 80, 793-797, 1999). Here we present a partial genome sequence for another SDV-like virus, NIMV, and a thorough phylogenetic analysis of the gene products encoded by SDV, CiMV, and NIMV to assess their relationships with picorna-like viruses infecting plants, insects, and vertebrates. The RdRp's of SDV-like viruses form a new lineage, separate from members of Como- and Sequiviridae families. Phylogenetic analysis suggests that SDV-like viruses may represent a new family of plant picorna-like viruses. Sequence analysis of the capsid proteins (CPs) encoded by the SDV-like viruses revealed a region of similarity to CPs of animal calici- and picornaviruses that encompasses the structural core of the eight-strand beta-barrel characteristic of picornaviral CPs. These data suggest that SDV and related bipartite viruses evolved separately from the viruses in the family Comoviridae and that the split of an ancestor, monopartite picorna-like virus genome might have occurred more than once.

Sequence diversity and interrelationships among isolates of satsuma dwarf-related viruses.

The 3'-region of RNA2 of three viruses (Natsudaidai dwarf virus (isolate ND-1), and two unidentified isolates (LB-1, Az-1)), which were related to Satsuma dwarf virus (SDV), were sequenced. Phylogenetic analysis including the previously reported SDV-related viruses (Citrus mosaic virus (CiMV, Ci-968), Navel orange infectious mottling virus (NIMV, NI-1)) showed that they were classified into three groups, SDV (S-58), CiMV (Ci-968, LB-1, Az-1, ND-1), and NIMV (NI-1). The results suggested these groups might correspond to the three distinct virus species. ND-1, LB-1, and Az-1 were considered strains of CiMV, although they do not induce citrus mosaic on the fruit rind.

Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2.

The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3'-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5' to 3', potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3' terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family CLOSTEROVIRIDAE: the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.