Complete nucleotide sequence of a new carlavirus infecting Aconitum carmichaelii in China.

A novel virus was identified in aconite (Aconitum carmichaelii Debx.) in China by high-throughput sequencing (HTS) and tentatively named "aconite virus A" (AcVA). The genomic RNA of AcVA consists of 8,844 nucleotides, excluding the poly(A) at the 3' end. Analysis of the genomic organization of AcVA indicated that it possesses a genomic structure that is typical of carlaviruses and contains six putative open reading frames (ORFs). Pairwise analysis revealed that the replicase and coat protein of AcVA share the highest amino acid sequence identity (43.78% and 57.01%) with those of coleus vein necrosis virus (CVNV) and butterbur mosaic virus (ButMV), respectively. Based on the current classification criteria for carlaviruses, AcVA should be considered a distinct member of the genus Carlavirus.

Complete genome sequence of a divergent strain of potato virus P isolated from Solanum tuberosum in Russia.

Contigs with sequence similarity to potato virus P (PVP), which belongs to the genus Carlavirus, were identified by high-throughput sequencing analysis in potato tubers collected from a farmer's potato production field in Surazhevka, Artyom, Primorskiy Krai (Russia) in 2018. The complete genome sequence of this virus consisted of 8,394 nucleotides, excluding the poly(A) tail. This is the first report of PVP being detected outside South America. The isolate had high sequence similarity to PVP isolates from Argentina and Brazil, but low sequence similarity was observed in the genes encoding the RNA-dependent RNA polymerase (69% nucleotide sequence identity and 80% amino acid sequence identity) and coat protein (78% nucleotide sequence identity and 89% amino acid sequence identity). Phylogenetic analysis revealed that this PVP-like virus clustered with known PVP isolates but was distinct from them. Comparison of the sequences using the classification criteria of the ICTV indicated that this PVP-like virus is a strain of PVP.

Phylogenetic relationships of two Ukrainian tomato isolates of potato virus M and genetic variability analysis of its population.

The aim of this study was to investigate biological and molecular properties of two Ukrainian tomato isolates of potato virus M (PVM), K-16 and Pol-14, to determine their phylogenetic relationships and the genetic variability of PVM isolates. Study of phylogenetic relationships of two Ukrainian tomato PVM isolates with 35 isolates represented in GenBank was conducted. It was found that the coat protein (CP) gene sequence identity between two Ukrainian PVM isolates is 94.3% at the nucleotide level and 100% at the amino acid level. The highest level of the sequence identity (97.0% and 96.5% nt and 100% aa) have the isolates K-16 and Pol-14 with the German potato isolate DSMZ PV0273, Indian potato isolates Del 123, Del 134, Del 147, M 34 and Chinese isolate from pepino GS-6-2 (isolate K-16), which testifies about their common origin. Ukrainian tomato isolates K-16 and Pol-14 belong together with all European, Chinese, Iranian, Indian isolates to PVM-o clade or group I. It was found that the nucleotide substitutions in the capsid protein gene of all tomato PVM isolates (except the Italian) are synonymous. Analysis showed that the global dN/dS ratio for the entire CP gene sequences used in the study was 0.041 (p Keywords: potato virus M; Solanum lycopersicum; phylogenetic analysis; genetic variability; selection pressure.

Genetic variation in potato virus M isolates infecting pepino (Solanum muricatum) in China.

Potato virus M (PVM, genus Carlavirus, family Betaflexviridae) is considered to be one of most economically important pathogens of pepino in China. However, the details and the mechanisms underlying PVM evolution are unknown. In this study, we determined and analyzed 40 TGB 1 gene sequences, 67 TGB 2 and TGB 3 gene sequences, and 88 CP and NABP gene sequences from viruses isolated from 19 samples of pepino (Solanum muricatum) and one sample of tomato (S. lycopersicum) collected from different areas of China. Recombination analysis identified only one clear recombinant in the TGB2-TGB3-CP region, but no recombinants were detected for each of the five individual genes. Phylogenetic analysis showed that all PVM isolates could be divided into at least two lineages in trees derived from the TGB 2, CP, and NABP gene sequences, and the lineages seemed to reflect geographical origin. The five PVM genes in this study were found to be under strong negative selection pressure. The PVM isolates examined showed frequent gene flow between the Chinese and European populations, and also within the Chinese population. Clear star phylogenies and the neutral equilibrium model test showed that pepino isolates of PVM appear to be experiencing a new expansion after a recent introduction into China, and these isolates display low levels of genetic diversity. To our knowledge, this study is the first report describing genetic structure, recombination, and gene flow in PVM populations, and it provides strong evolutionary evidence for the virus populations from different geographic regions of China.

Genetic structure and molecular variability of potato virus M populations.

To investigate the genetic diversity of potato virus M (PVM; genus Carlavirus, family Betaflexiviridae), the complete nucleotide sequence of the coat protein gene of 30 PVM isolates from a major potato-growing region in Iran were determined. Phylogenetic analysis of these Iranian PVM isolates together with those available in the GenBank database suggested two divergent evolutionary lineages that did not reflect the origin of the isolates, and these were designated as PVM-o and PVM-d. Examination of the genetic variability of the coat protein of Iranian isolates and their counterparts whose sequences are available in the Genbank database revealed 16 genotype groups in the PVM population. Analysis of the synonymous-tononsynonymous ratio showed strong purifying selection in the CP gene in the genotype groups of divergent clades.

Molecular characterization of domestic and exotic potato virus S isolates and a global analysis of genomic sequences.

Five potato virus S (PVS) isolates from the USA and three isolates from Chile were characterized based on biological and molecular properties to delineate these PVS isolates into either ordinary (PVS(O)) or Andean (PVS(A)) strains. Five isolates - 41956, Cosimar, Galaxy, ND2492-2R, and Q1 - were considered ordinary strains, as they induced local lesions on the inoculated leaves of Chenopodium quinoa, whereas the remaining three (FL206-1D, Q3, and Q5) failed to induce symptoms. Considerable variability of symptom expression and severity was observed among these isolates when tested on additional indicator plants and potato cv. Defender. Additionally, all eight isolates were characterized by determining the nucleotide sequences of their coat protein (CP) genes. Based on their biological and genetic properties, the 41956, Cosimar, Galaxy, ND2492-2R, and Q1 isolates were identified as PVS(O). PVS-FL206-1D and the two Chilean isolates (PVS-Q3 and PVS-Q5) could not be identified based on phenotype alone; however, based on sequence comparisons, PVS-FL206-1D was identified as PVS(O), while Q3 and Q5 clustered with known PVS(A) strains. C. quinoa may not be a reliable indicator for distinguishing PVS strains. Sequences of the CP gene should be used as an additional criterion for delineating PVS strains. A global genetic analysis of known PVS sequences from GenBank was carried out to investigate nucleotide substitution, population selection, and genetic recombination and to assess the genetic diversity and evolution of PVS. A higher degree of nucleotide diversity (π value) of the CP gene compared to that of the 11K gene suggested greater variation in the CP gene. When comparing PVS(A) and PVS(O) strains, a higher π value was found for PVS(A). Statistical tests of the neutrality hypothesis indicated a negative selection pressure on both the CP and 11K proteins of PVS(O), whereas a balancing selection pressure was found on PVS(A).

[Detection and molecular characterization of Potato virus S (PVS, Carlavirus) from Colombia]. / Detección serológica y caracterización molecular de Potato virus S (PVS, Carlavirus) en cultivos de papa de Colombia.

In Colombia, potato crops are affected by a wide variety of viruses such as PVY, PLRV, PVX, PMTV and PVS. Unfortunately, there are very few studies on the biology, distribution and pathogenicity of these viruses; this situation is even worse for the latent virus PVS. In this work, we evaluated the presence ofPVS in four Colombian provinces (Antioquia, Boyacá, Cundinamarca, Nariño) by the use of ELISA. We also studied the degree of molecular variation by sequence comparison of a segment of the gene encoding for the viral coat protein. In average, PVS was detected in 40% of 320 analyzed samples of potato leaves; the highest levels were observed in the East ofAntioquia (49%) and Pasto (Nariño) (47%), while in the other regions ranged between 35% and 42%. Analysis of sequence revealed the presence of two PVS strains in Colombia: three isolates were associated to PVSo (Ordinary) and twelve belonged to PVSA (Andean). A high diversity was observed among PVSA strains with percent identities in the range of 88-99%. These findings highlight the importance of strengthening seed certification programs and quarantine measures in Colombia for viruses like PVS, which can cause losses of up to 20% in potato crops and even higher in mixed virus infection.

Discovery and Characterization of a Novel Carlavirus Infecting Potatoes in China.

A new carlavirus, tentatively named Potato virus H (PVH), was found on potato plants with mild symptoms in Hohhot, Inner Mongolia Autonomous Region, China. PVH was confirmed by genome sequencing, serological reactions, electron microscopy, and host index assays. The PVH particles were filamentous and slightly curved, with a modal length of 570 nm. Complete RNA genomic sequences of two isolates of PVH were determined using reverse transcription-PCR (RT-PCR) and the 5' rapid amplification of cDNA ends (5' RACE) method. Sequence analysis revealed that PVH had the typical genomic organization of members of the genus Carlavirus, with a positive-sense single-stranded genome of 8410 nt. It shared coat protein (CP) and replicase amino acid sequence identities of 17.9-56.7% with those of reported carlaviruses. Phylogenetic analyses based on the protein-coding sequences of replicase and CP showed that PVH formed a distinct branch, which was related only distantly to other carlaviruses. Western blotting assays showed that PVH was not related serologically to other potato carlaviruses (Potato virus S, Potato virus M, and Potato latent virus). PVH systemically infected Nicotianaglutinosa but not Nicotiana tabacum, Nicotianabenthamiana, or Chenopodiumquinoa, which is in contrast with the other potato carlaviruses. These results support the classification of PVH as a novel species in the genus Carlavirus. Preliminary results also indicated that a cysteine-rich protein encoded by the smallest ORF located in the 3' proximal region of the genome suppressed local RNA silencing and enhanced the pathogenicity of the recombinant PVX.

Detección serológica y caracterización molecular de Potato virus S (PVS, Carlavirus) en cultivos de papa de Colombia / Detection and molecular characterization of Potato virus S (PVS, Carlavirus) from Colombia

In Colombia, potato crops are affected by a wide variety of viruses such as PVY, PLRV, PVX, PMTV and PVS. Unfortunately, there are very few studies on the biology, distribution and pathogenicity of these viruses; this situation is even worse for the latent virus PVS. In this work, we evaluated the presence of PVS in four Colombian provinces (Antioquia, Boyacá, Cundinamarca, Nariño) by the use of ELISA. We also studied the degree of molecular variation by sequence comparison of a segment of the gene encoding for the viral coat protein. In average, PVS was detected in 40% of 320 analyzed samples of potato leaves; the highest levels were observed in the East of Antioquia (49%) and Pasto (Nariño) (47%), while in the other regions ranged between 35% and 42%. Analysis of sequence revealed the presence of two PVS strains in Colombia: three isolates were associated to PVS O (Ordinary) and twelve belonged to PVS A (Andean). A high diversity was observed among PVS A strains with percent identities in the range of 88-99%. These findings highlight the importance of strengthening seed certification programs and quarantine measures in Colombia for viruses like PVS, which can cause losses of up to 20% in potato crops and even higher in mixed virus infection.

El cultivo de papa en Colombia es afectado por diversos virus, que incluyen PVY, PLRV, PVX, PMTV y PVS; aunque se han realizado pocos estudios sobre la biología, distribución y patogenicidad de dichos virus en Colombia, siendo especialmente escasa la información referente al PVS. En este trabajo se evaluó mediante pruebas de ELISA, la presencia del PVS en cuatro departamentos de Colombia, así como sus niveles de variación, a partir de la secuenciación de una porción del gen de la cápside viral. Los resultados indicaron una detección promedio del virus en el 40% de las 320 muestras analizadas, con zonas como el Oriente cercano de Antioquia (49%) y Pasto (Nariño) (47%), donde se detectó en mayor proporción el virus. Los análisis de variación molecular indicaron la presencia de las dos razas de PVS (Ordinaria y Andina) en Colombia, siendo los aislamientos de PVS A los más diversos, al pre- sentar un rango de identidad del 88 al 99%. Estos hallazgos indican que es imperativo el fortalecimiento de los programas de certificación de semilla y vigilancia cuarentenaria en el país, especialmente para virus como el PVS, que aunque puede ser asintomático, causa pérdidas hasta del 20% en cultivos de papa.

Molecular characterization of coat protein gene of Garlic common latent virus isolates from India: an evidence for distinct phylogeny and recombination.

The coat protein (CP) gene of five Indian Garlic common latent virus (GarCLV) isolates was sequenced and it was 960 bp long in all the five isolates, encoding a protein of 319 amino acids. Comparative nucleotide sequence analysis revealed diversity of 4.3% among the Indian isolates and of 11.9% among all isolates worldwide. Amino acid sequence comparison showed a significant variability in the N-terminal of CP of GarCLV. Various protein analysis tools identified thirteen conserved domains and motifs including Carlavirus and Potexvirus-specific Flexi CP and Flexi N CP. Phylogenetic analysis clustered GarCLV isolates in the subgroup II with isolates from Australia, Brazil, Japan, and South Korea. Intraspecies recombination study revealed that only one of the Indian isolates was a recombinant. Interspecies recombination study suggested the absence of genetic exchange from Carlavirus species to GarCLV; conversely, GarCLV was identified as a putative donor for at least two other Carlavirus species. This is the first report of molecular variability and recombination in GarCLV isolates.

Complete genome sequence of the first Andean strain of potato virus S from Brazil and evidence of recombination between PVS strains.

An isolate of the Andean strain of potato virus S (PVS), named BB-AND, was detected for the first time in a Brazilian potato crop, fully sequenced and analyzed. A comparison of BB-AND with other PVS isolates (Andean and Ordinary) showed that BB-AND is quite distinct. The lowest amino acid sequence identity to the only other fully sequenced Andean isolate was found in ORF 1 (82%) and ORF 6 (87%). Recombination analysis showed that the isolate Vltava (AJ863510), from Germany, is a recombinant between PVS(O) and PVS(A) isolates, with the recombination event located between nucleotides 6125 and 8324.

Analysis of Iranian Potato virus S isolates.

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS(O) isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS(O) strain and 3 genotypes for the PVS(A) strain.

Genomic variability in potato virus M and the development of RT-PCR and RFLP procedures for the detection of this virus in seed potatoes.

Potato virus M (PVM, Carlavirus) is considered to be one of the most common potato viruses distributed worldwide. Sequences of the coat protein (CP) gene of several Canadian PVM isolates were determined. Phylogenetic analysis indicated that all known PVM isolates fell into two distinct groups and the isolates from Canada and the US clustered in the same group. The Canadian PVM isolates could be further divided into two sub-groups. Two molecular procedures, reverse transcription - polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) were developed in this study for the detection and identification of PVM in potato tubers. RT-PCR was highly specific and only amplified PVM RNA from potato samples. PVM RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 dormant tubers. Restriction analysis of PCR amplicons with MscI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by RFLP analysis may be a useful approach for screening potato samples on a large scale for the presence of PVM.

[Complete sequence analysis on potato virus M infecting Solanum muricatum].

The complete sequence of Potato virus M from Solanum muricatum was determined. The liner, single-strand and positive sense genomic RNA was 8526 nucleotides long and six open reading frames were identified. The genome organization was typical feature of genus Carlavirus. Sequence analysis showed that PVM isolates shared 62.5% - 97.2% nucleotide and 60.9% - 97.4% amino acid identities. The coat protein gene was most conserved and TGB3 gene was greatly variable. Phylogenetic tree analysis suggested that the Idaho potato isolate (PVM-Id) was a distant strain, and other 4 isolates were grouped discriminatively based on CP and NABP amino acid sequences respectively. This is the first report of PVM from Solanum muricatum.

Nucleotide sequence analysis of the 3'-terminal region of two Korean isolates of lily symptomless Carlavirus and expression of the coat protein in E. coli.

The 3'-terminal regions of the genomic RNAs of two Korean isolates of the lily symptomless Carlavirus (LSV), LSV-Ko and LSV-KII, were cloned and their nucleotide sequences were determined. The nucleotide sequence analysis and protein analysis by the Western blot revealed that E. coli expressed a 32-kDa protein that is the viral coat protein (CP) for the LSV. The two Korean strains share 98.4% and 98.3% sequence identities at the nucleotide and amino acid levels, respectively. The CP gene of LSV-Ko showed 99.1% and 87.0% nucleotide sequence identities, and 99.0% and 96.6% amino acid sequence identities with those of the Netherlands and the Japanese LSV strains, respectively. A pairwise amino acid sequence comparison revealed a sequence similarity of 29.6% to 69.8% between LSV-Ko and other species of the carlavirus. The 16 kDa protein of LSV-Ko shares 17.6% to 42.7% amino acid similarity with those of 8 other the carlaviruses, and they are variable in the N-terminal region. The Cys repeated zinc finger nucleic acid binding domain was found in the 16 kDa protein for all of the LSV strains. Sequence comparisons of the 7 kDa protein of LSV in the strain level showed significant identities from 100.0% to 98.4%. LSV-Ko shares 21.9% to 42.2% amino acid similarity with those of 8 other carlaviruses, 4 members of the potexviruses, and a closterovirus. LSV is closely related to blueberry scorch virus (BISV) based upon the phylogenetic tree analyses of the three proteins, indicating LSV to be a quite distinct member of the genus Carlavirus.

Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus.

Two strains of whitefly-transmitted cowpea mild mottle virus (CPMMV) causing severe (CPMMV-S) and mild (CPMMV-M) disease symptoms in peanuts were collected from two distinct agro-ecological zones in India. The host-range of these strains was restricted to Leguminosae and Chenopodiaceae, and each could be distinguished on the basis of symptoms incited in different hosts. The 3'-terminal 2500 nucleotide sequence of the genomic RNA of both the strains was 70% identical and contains five open reading frames (ORFs). The first three (P25, P12 and P7) overlap to form a triple gene block of proteins, P32 encodes the coat protein, followed by P12 protein located at the 3' end of the genome. Genome organization and pair-wise comparisons of amino acid sequences of proteins encoded by these ORFs with corresponding proteins of known carlaviruses and potexviruses suggest that CPMMV-S and CPMMV-M are closely related to viruses in the genus Carlavirus. Based on the data, it is concluded that CPMMV is a distinct species in the genus Carlavirus.