Watermelon Silver Mottle Virus Serogroup Research Articles

Molecular identification and characterization of groundnut bud necrosis virus and its associated thrips vector from Gerbera jamesonii in India

Gerbera is an important cut flower crop popular throughout the world. In summer months of 2016, gerberas grown in polyhouses of RHREC, GKVK, Bengaluru, Karnataka, India showed heavy infestation of thrips and displayed symptoms of viral infections. Infected plants displayed irregular chlorotic or necrotic spots or blotches, necrosis of veins, bronzing, crinkling and deformations of leaves; necrotic streaks on flower stalks; chlorosis or necrosis of flower petals; tip necrosis or death of young plants. The virus was identified as groundnut bud necrosis virus based on DAC-ELISA, RT-PCR and transmission studies, and it was characterized by cloning and sequencing of N gene and 3′ UTRs. Thrips associated was identified as Scirtothrips dorsalis and the virus was detected in individual thrips using DAC-ELISA. The virus could be efficiently transmitted to cowpea variety C-152 both by mechanical sap inoculation and vector transmission and to tulsi plants by vector transmission. Sequence of S-RNA segment of the virus from two independent gerbera samples was submitted under the accession numbers KX832986.1 and KX832987.1. The two isolates showed per cent identities of 97.1 and 98.5 at N gene and at 3′ UTRs, respectively, with each other and 92.9–99.1% at N gene with other GBNV isolates. Phylogenetically they clustered along with other GBNV isolates reported from different hosts and locations within the major clade of watermelon silver mottle virus serogroup. Differential response was observed in gerbera varieties with respect to incidence of GBNV disease (19.6–80.6%) and thrips (24.6–54.0 thrips/leaf) with the least incidence in the varieties Esmara and Orinaco.

Characterization of a New Orthotospovirus from Chilli Pepper in Yunnan Province, China.

Chilli pepper (Capsicum annuum L.) is one of the most important crops in Yunnan Province, China. An orthotospovirus isolate 14YV855 was isolated from a diseased chilli pepper plant exhibiting yellow ringspots and necrosis on leaves in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province in 2014. The complete genome sequence of 14YV855 was determined. The small, medium, and large RNAs are 3,428, 4,781, and 8,917 nucleotides long, respectively. The complete nucleocapsid (N) protein of 14YV855 shares a high amino acid identity of 84.8 to 89.9% to that of Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV), Watermelon bud necrosis virus (WBNV), and Watermelon silver mottle virus (WSMoV), which is slightly less than the 90% identity threshold for the demarcation of new Orthotospovirus sp. Phylogenetic analyses revealed that the N protein and RNA-dependent RNA polymerase of 14YV855 are the most related to WSMoV, while the NSs, NSm, and Gn/Gc proteins are similar to those of GBNV. As expected, 14YV855 is serologically related to CaCV, GBNV, WBNV, and WSMoV when the monoclonal antibody against the N protein of WSMoV was used; however, 14YV855 can be distinguished from other orthotospoviruses by reverse-transcription PCR using the specific primers. Our results indicate that 14YV855 is a new Orthotospovirus sp. belonging to the WSMoV serogroup and is provisionally named Chilli yellow ringspot virus.

Characterization of a new isolate of pepper chlorotic spot virus from Yunnan province, China.

A new isolate, 14YV733, of pepper chlorotic spot virus (PCSV) from chili peppers in Yunnan province of China was identified. The typical tospoviral particles of 80-120 nm in diameter were observed by electron microscopy. The virus caused systemic symptoms in several solanaceous plants and the Brassica rapa L. Chinensis group with mechanical inoculation. The sap from infected leaves reacted positively to a rabbit antibody to the N protein of watermelon silver mottle virus (WSMoV) in immunoblotting. The S, M, and L RNAs of PCSV-14YV733 are 3310 nts, 4711 nts, and 8913 nts long, respectively. This is the first report of complete sequences of PCSV in mainland China. Phylogenetic analysis of all tospoviral proteins indicated that PCSV-14YV733 is closely related to members of the WSMoV serogroup.

Full-length M and L RNA sequences of tospovirus isolate 2009-GZT, which causes necrotic ringspot on tomato in China.

The virus isolate 2009-GZT, collected from tomato in Guizhou province of China, was identified as a new member of the genus Tospovirus based on its S RNA sequence. Because its provisional name, "tomato necrotic spot virus" (TNSV), was identical to an already existing member of the genus Ilarvirus, 2009-GZT was renamed "tomato necrotic spot-associated virus" (TNSaV). In this study, the full-length sequences of the genomic M and L RNAs of TNSaV were determined and analyzed. The M RNA has 4,773 nucleotides (nt), encoding the NSm protein of 309 aa (34.4 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1123 aa (128 kDa) in the viral complementary (vc) strand. The NSm and Gn/Gc of TNSaV share the highest aa sequence identity (86.2 % and 86.9 %, respectively) with those of tomato zonate spot virus. The L RNA contains 8,908 nt and codes for the putative RNA-dependent RNA polymerase (RdRp) of 2885 aa (332 kDa) in the vc strand. The RdRp of TNSaV shares the highest aa sequence identity (85.2 %) with that of calla lily chlorotic spot virus (CCSV). Serological assays showed that TNSaV cross-reacts with rabbit antisera against watermelon silver mottle virus (WSMoV) NP and CCSV NP, indicating that TNSaV is a member of the WSMoV serogroup.

Complete Genome Sequence of Mulberry Vein Banding Associated Virus, a New Tospovirus Infecting Mulberry.

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5’-/3’-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.

DETECTION OF EIGHT DIFFERENT TOSPOVIRUS SPECIES BY A MONOCLONAL ANTIBODY AGAINST THE COMMON EPITOPE OF NSS PROTEIN

Rabbit antisera against the nucleocapsid protein (NP) have been commonly used for detection of tospoviruses and classification into serogroups or serotypes. Mouse monoclonal antibodies (MAbs) with high specificity to the NPs have also been widely used to identify tospovirus species. Recently, a serogroup-specific MAb against the NSs protein of Watermelon silver mottle virus (WSMoV) was produced by our laboratory to react with five members of WSMoV serogroup, i.e., WSMoV, Capsicum chlorosis virus (CaCV), Calla lily chlorotic spot virus (CCSV), Peanut bud necrosis virus (PBNV) and Watermelon bud necrosis virus (WBNV). The epitope recognized by the NSs MAb was determined and the comparison with the reported sequences of tospoviral NSs proteins revealed that the epitope is highly conserved at the N-terminal region of NSs proteins among members of WSMoV and Iris yellow spot virus (IYSV) serogroups, and Melon yellow spot virus (MYSV) serotype. When the NSs MAb was further used to react with the crude antigens of MYSV serotype, IYSV and Tomato yellow ring virus (TYRV) of IYSV serogroup, strong serological reactions, both in ELISA and western blotting, were observed. Thus, our results indicated that the NSs MAb is a useful and convenient tool for detection of the eight tospovirus species. It is also suggested that these eight Asian-type tospoviruses, i.e., WSMoV, CaCV, CCSV, PBNV, WBNV, MYSV, IYSV and TYRV, may share a common evolutionary ancestor.

Serological relationship between Melon yellow spot virus and Watermelon silver mottle virus and differential detection of the two viruses in cucurbits

Melon yellow spot virus (MYSV), a tentative member of the genus Tospovirus, is considered a distinct serotype due to the lack of a serological relationship with other tospoviruses in its nucleocapsid protein (NP). Recently, a virus isolate collected from diseased watermelon in central Taiwan (MYSV-TW) was found to react with a rabbit antiserum (RAs) prepared against the NP of Watermelon silver mottle virus (WSMoV), and a monoclonal antibody (MAb) prepared against the common epitope of the NSs proteins of WSMoV-serogroup tospoviruses, but not with the WSMoV NP-specific MAb, in both enzyme-linked immunosorbent assay (ELISA) and western blotting. In this investigation, both RAs and MAb against MYSV-TW NP were produced. Results of serological tests revealed that the RAs to MYSV-TW NP reacted with the homologous antigen and the crude antigens of members of the WSMoV serogroup, including members of the formal species WSMoV and Peanut bud necrosis virus, and members of three tentative species, Watermelon bud necrosis virus, Capsicum chlorosis virus and Calla lily chlorotic spot virus. The MAb to MYSV-TW NP reacted only with the homologous antigen and the other geographic isolates of MYSV from Japan (JP) and Thailand (TH). Our results of reciprocal tests indicate that the NP and the NSs protein of MYSV are serologically related to those of WSMoV-serogroup tospoviruses. Furthermore, we show that both the MYSV NP MAb and the WSMoV NP MAb are reliable tools for identification of MYSV and WSMoV from single or mixed infection in field surveys, as verified using species-specific primers in reverse transcription-polymerase chain reaction.

First Report of Capsicum chlorosis virus Causing Yellow Stripes on Calla Lilies in Taiwan.

Tomato spotted wilt virus (TSWV) and Calla lily chlorotic spot virus (CCSV) are two recognized species of the Tospovirus genus in the family Bunyaviridae infecting calla lily (Zantedeschia spp.). During 2005, 15 virus isolates were collected from different calla lily plants exhibiting yellow stripes on their leaves in Ho-Li, a major calla lily-production township in Taiwan. After three successive local lesion passages on Chenopodium quinoa Willd., diseased leaf tissues individually infected by these isolates were preserved in liquid nitrogen and used for subsequent identification studies. Using the tospovirus genus-specific primers gL3637 and gL4435c designed from the L RNA, an 800-bp DNA fragment was amplified in reverse transcription-PCR from all 15 isolates. Moreover, leaf extracts of the diseased calla lilies and the C. quinoa plants inoculated with the 15 virus isolates reacted with antisera against the nucleocapsid proteins (NP) of Capsicum chlorosis virus (CaCV)-gloxinia and Watermelon silver mottle virus (WSMoV), but not to monoclonal antibodies against the NP of TSWV, CCSV, Peanut chlorotic fan-spot virus (PCFV), or Impatiens necrotic spot virus (INSV) in indirect ELISA. These results indicate that the 15 virus isolates are tospoviruses belonging to the WSMoV serogroup. Additionally, we amplified and sequenced the full-length N gene from these tospovirus isolates using primers WN2328 (5'-CCATTGGTTTGCCTCCG-3') and WN3534 (5'-CGTCGACAGAGCAATCGAGGC-3') designed from the S RNA of WSMoV. The deduced amino acid sequences of the N protein from these 15 tospovirus isolates showed a greater than 92% identity to that of CaCV (GenBank Accession No. NC-008301). Furthermore, results of phylogenetic analysis of the 15 isolates on the basis of amino acids sequences, both genetic distance and parsimony trees indicated that they were all genetically clustered within CaCV using INSV, TSWV, and WSMoV as outgroups. The results indicate that the virus causing yellow stripes in calla lilies is a strain of CaCV. To our knowledge, this is the first evidence that CaCV can naturally infect calla lilies and cause yellow stripe symptoms. Reference: (1) F.-H. Chu et al. Phytopathology 91:361, 2001.

SEQUENCE ANALYSIS OF S RNA OF CALLA LILY CHLOROTIC SPOT VIRUS, A NEW TOSPOVIRUS

Calla lily chlorotic spot virus (CCSV), a tospovirus infecting Calla lily (Zantedeschia spp.) in Taiwan, with symptoms of chlorotic spots on leaves and stems, was previously reported serologically but distantly related to Watermelon silver mottle virus (WSMoV). To further characterize the virus, DNA fragments corresponding to the S RNA of the virus were amplified from total RNA extracted from CCSV-infected plants of Nicotiana benthamiana, cloned and sequenced. The full-length viral strand of the S RNA was determined as 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA was found identical to those of other tospoviruses, confirming that CCSV belongs to the genus Tospovirus. The N and the NSs proteins of CCSV share low amino acid identities, 20.9 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. The phylogenetic dendrogram of the N protein of CCSV compared with those of other tospoviruses in WSMoV serogroup indicates that CCSV is a distinct tospovirus. Thus, based on the results of molecular characterization of S RNA, we conclude that CCSV is a new tospovirus species belonging to WSMoV serogroup.

Serological Comparison and Molecular Characterization for Verification of Calla lily chlorotic spot virus as a New Tospovirus Species Belonging to Watermelon silver mottle virus Serogroup

ABSTRACT Calla lily chlorotic spot virus (CCSV) isolated from central Taiwan was recently identified as a tospovirus serologically but distantly related to Watermelon silver mottle virus (WSMoV). To clarify the serological relationship between the two viruses, rabbit polyclonal antibody (PAb) to CCSV and mouse monoclonal antibodies (MAbs) to WSMoV NP or CCSV NP were produced in this investigation, using purified nucleocapsid protein (NP) as immunogens. The PAb to CCSV NP reacted stronger with the homologous antigen than with the heterologous antigen, with much lower A(405) readings in indirect enzyme-linked immunosorbent assay (ELISA) and low-intensity banding in immunoblotting. MAbs produced to CCSV NP or WSMoV NP reacted specifically with the homologous antigens but not with the heterologous antigens in both ELISA and immunoblot analyses. The CCSV S RNA was determined to be 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames encoding the NP and a nonstructural (NSs) protein in an ambisense arrangement. A typical 3'-terminal sequence (5'-AUUGCUCU-3') that is shared by all members of the genus Tospovirus also is present in the CCSV S RNA. The CCSV NP and NSs protein share low amino acid identities of 20.1 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. Phylogenetic dendrogram analysis indicates that CCSV is a distinct member in the genus Tospovirus. The results provide evidence that CCSV is a new species in the genus Tospovirus and belongs to WSMoV serogroup.

First Report of Natural Infection of Luffa acutangula by Watermelon bud necrosis virus in India.

In August 2002, ridge gourd (Luffa acutangula) plant samples exhibiting yellowing of leaves were collected from the experimental farm of Tamil Nadu Agricultural University, Coimbatore, India. Mechanical inoculations of ridge gourd seedlings using the above samples resulted in chlorotic spots on inoculated leaves and vein clearing followed by chlorotic rings and yellow netting along veins of newly developed leaves. The virus was reisolated from the chlorotic spots of the seedlings. Extracts from the field samples as well as mechanically inoculated seedlings reacted with antiserum to Watermelon silver mottle virus (WSMoV) in direct antigen coating enzyme-linked immunosorbent assay, suggesting the association of a tospovirus belonging to WSMoV serogroup (2). Symptomatic leaves from the second mechanical passage exhibiting yellow netting along the veins were subjected to reverse transcription-polymerase chain reaction (1) to identify the genus Tospovirus. Using the primer pair (5'TCTGTCCT(C/T) TTGAA (G/T) GTCCA3' and 5'AGAGCAATCGAGGCGCT3') derived from the conserved sequences of Groundnut bud necrosis virus (GBNV) and WSMoV, part of the nucleocapsid (N) protein gene and the complete noncoding region upstream of the N gene's coding sequence were cloned and sequenced. Comparative sequence analysis of 291-bp region of the N gene revealed that the genus Tospovirus infecting ridge gourd shared maximum identity both at nucleotide (94%) and amino acid (97%) levels with the corresponding region of Watermelon bud necrosis virus (WBNV), which is a distinct species of WSMoV serogroup infecting watermelon in India (1). In contrast, only 76 to 81% and 82% identity at nucleotide and amino acid levels, respectively, was observed with the corresponding region of the N genes of GBNV and WSMoV. Natural infection of WBNV in cucurbits except watermelon in India is not known. To our knowledge, this is the first report of natural infection of ridge gourd by WBNV.