The complete nucleotide sequence and genome organization of a novel carmovirus--honeysuckle ringspot virus isolated from honeysuckle.

A virus associated with yellow-to-purple ringspot on honeysuckle plants has been detected and tentatively named honeysuckle ringspot virus (HnRSV). The complete nucleotide sequence of HnRSV from infected honeysuckle has been determined. The genomic RNA of HnRSV is 3,956 nucleotides in length and is predicted to contain five open reading frames (ORFs). Comparisons of the amino acid sequences of the ORFs of HnRSV with those of members of the family Tombusviridae show that HnRSV is closely related to members of the genus Carmovirus. Phylogenetic analysis based on the amino acid sequences of RdRp and coat protein and nucleotide sequences of the whole genome revealed that HnRSV forms a subgroup with the carmoviruses. Together, our results support the classification of HnRSV as a member of a new species in the genus Carmovirus, family Tombusviridae.

Complete nucleotide sequence and genome organization of Calibrachoa mottle virus (CbMV)--a new species in the genus Carmovirus of the family Tombusviridae.

Complete genomic sequence of the viral RNA of Calibrachoa mottle virus (CbMV) has been determined. The CbMV genome has a positive-sense single-stranded RNA of 3919 nucleotides in length and encodes five open reading frames (ORFs). ORF1 encodes a protein with predicted molecular weight of 28 kDa (p28). ORF2 extends through the amber stop codon of ORF1 to give a protein with a predicted molecular weight of 87 kDa (p87). The readthrough domain of p87 contains the GDD motif common to RNA-dependent RNA polymerases (RdRp). ORF3 and ORF4 encode two small overlapping polypeptides of 8 kDa (p8) and 9 kDa (p9), respectively. The 3'-proximal ORF5 encodes a capsid protein (CP) of 37 kDa (p37). The untranslated 5'- and 3'-terminal regions are composed of 34 and 234 non-coding nucleotides, respectively. Comparisons of amino acid sequences of the ORFs of CbMV with members of Tombusviridae show that CbMV is closely related to members of the genus Carmovirus. Phylogenetic analyses based on the amino acid sequences of RdRp and coat protein and nucleotide sequences of the whole genome reveal that CbMV forms a subgroup with several carmoviruses. Therefore, the genome organization, physico-chemical properties, sequence alignments and phylogenetic analysis support the classification of CbMV as a new species in the genus Carmovirus, family Tombusviridae.

The complete nucleotide sequence and genome organization of tomato infectious chlorosis virus: a distinct crinivirus most closely related to lettuce infectious yellows virus.

The complete nucleotide sequence of tomato infectious chlorosis virus (TICV) was determined and compared with those of other members of the genus Crinivirus. RNA 1 is 8,271 nucleotides long with three open reading frames and encodes proteins involved in replication. RNA 2 is 7,913 nucleotides long and encodes eight proteins common within the genus Crinivirus that are involved in genome protection, movement and other functions yet to be identified. Similarity between TICV and other criniviruses varies throughout the genome but TICV is related more closely to lettuce infectious yellows virus than to any other crinivirus, thus identifying a third group within the genus.

Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis.

Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3mg refolded protein per 200mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.

Co-infection by two criniviruses alters accumulation of each virus in a host-specific manner and influences efficiency of virus transmission.

Tomato chlorosis virus (ToCV), and Tomato infectious chlorosis virus (TICV), family Closteroviridae, genus Crinivirus, cause interveinal chlorosis, leaf brittleness, and limited necrotic flecking or bronzing on tomato leaves. Both viruses cause a decline in plant vigor and reduce fruit yield, and are emerging as serious production problems for field and greenhouse tomato growers in many parts of the world. The viruses have been found together in tomato, indicating that infection by one Crinivirus sp. does not prevent infection by a second. Transmission efficiency and virus persistence in the vector varies significantly among the four different whitefly vectors of ToCV; Bemisia tabaci biotypes A and B, Trialeurodes abutilonea, and T. vaporariorum. Only T. vaporariorum can transmit TICV. In order to elucidate the effects of co-infection on Crinivirus sp. accumulation and transmission efficiency, we established Physalis wrightii and Nicotiana benthamiana source plants, containing either TICV or ToCV alone or both viruses together. Vectors were allowed to feed separately on all virus sources, as well as virus-free plants, then were transferred to young plants of both host species. Plants were tested by quantitative reverse-transcription polymerase chain reaction, and results indicated host-specific differences in accumulation by TICV and ToCV and alteration of accumulation patterns during co-infection compared with single infection. In N. benthamiana, TICV titers increased during co-infection compared with levels in single infection, while ToCV titers decreased. However, in P. wrightii, titers of both TICV and ToCV decreased during mixed infection compared with single infection, although to different degrees. Vector transmission efficiency of both viruses corresponded with virus concentration in the host in both single and mixed infections. This illustrates that Crinivirus epidemiology is impacted not only by vector transmission specificity and incidence of hosts but also by interactions between viruses and efficiency of accumulation in host plants.