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Molecular analysis of capsid protein of Homalodisca coagulata Virus-1, a new leafhopper-infecting virus from the glassy-winged sharpshooter, Homalodisca coagulata.

Hunter WB, Katsar CS, Chaparro JX - J. Insect Sci. (2006)

Bottom Line: Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI.Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades.The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, US Horticultural Research Laboratory, Fort Pierce, FL 34945, USA. whunter@ushrl.ars.usda.gov

ABSTRACT
A new virus that infects and causes increased mortality in leafhoppers was isolated from the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The virus, named Homalodisca coagulata virus -1, HoCV-1, was associated with increased mortality of cultured 5(th) instar H. coagulata. To identify the presence of H. coagulata viral pathogens, cDNA expression libraries were made from adult and nymphs. Analysis using reverse transcriptase PCR demonstrated that the virus was present in midgut tissues. As the viral capsid proteins are commonly used in classification of newly discovered viruses, the capsid proteins (CP) of the virus discovered in H. coagulata was examined. The order of the polyprotein subunits of HoCV-1 capsid proteins was determined to be CP2, CP4, CP3, and CP1. The CP4/CP3 (AFGL/GKPK) cleavage boundary site was clearly identified when the sequences were aligned. The putative CP3/CP1 (ADVQ/SAFA) cleavage site and the putative CP2/CP4 (VTMQ/EQSA) cleavage site of HoCV-1, respectively, were located in the same region as that of the other viruses. After alignment, the CP3/CP1 cleavage sites and CP2/CP4 cleavage sites of the viruses analyzed fell within 50 amino acids of one another. As with the cricket paralysis virus, HoCV-1 was found to be mainly comprised of beta-sandwiches in CP1-3 with a jelly roll topological motif. CP4 of HoCV-1 appeared to be mainly alpha-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus coat proteins as was demonstrated by the results of the BLASTP and PSI-BLAST tests, and is strongly supported by the structural modeling. While sequence homology between the cricket paralysis virus and HoCV-1 was low, the global structure of the proteins was conserved. Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI. Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades. Clade 1 consisted of Drosophila C virus, Clade 2 consisted of cricket paralysis virus, Clade 3 of Triatoma virus, Plautia stali intestine virus, Himetobi P virus, black queen cell virus, and HoCV-1. Clade 4 encompassed acute bee paralysis virus and Kashmir bee virus, and Clade 5 consisted of Rhopalosiphum padi virus. Analysis of the capsid protein of this new leafhopper virus provided significant evidence that it is related to other ssRNA insect viruses within the Family, Dicistroviridae. The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

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Gel of detected virus from salivary gland (Sg) and midgut (Mg) tissues of Homalodisca coagulata adults tested for presence of HoCV-1, using rtPCR. Both types of tissues from individual insects were dissected and analyzed in a pairwise fashion. Only midgut tissues were shown to test positively for virus presence. M = ladder wide-range DNA marker (16 fragments 50-10,000 bp), Sg = salivary glands, Mg = midgut tissue. Amplified fragment ~ 443 bp, was validated by sequencing of the amplified product and Blast analysis.
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i1536-2442-6-28-1-f01: Gel of detected virus from salivary gland (Sg) and midgut (Mg) tissues of Homalodisca coagulata adults tested for presence of HoCV-1, using rtPCR. Both types of tissues from individual insects were dissected and analyzed in a pairwise fashion. Only midgut tissues were shown to test positively for virus presence. M = ladder wide-range DNA marker (16 fragments 50-10,000 bp), Sg = salivary glands, Mg = midgut tissue. Amplified fragment ~ 443 bp, was validated by sequencing of the amplified product and Blast analysis.

Mentions: A total of 8,600 random EST sequences were generated from H. coagulata. Viral sequences were initially discovered through analysis of the cDNA library generated from whole bodies of adults. Approximately 5.8% (500) of the clones were homologous to viral sequences as indicated by BLASTX results. Analysis using rtPCR showed that the virus was in the midgut tissues, but not in salivary glands (Fig. 1). Blast analysis of the viral capsid protein indicated that the amino acid sequence had homology to the capsid proteins of insect picorna-like viruses, within the Family Dicistroviridae. The capsid protein sequence of HoCV-1 was submitted into GenBank (accession number: DQ308403), and was used to perform TBLASTX search of the NCBI database. Sequences with significant amino acid identity were downloaded for comparison to the HoCV-1 sequence. Comparisons were made to capsid protein sequences of: cricket paralysis virus (CrPV) [NC_003924], Drosophila C virus (DCV) [NC_001834], Triatoma virus (TrV) [NC_003783], Himetobi P virus (HiPV) [NC_003782], Plautia stali intestine virus (PSIV) [NC_003779], black queen cell virus (NC_003784), acute bee paralysis virus (ABPV) (NC_002548), Kashmir bee virus (KBV) [NC_004807], and Rhopalosiphum padi virus (RhPV) [NC_001874] (Table 1).


Molecular analysis of capsid protein of Homalodisca coagulata Virus-1, a new leafhopper-infecting virus from the glassy-winged sharpshooter, Homalodisca coagulata.

Hunter WB, Katsar CS, Chaparro JX - J. Insect Sci. (2006)

Gel of detected virus from salivary gland (Sg) and midgut (Mg) tissues of Homalodisca coagulata adults tested for presence of HoCV-1, using rtPCR. Both types of tissues from individual insects were dissected and analyzed in a pairwise fashion. Only midgut tissues were shown to test positively for virus presence. M = ladder wide-range DNA marker (16 fragments 50-10,000 bp), Sg = salivary glands, Mg = midgut tissue. Amplified fragment ~ 443 bp, was validated by sequencing of the amplified product and Blast analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2990318&req=5

i1536-2442-6-28-1-f01: Gel of detected virus from salivary gland (Sg) and midgut (Mg) tissues of Homalodisca coagulata adults tested for presence of HoCV-1, using rtPCR. Both types of tissues from individual insects were dissected and analyzed in a pairwise fashion. Only midgut tissues were shown to test positively for virus presence. M = ladder wide-range DNA marker (16 fragments 50-10,000 bp), Sg = salivary glands, Mg = midgut tissue. Amplified fragment ~ 443 bp, was validated by sequencing of the amplified product and Blast analysis.
Mentions: A total of 8,600 random EST sequences were generated from H. coagulata. Viral sequences were initially discovered through analysis of the cDNA library generated from whole bodies of adults. Approximately 5.8% (500) of the clones were homologous to viral sequences as indicated by BLASTX results. Analysis using rtPCR showed that the virus was in the midgut tissues, but not in salivary glands (Fig. 1). Blast analysis of the viral capsid protein indicated that the amino acid sequence had homology to the capsid proteins of insect picorna-like viruses, within the Family Dicistroviridae. The capsid protein sequence of HoCV-1 was submitted into GenBank (accession number: DQ308403), and was used to perform TBLASTX search of the NCBI database. Sequences with significant amino acid identity were downloaded for comparison to the HoCV-1 sequence. Comparisons were made to capsid protein sequences of: cricket paralysis virus (CrPV) [NC_003924], Drosophila C virus (DCV) [NC_001834], Triatoma virus (TrV) [NC_003783], Himetobi P virus (HiPV) [NC_003782], Plautia stali intestine virus (PSIV) [NC_003779], black queen cell virus (NC_003784), acute bee paralysis virus (ABPV) (NC_002548), Kashmir bee virus (KBV) [NC_004807], and Rhopalosiphum padi virus (RhPV) [NC_001874] (Table 1).

Bottom Line: Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI.Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades.The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, US Horticultural Research Laboratory, Fort Pierce, FL 34945, USA. whunter@ushrl.ars.usda.gov

ABSTRACT
A new virus that infects and causes increased mortality in leafhoppers was isolated from the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The virus, named Homalodisca coagulata virus -1, HoCV-1, was associated with increased mortality of cultured 5(th) instar H. coagulata. To identify the presence of H. coagulata viral pathogens, cDNA expression libraries were made from adult and nymphs. Analysis using reverse transcriptase PCR demonstrated that the virus was present in midgut tissues. As the viral capsid proteins are commonly used in classification of newly discovered viruses, the capsid proteins (CP) of the virus discovered in H. coagulata was examined. The order of the polyprotein subunits of HoCV-1 capsid proteins was determined to be CP2, CP4, CP3, and CP1. The CP4/CP3 (AFGL/GKPK) cleavage boundary site was clearly identified when the sequences were aligned. The putative CP3/CP1 (ADVQ/SAFA) cleavage site and the putative CP2/CP4 (VTMQ/EQSA) cleavage site of HoCV-1, respectively, were located in the same region as that of the other viruses. After alignment, the CP3/CP1 cleavage sites and CP2/CP4 cleavage sites of the viruses analyzed fell within 50 amino acids of one another. As with the cricket paralysis virus, HoCV-1 was found to be mainly comprised of beta-sandwiches in CP1-3 with a jelly roll topological motif. CP4 of HoCV-1 appeared to be mainly alpha-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus coat proteins as was demonstrated by the results of the BLASTP and PSI-BLAST tests, and is strongly supported by the structural modeling. While sequence homology between the cricket paralysis virus and HoCV-1 was low, the global structure of the proteins was conserved. Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI. Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades. Clade 1 consisted of Drosophila C virus, Clade 2 consisted of cricket paralysis virus, Clade 3 of Triatoma virus, Plautia stali intestine virus, Himetobi P virus, black queen cell virus, and HoCV-1. Clade 4 encompassed acute bee paralysis virus and Kashmir bee virus, and Clade 5 consisted of Rhopalosiphum padi virus. Analysis of the capsid protein of this new leafhopper virus provided significant evidence that it is related to other ssRNA insect viruses within the Family, Dicistroviridae. The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

Show MeSH
Related in: MedlinePlus