ABSTRACT
Two large contigs with sequence similarities to different carlaviruses were identified by high-throughput sequencing in samples from a cactus plant. The complete genomes of the two viruses, tentatively named "cactus carlavirus 1" (CCV-1) and "cactus carlavirus 2" (CCV-2), were determined to be 8,441 and 8,396 nucleotides long, respectively, excluding the poly(A) tail. These viruses have the typical genomic organization of members of the genus Carlavirus. CCV-1 appears to be a cactus isolate of the carlavirus HSO-2016a, with 90.1% nucleotide sequence identity between the two virus genomes, whereas CCV-2 may be classified as a member of a new species. The sequences of CCV-2 and other carlaviruses are 48.9-60.0% identical at the whole-genome level.
Subject(s)
Cactaceae/virology , Carlavirus/genetics , Carlavirus/isolation & purification , Genome, Viral/genetics , Plant Diseases/virology , RNA, Viral/genetics , Base Sequence , Carlavirus/classification , High-Throughput Nucleotide Sequencing , PhylogenyABSTRACT
A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.
Subject(s)
Nucleic Acids/isolation & purification , Plant Diseases/microbiology , Plant Diseases/virology , Polymerase Chain Reaction/methods , DNA, Bacterial/isolation & purification , DNA, Viral/isolation & purification , Plants , RNA, Viral/isolation & purification , Viroids/isolation & purificationABSTRACT
An insect iridescent virus has been isolated from diseased velvetbean caterpillars, Anticarsia gemmatalis, found in Argentina. The cytopathology is similar to that reported for other iridescent viruses with infected larvae exhibiting blue-purple opalescence. Icosahedral particles (n = 119) purified by sucrose gradient rate zonal centrifugation had dimensions of 145 +/- 7 nm (point-to-point) and 136 +/- 12 nm (side-to-side). The sedimentation coefficient was 2250 +/- 10 S20,w when the virus was suspended in phosphate buffer. Characterization of proteins by SDS-polyacrylamide gel electrophoresis revealed 24 polypeptides with a single major species of 53.6 kDa. Purified viral DNA had a density of 1.6902 g cm-3 in equilibrium ultracentrifugation, corresponding to a guanine:cytosine content of 32.2%. Analysis of digests of this DNA with two restriction enzymes revealed an average molecular size of 181.8 +/- 5.6 kbp. A 499-bp fragment was amplified from the genome of this isolate using the polymerase chain reaction and then sequenced; data suggest this virus is very closely related to IV 1, originally isolated from Tipula paludosa. These properties indicate this virus is an isolate of the genus Iridovirus.
Subject(s)
Iridoviridae/genetics , Moths/virology , Amino Acid Sequence , Animals , Base Sequence , DNA, Viral , Electrophoresis, Polyacrylamide Gel , Iridoviridae/growth & development , Iridoviridae/isolation & purification , Microscopy, Electron , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
A general population sample of bite marks in wax is used to demonstrate mathematically the individuality of the human dentition. The general principles of probability are discussed and applied to the analysis of teeth using a precise method of measurement. The unique nature of the human dentition is confirmed.
