RdRp or RT, That is the Question.

The RNA-dependent RNA polymerase (RdRp) of all known double-stranded RNA viruses is located within the viral particle and is responsible for the transcription and replication of the viral genome. Through an RT-PCR assay, we determined that purified virions, in vitro translated RdRp proteins, and purified recombinant RdRp proteins of partitiviruses also have reverse transcriptase (RT) function. We show that partitivirus RdRps 1) synthesized DNA from homologous and heterologous dsRNA templates; 2) are active using both ssRNA and dsRNA templates; and 3) are active at lower temperatures compared to an optimal reaction temperature of commercial RT enzymes. This finding poses an intriguing question: why do partitiviruses, with dsRNA genomes, have a polymerase with RT functions? In comparison, 3Dpol, the RdRp of poliovirus, did not show any RT activity. Our findings lead us to propose a new evolutionary model for RNA viruses where the RdRp of dsRNA viruses could be the ancestor of RdRps.

A 1,000-Year-Old RNA Virus.

Only a few RNA viruses have been discovered from archaeological samples, the oldest dating from about 750 years ago. Using ancient maize cobs from Antelope house, Arizona, dating from ca. 1,000 CE, we discovered a novel plant virus with a double-stranded RNA genome. The virus is a member of the family Chrysoviridae that infect plants and fungi in a persistent manner. The extracted double-stranded RNA from 312 maize cobs was converted to cDNA, and sequences were determined using an Illumina HiSeq 2000. Assembled contigs from many samples showed similarity to Anthurium mosaic-associated virus and Persea americana chrysovirus, putative species in the Chrysovirus genus, and nearly complete genomes were found in three ancient maize samples. We named this new virus Zea mays chrysovirus 1. Using specific primers, we were able to recover sequences of a closely related virus from modern maize and obtained the nearly complete sequences of the three genomic RNAs. Comparing the nucleotide sequences of the three genomic RNAs of the modern and ancient viruses showed 98, 96.7, and 97.4% identities, respectively. Hence, in 1,000 years of maize cultivation, this virus has undergone about 3% divergence.IMPORTANCE A virus related to plant chrysoviruses was found in numerous ancient samples of maize, with nearly complete genomes in three samples. The age of the ancient samples (i.e., about 1,000 years old) was confirmed by carbon dating. Chrysoviruses are persistent plant viruses. They infect their hosts from generation to generation by transmission through seeds and can remain in their hosts for very long time periods. When modern corn samples were analyzed, a closely related chrysovirus was found with only about 3% divergence from the ancient sequences. This virus represents the oldest known plant virus.

Characterizing Mycoviruses.

A variety of methods for the detection and characterization of fungal viruses are available. For many years, serological and biological assays were used for virus detection. Today, more sensitive methods like polymerase chain reaction, together with sequencing, are widely used to study viruses. Extracting double-stranded (ds) RNA can be a useful approach to detect and study mycoviruses from fungal tissues, as dsRNAs accumulate in infected cells as copies of viral genomes or as replicative intermediates of single-stranded RNA genomes. Here we present a basic protocol for growing fungal strains and isolating dsRNA using cellulose chromatography, followed by molecular diagnostic methods including cDNA synthesis, sequencing, and determination of 5' ends by primer ligation.

Molecular Characterization of a Novel Putative Partitivirus Infecting Cytospora sacchari, a Plant Pathogenic Fungus.

Three double-stranded RNAs (dsRNAs), approximately 1.85, 1.65 and 1.27 kb in size, were detected in an isolate of Cytospora sacchari from Iran. Partial nucleotide sequence revealed a 1,284 bp segment containing one ORF that potentially encodes a 405 aa protein. This protein contains conserved motifs related to RNA dependent RNA polymerases (RdRp) that showed similarity to RdRps of partitiviruses. The results indicate that these dsRNAs represent a novel Partitivirus that we tentatively designate Cytospora sacchari partitivirus (CsPV). Treatment of the fungal strain by cyclohexamide and also hyphal tip culture had no effect on removing the putative virus. Phylogenetic analysis of putative RdRp of CsPV and other partitiviruses places CsPV as a member of the genus Partitivirus in the family Partitiviridae, and clustering with Aspergillus ochraceous virus 1.