The hidden RNA viruses in Blattodea (cockroaches and termites).

The insect order Blattodea (cockroaches and termites) has drawn substantial research attention for their dietary habits and lifestyle of living with or around humans. In the present study, we focused on the discovery of RNA viruses hidden in Blattodea insects using the publicly available RNA sequencing datasets. Overall, 136 distinctive RNA viruses were identified from 36 Blattodea species, of which more than 70 % were most closely related to the invertebrate-associated viral groups within Picornavirales, Sobelivirales, Bunyaviricetes, Jingchuvirales, Durnavirales, Lispiviridae, Orthomyxoviridae, Permutotetraviridae, Flaviviridae and Muvirales. Several viruses were associated with pathogens of vertebrates (Paramyxoviridae), plants (Tymovirales), protozoa (Totiviridae), fungi (Narnaviridae) and bacteria (Norzivirales). Collectively, 93 complete or near-complete viral genomes were retrieved from the datasets, and several viruses appeared to have remarkable temporal and spatial distributions. Interestingly, the newly identified Periplaneta americana dicistrovirus displayed a remarkable distinct bicistronic genome arrangement from the well-recognized dicistroviruses with the translocated structural and non-structural polyprotein encoding open reading frames over the genome. These results significantly enhance our knowledge of RNA virosphere in Blattodea insects, and the novel genome architectures in dicistroviruses and other RNA viruses may break our stereotypes in the understanding of the genomic evolution and the emergence of potential novel viral species.

Novel cyclovirus discovered in the Florida woods cockroach Eurycotis floridana (Walker).

A novel cyclovirus (proposed genus "Cyclovirus", family Circoviridae) was discovered in a specimen of Eurycotis floridana (Walker), also known as the Florida woods cockroach or palmetto bug, collected from Tarpon Springs, Florida. The Florida woods cockroach-associated cyclovirus GS140 (FWCasCyV-GS140) was obtained through a degenerate PCR assay and showed 64 % genome-wide pairwise identity to a cyclovirus identified in bat feces. This finding supports recent reports suggesting that Circoviridae members, traditionally thought to only infect vertebrates, are present within insect populations.

Expression strategy of densonucleosis virus from the German cockroach, Blattella germanica.

Blattella germanica densovirus (BgDNV) is an autonomous parvovirus that infects the German cockroach. BgDNV possesses three mRNAs for NS proteins, two of which are splice variants of the unspliced transcript. The unspliced variant encodes open reading frame 5 (ORF5) (NS3), while NSspl1 encodes ORF3 (NS1) and ORF4 (NS2) and NSspl2 encodes the C-proximal half of NS1. BgDNV possesses three VP transcripts, one of which (VP) is unspliced, while the other two (VPspl1 and VPspl2) are generated by alternative splicing. The unspliced VP transcript contains both ORF1 and ORF2, while in VPspl1, ORF1 and ORF2 are joined in frame. The transcription of NS genes begins at an earlier stage of the virus life cycle than the transcription of VP genes. NS and VP transcripts overlap by 48 nucleotides (nt). BgDNV is characterized by two additional NS transcripts overlapping by more than 1,650 nt with VP-coding transcripts. Four different bands (97, 85, 80, and 57 kDa) corresponding to three BgDNV capsid proteins were detected on SDS-PAGE. Mass spectrometry analysis showed that the amino acid composition of the 85-kDa and 80-kDa proteins is the same. Moreover, both of these proteins are ubiquitinated. The BgDNV PLA(2) domain, which is critical for cellular uptake of the virus, is located in ORF2 and is present only in VP1. In contrast to all of the parvoviruses studied in this respect, VP2 has a unique N terminus that is not contained within VP1 and VP3. In situ recognition with NS1- and VP-specific antibodies revealed an uneven pattern of NS1 expression resembling a halo within the nuclear membrane.

House fly vector for porcine circovirus 2b on commercial pig farms.

We investigated the on-farm potential of common farm invertebrates to transmit porcine circovirus genotype 2 (PCV2) and other non-enveloped viruses. In 2007 (pre-PCV2 vaccination) and 2008 (post-PCV2 vaccination), invertebrate communities were trap-collected (8 trap-dates per year), counted and sorted into genus and species groups on 5 farm study sites within England. Total DNA was extracted from feces of representational cross-sections of pigs on each farm in each year and also from intact samples of Diptera flies (ca. 20 flies per trap) and dissected viscera of any cockroaches (ca. 5 per trap). Each DNA sample was tested for the presence of PCV2 DNA by separate PCRs for ORF1 and ORF2. Positive samples were sub-typed via DNA sequencing of PCR products. The pig-associated Diptera fly community was dominated by Musca domestica (house fly) in both years on all 5 farms; numerous Blatta orientalis cockroaches were only noted on 1 farm throughout. Specific PCV2b DNA elements were routinely detected (25-60% of samples) in weaner/nursery pig feces in 2007, but not in other age groups. Musca collected on 4 of the 5 farms in 2007 was also positive for PCV2b DNA elements. Comparison of ORF2 sequences indicated that ORF2 sequences indicating PCV2b genotype were identical in pigs and flies. Minor changes were noted in ORF1 sequences from different samples. Flies collected in the weaner/nursery area were most likely to be positive (22-50% of fly-trap samples). DNA extracted from all cockroaches (2007 and 2008) and all flies and pig feces in 2008 were also negative throughout. We suggest that Musca flies have the most likely on-farm potential to carry and transmit PCV2b due to their life cycle incorporating stages in close association with pigs and their habitat. Vaccination appeared to reduce environmental load of PCV2b.