Defective viral genomes are key drivers of the virus-host interaction.

Viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. We are beginning to appreciate the surprising versatility of viral genomes and how replication-competent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. This Review summarizes current knowledge of the types of defective viral genomes generated during the replication of RNA viruses and the functions that they carry out. We highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools.

Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm.

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.

A naturally occurring defective DNA satellite associated with a monopartite begomovirus: evidence for recombination between alphasatellite and betasatellite.

Monopartite begomoviruses and their associated satellites form unique disease complexes that have emerged as a serious threat to agriculture worldwide. It is well known that frequent recombination contributes to the diversification and evolution of geminiviruses. In this study, we identified a novel defective satellite molecule (RecSat) in association with Tobacco leaf curl Yunnan virus (TbLCYNV) in a naturally infected tobacco plant. Sequence analysis showed that Recsat comprises 754 nucleotides in size and is a chimera involving alphasatellite and betasatellite sequences, containing both betasatellite-conserved region and alphasatellite stem-loop structure. Recombination analysis revealed that RecSat has arisen from three independent recombination events likely involving Tomato yellow leaf curl China betasatellite, Ageratum yellow vein China betasatellite and Tobacco curly shoot alphasatellite. Co-inoculation of RecSat with TbLCYNV induced symptoms indistinguishable from those induced by TbLCYNV alone in Nicotiana benthamiana. Southern blot hybridization showed that RecSat could be trans-replicated stably in N. benthamiana plants by TbLCYNV, and impaired the accumulation of helper virus and co-inoculated alphasatellite. Our results provide the first evidence for recombination between two distinct types of satellites among geminivirus complex and highlight recombination as a driving force for geminivirus evolution.

Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra- and inter-species recombination.

We report the isolation, purification, genome-sequencing and characterization of a picorna-like virus from dead bees in Israel. Sequence analysis indicated that IAPV (Israeli acute paralysis virus) is a distinct dicistrovirus. It is most homologous to Kashmir bee virus and acute bee paralysis virus. The virus carries a 9487 nt RNA genome in positive orientation, with two open reading frames separated by an intergenic region, and its coat comprises four major proteins, the sizes of which suggest alternate processing of the polyprotein. IAPV virions also carry shorter, defective-interfering (DI)-like RNAs. Some of these RNAs are recombinants of different segments of IAPV RNA, some are recombinants of IAPV RNA and RNA from another dicistrovirus, and yet others are recombinants of IAPV and non-viral RNAs. In several of the DI-like RNAs, a sense-oriented fragment has recombined with its complement, forming hairpins and stem-loop structures. In previous reports, we have shown that potyviral and IAPV sequences are integrated into the genome of their respective hosts. The dynamics of information exchange between virus and host and the possible resistance-engendering mechanisms are discussed.

Molecular characterization of banana virus X (BVX), a novel member of the Flexiviridae family.

A novel virus was identified in banana (Musa spp). Analysis of the last 2917 nucleotides of its positive strand genomic RNA showed five open reading frames corresponding, from 5' to 3', to a truncated ORF coding for a replication-associated protein, three ORFs coding for a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This genome organization is similar to that of some members of the Flexiviridae family such as potexviruses and foveaviruses. This virus was named Banana virus X (BVX). Comparative sequence analysis showed that BVX is only distantly related to other members of the Flexiviridae family, in which it appears to define a new genus. BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized, accounting for deletions of 1268, 1358 and 1503 nucleotides. Contrary to the situation encountered for Potexviruses, these recombination sites are located within the TGB1 and CP genes and result in a truncated TGB1 protein.

Intranasal immunisation with defective adenovirus serotype 5 expressing the Venezuelan equine encephalitis virus E2 glycoprotein protects against airborne challenge with virulent virus.

There is no vaccine licensed for human use to protect laboratory or field workers against infection with Venezuelan equine encephalitis virus (VEEV). Infection of these groups is most likely to occur via the airborne route and there is evidence to suggest that protection against airborne infection may require high antibody levels and the presence of antibody on the mucosal surface of the respiratory tract. Recombinant defective type 5 adenoviruses, expressing the E3E26K structural genes of VEEV were examined for their ability to protect mice against airborne challenge with virulent virus. After intranasal administration, good protection was achieved against the homologous serogroup 1A/B challenge virus (strain Trinidad donkey). There was less protection against enzootic serogroup II and III viruses, indicating that inclusion of more than one E3E26K sequence in a putative vaccine may be necessary. These studies confirm the potential of recombinant adenoviruses as vaccine vectors for VEEV and will inform the development of a live replicating adenovirus-based VEEV vaccine, deliverable by a mucosal route and suitable for use in humans.

Injury-associated induction of two novel and replication-defective murine retroviral RNAs in the liver of mice.

Injury can alter the expression of numerous genes in affected tissues as well as in distant organs. The mouse genome harbors numerous copies of endogenous murine leukemia virus (MuLV)-related retroviral sequences. Mouse liver tissues harvested after burn injury were subjected to RT-PCR analysis to investigate the regulation of MuLV-related sequences using a primer set capable of amplifying the full-length transcript. A doublet of approximately 5-kb was transiently up-regulated at 3 and 6 h after injury. Sequence analyses revealed that these are novel defective endogenous retroviral sequences (MuLV(LI-8) and MuLV(LI-12)), which are predominantly characterized by major deletions in pol and env genes. The MuLV(LI-8) clone is 4.85 kb long and the deduced gag polypeptide sequence was almost identical to a previously reported replication-defective retroviral sequence associated with immunesuppression. In the MuLV(LI-12) clone of 5.06 kb, there were two truncated gag open reading frames (ORFs) and 1 pol ORF fused to the C-terminus of the env p15E. Furthermore, the ORFs for the unique gag p12 presumed to be responsible for the immunesuppression were present in both clones. These novel replication-defective MuLVs may participate in the pathogenesis of distant organs after injury.

[Defective proviruses of the human T-cell leukemia virus: structure and classification]. / Defektnye provirusy virusa T-kletochnogo leikoza cheloveka: struktura i klassifikatsiia.

Peripheral blood mononuclear cells of 24-70% individuals infected with HTLV-1 contain defective proviruses (dp) in addition to the full size ones. Most of them remain silent lacking regions sufficient for viral genes transcription except those activated under cell promoter or retaining viral open reading frames (orfs). It is still unclear whether these proviruses are associated with the development of T-cell leukemia in adults, tropic spastic paresis, or myelopathy. Classification of previously reported dp is presented, their origin and possible function in human HTLV-1 associated diseases are discussed.

Case report and molecular analysis of subacute sclerosing panencephalitis in a South African Child.

This is the first case of subacute sclerosing panencephalitis from South Africa in which the molecular characteristics of the causative measles virus were examined. The virus found is classified as genotype D3, which has not previously been found in Africa and was last circulating in the United States before 1992.

Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.

Characterization of cassava common mosaic virus and a defective RNA species.

The genome of cassava common mosaic potexvirus (CsCMV) has been sequenced from cDNA clones and consists of 6376 nucleotides (nt). A 76 nt untranslated region (UTR) at the 5' terminus was followed by ORF1 which potentially encodes a protein of 1449 amino acids (aa). ORFs 2, 3, and 4 were predicted to encode proteins of 231, 112 and 97 aa, respectively. ORF5 potentially encodes a 229 aa protein of 25 kDa that is similar to the coat proteins of other potexviruses. The 3'-terminal UTR of 114 nt was followed by a poly(A) tail. The genomic organization of the CsCMV genome is similar to that of other potexviruses. A cDNA clone that was apparently obtained from a defective RNA species contained both the 5' and 3' UTRs and an ORF that potentially encodes the first 263 aa of ORF1 and the last 33 aa of the coat protein. Defective RNA species were found both in purified preparations of the virus and in total nucleic acid isolated from CsCMV-infected plants.

Round cell variant of measles virus: spontaneous conversion from productive to cell-associated state of infection.

A subacute sclerosing panencephalitis strain of measles virus was found to be composed of two viral variants distinguishable by their cytopathic effects in Vero cells. One of the variants was similar to defective cell-associated measles virus strains, whereas the other was highly productive of viral progeny but did not induce cell fusion. Cloning of the variants by an agarose plaque procedure revealed a consistent and rapid interconversion of the variants into one another. While the mechanism of this interconversion remains unknown, analysis of the expression of viral antigens by the variants using indirect immunofluorescence with monoclonal antibodies specific for measles structural antigens suggested that the interconversion involved modulation of the expression of the viral matrix or M antigen.