Propidium monoazide (PMA) and ethidium bromide monoazide(EMA) improve DNA array and high-throughput sequencing ofporcine reproductive and respiratory syndrome virus identification

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.

Deoxynivalenol (DON) naturally contaminated feed impairs the immune response induced by porcine reproductive and respiratory syndrome virus (PRRSV) live attenuated vaccine

Cereal commodities are frequently contaminated with mycotoxins produced by the secondary metabolism of fungal infection. Among these contaminants, deoxynivalenol (DON), also known as vomitoxin, is the most prevalent type B trichothecene mycotoxin worldwide. Pigs are very sensitive to the toxic effects of DON and are frequently exposed to naturally contaminated feed. Recently, DON naturally contaminated feed has been shown to decrease porcine reproductive and respiratory syndrome virus (PRRSV) specific antibody responses following experimental infection. The objective of this study was to determine the impact of DON naturally contaminated feed on the immune response generated following vaccination with PRRSV live attenuated vaccine. Eighteen pigs were randomly divided into three experimental groups of 6 animals based on DON content of the diets (0, 2.5 and 3.5 mg DON/kg). They were fed these rations one week prior to the vaccination and for all the duration of the immune response evaluation. All pigs were vaccinated intra-muscularly with one dose of Ingelvac® PRRSV modified live vaccine (MLV). Blood samples were collected at day −1, 6, 13, 20, 27 and 35 post vaccination (pv) and tested for PRRSV RNA by RT-qPCR and for virus specific antibodies by ELISA. Results showed that ingestion of DON-contaminated diets significantly decreased PRRSV viremia. All pigs fed control diet were viremic while only 1 (17%) and 3 (50%) out of 6 pigs were viremic in the groups receiving 3.5 and 2.5 mg of DON/kg, respectively. Subsequently, all pigs fed control diet developed PRRSV specific antibodies while only viremic pigs that were fed contaminated diets have developed PRRSV specific antibodies. These results suggest that feeding pigs with DON-contaminated diet could inhibit vaccination efficiency of PRRSV MLV by severely impairing viral replication.

Molekularbiologische Charakterisierung der Argonauten Proteine AgnA und AgnB innerhalb der RNA vermittelten Genregulation in D. discoideum

Argonauten Proteine übernehmen vielfältige Funktionen in RNA vermittelten Signalwegen zur Genregulation und sind in eukaryotischen Organismen hoch konserviert. Obwohl das Repertoire an kleinen regulatorischen RNAs in D. discoideum schon früh untersucht wurde und dabei sowohl siRNAs als auch miRNAs identifiziert werden konnten, war die Funktion der fünf kodierten Argonauten Proteine zu Beginn meiner Arbeit noch völlig unbekannt. Im Fokus meiner Untersuchung standen die zwei Homologe AgnA und AgnB. Die molekularbiologische Charakterisierung von AgnA hat gezeigt, dass das Protein eine essentielle Funktion bei der posttranskriptionellen Regulation des Retrotransposons DIRS-1 hat. AgnA wird für die Generierung von über 90 % der DIRS-1 siRNAs benötigt, wobei unklar ist, ob die Slicer-Aktivität des Proteins relevant ist oder ob AgnA andere Proteine zur Generierung der kleinen RNAs rekrutiert. Mit Hilfe der Deep Sequencing Analyse kleiner RNAs im AgnA KO konnte die Abreicherung der DIRS-1 siRNAs bestätigt werden. Die Anreicherung von DIRS-1 sense und antisense Transkripten weist deutlich auf eine Deregulation des Retrotransposons bei Abwesenheit von AgnA hin. Der Verlust der AgnA abhängigen Regulationsebene ist nicht nur auf RNA- sondern auch auf DNA-Ebene nachweisbar, da im AgnA Knockout einzelsträngige extrachromosomale DIRS-1 Intermediate nachweisbar sind. Die Analyse dieser Strukturen mit Hilfe von Rasterkraftmikroskopie zeigt, dass die extrachromosomale DNA mit Proteinen assoziiert ist. Das Erscheinungsbild legt die Vermutung nahe, dass es sich um Virus ähnliche Partikel handeln könnte. Die Transposition der DIRS-1 Elemente konnte nicht nachgewiesen werden. Sie schlägt vermutlich fehl, da der zur Integration notwendige DNA-Doppelstrang nicht gebildet wird. Auch wenn der genaue Mechanismus der AgnA abhängigen DIRS-1 Regulation nicht vollständig aufgeklärt werden konnte, weisen die Ergebnisse darauf hin, dass AgnA nicht nur an der Biogenese der kleinen DIRS-1 siRNAs beteiligt ist, sondern auch weiter downstream, vermutlich innerhalb von Effektorkomplexen, als Regulator aktiv ist. AgnB ist nicht an der negativen Regulation des DIRS-1 Retrotransposons beteiligt. Im Gegenteil haben Experimente gezeigt, dass das Protein die Transkription des Elementes und die Bildung von DNA-Intermediaten eher positiv beeinflusst. Im Fall des Retrotransposons Skipper ist unklar, ob die wenigen siRNAs, die identifiziert worden sind, tatsächlich für die Regulation dieses Elementes genutzt werden. Der Knockout von AgnA hat eine Anreicherung der Skipper siRNAs zur Folge, wobei diese sehr variabel ist. Es konnten Skipper Transkripte nachgewiesen werden (Hinas et al., 2007), die wahrscheinlich die Vorläufermoleküle der siRNAs darstellen. Die Menge dieser Transkripte unterscheidet sich allerdings im Wildtyp und den untersuchten Knockout-Stämmen nicht. Bei der Untersuchung der miRNAs zeigte sich eine signifikante Anreicherung dieser regulatorischen RNAs im AgnA Knockout. Die Akkumulation kann durch die Expression von rekombinantem AgnA wieder auf Wildtyp Niveau gebracht werden. Die genaue Funktion von AgnA im miRNA Signalweg konnte aber nicht näher spezifiziert werden. Im Fall der beiden miRNAs konnte im Rahmen dieser Arbeit nachgewiesen werden, dass sie keine 2‘-O Methylierung besitzen und fast ausschließlich im Cytoplasma der Zelle vorliegen. Letzteres weist darauf hin, dass die untersuchten miRNAs ihre Zielgene vermutlich posttranskriptionell regulieren. Die Akkumulation von miRNAs im AgnA KO konnte ebenfalls durch Deep Sequencing Analysen verifiziert werden. Weiterhin wurden tRNA Fragmente gefunden, die im AgnA KO wesentlich stärker vertreten sind. Northern Blot Analysen haben gezeigt, dass ein zusätzliches Fragment der tRNA Asp akkumuliert, wenn AgnA nicht exprimiert wird. Möglicherweise ist AgnA am Umsatz der tRNA beteiligt. Die biologische Funktion der tRNA Fragmente in D. discoideum ist jedoch bisher ungeklärt. Bei der Suche nach putativen Interaktionspartnern konnte im Fall von AgnA das Protein DDB_G0268914 mittels Massenspektrometrie als putativer Interaktionspartner identifiziert werden. Dieses Protein zeigt Homologien zu MOV10 aus H. sapiens, das ebenfalls mit Argonauten Proteinen interagiert (Hock et al., 2007) und die Replikation von Retroviren unterdrückt (Burdick et al., 2010). Die Interaktion zwischen AgnA und dem MOV10 Homolog konnte bisher nicht mit anderen Ansätzen bestätigt werden. Darüber hinaus bleibt zu klären, ob der putative Interaktionsparter ebenfalls an der Regulation des Retrotransposons DIRS-1 beteiligt ist.

Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission

The 3' untranslated regions (3'UTRs) of flaviviruses are reviewed and analyzed in relation to short sequences conserved as direct repeats (DRs). Previously, alignments of the 3'UTRs have been constructed for three of the four recognized flavivirus groups, namely mosquito-borne, tick-borne, and nonclassified flaviviruses (MBFV, TBFV, and NCFV, respectively). This revealed (1) six long repeat sequences (LRSs) in the 3'UTR and open-reading frame (ORF) of the TBFV, (2) duplication of the 3'UTR of the NCFV by intramolecular recombination, and (3) the possibility of a common origin for all DRs within the MBFV. We have now extended this analysis and review it in the context of all previous published analyses. This has been achieved by constructing a robust alignment between all flaviviruses using the published DRs and secondary RNA structures as "anchors" to reveal additional homologies along the 3'UTR. This approach identified nucleotide regions within the MBFV, NKV (no-known vector viruses), and NCFV 3'UTRs that are homologous to different LRSs in the TBFV 3'UTR and ORF. The analysis revealed that some of the DRs and secondary RNA structures described individually within each flavivirus group share common evolutionary origins. The 3'UTR of flaviviruses, and possibly the ORF, therefore probably evolved through multiple duplication of an RNA domain, homologous to the LRS previously identified only in the TBFV. The short DRs in all virus groups appear to represent the evolutionary remnants of these domains rather than resulting from new duplications. The relevance of these flavivirus DRs to evolution, diversity, 3'UTR enhancer function, and virus transmission is reviewed.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Investigation on Cacao swollen shoot virus (CSSV) pollen transmission through cross-pollination

DNA- and RNA-based polymerase chain reaction (PCR) systems were used with Cacao swollen shoot virus (CSSV) primers designed from conserved regions of the six published genomic sequences of CSSV to investigate whether the virus is transmissible from infected trees through cross-pollination to seeds and seedlings. Pollen was harvested from CSSV infected cocoa trees and used to cross-pollinate flowers of healthy cocoa trees (recipient parents) to generate enough cocoa seeds for the PCR screening. Adequate precautions were taken to avoid cross-contamination during duplicated DNA extractions and only PCR results accompanied by effective positive and negative controls were scored. Results from the PCR analyses showed that samples of cocoa pod husk, mesocarp and seed tissues (testa, cotyledon and embryo) from the cross-pollinations were PCR negative for CSSV DNA. Sequential DNA samples from new leaves of seedlings resulting from the cross-pollinated trees were consistently PCR negative for presence of portions of CSSV DNA for over 36 months after germination. A reverse transcription-PCR analysis performed on the seedlings showed negative results, indicating absence of functional CSSV RNA transcripts in the seedlings. None of the seedlings exhibited symptoms characteristic of the CSSV disease, and all infectivity tests on the seedlings were also negative. Following these results, the study concluded that although CSSV DNA was detected in pollen from CSSV infected trees, there was no evidence of pollen transmission of the virus through cross-pollination from infected cocoa parents to healthy cocoa trees. Keywords:badnavirus;CSSV;PCR;pollen;seed transmission;Theobroma cacao

Investigation on Cacao swollen shoot virus (CSSV) pollen transmission through cross-pollination

DNA- and RNA-based polymerase chain reaction (PCR) systems were used with Cacao swollen shoot virus (CSSV) primers designed from conserved regions of the six published genomic sequences of CSSV to investigate whether the virus is transmissible from infected trees through cross-pollination to seeds and seedlings. Pollen was harvested from CSSV infected cocoa trees and used to cross-pollinate flowers of healthy cocoa trees (recipient parents) to generate enough cocoa seeds for the PCR screening. Adequate precautions were taken to avoid cross-contamination during duplicated DNA extractions and only PCR results accompanied by effective positive and negative controls were scored. Results from the PCR analyses showed that samples of cocoa pod husk, mesocarp and seed tissues (testa, cotyledon and embryo) from the cross-pollinations were PCR negative for CSSV DNA. Sequential DNA samples from new leaves of seedlings resulting from the cross-pollinated trees were consistently PCR negative for presence of portions of CSSV DNA for over 36 months after germination. A reverse transcription-PCR analysis performed on the seedlings showed negative results, indicating absence of functional CSSV RNA transcripts in the seedlings. None of the seedlings exhibited symptoms characteristic of the CSSV disease, and all infectivity tests on the seedlings were also negative. Following these results, the study concluded that although CSSV DNA was detected in pollen from CSSV infected trees, there was no evidence of pollen transmission of the virus through cross-pollination from infected cocoa parents to healthy cocoa trees.

Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

The proteome of the infectious bronchitis virus Beau-R Virion

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus IBV. It was thought that coronavirus virions were composed of three major viral structural proteins, until investigations of other coronaviruses showed that coronavirus virions also include viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation and analysed by mass spectrometry proteomic. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus thirty-five additional virion-associated host proteins. Virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in SARS-CoV virions, and also virions from a number of other RNA and DNA viruses. Together these results demonstrate that coronaviruses have the capacity to incorporate a substantial variety of host protein, which may have implications for the disease process.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ~319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.

Bayesian coalescent analysis reveals a high rate of molecular evolution in GB virus C

GB virus C/hepatitis G (GBV-C) is an RNA virus of the family Flaviviridae. Despite replicating with an RNA-dependent RNA polymerase, some previous estimates of rates of evolutionary change in GBV-C suggest that it fixes mutations at the anomalously low rate of similar to 100(-7) nucleotide substitution per site, per year. However, these estimates were largely based on the assumption that GBV-C and its close relative GBV-A (New World monkey GB viruses) codiverged with their primate hosts over millions of years. Herein, we estimated the substitution rate of GBV-C using the largest set of dated GBV-C isolates compiled to date and a Bayesian coalescent approach that utilizes the year of sampling and so is independent of the assumption of codivergence. This revealed a rate of evolutionary change approximately four orders of magnitude higher than that estimated previously, in the range of 10(-2) to 10(-3) sub/site/year, and hence in line with those previously determined for RNA viruses in general and the Flaviviridae in particular. In addition, we tested the assumption of host-virus codivergence in GBV-A by performing a reconciliation analysis of host and virus phylogenies. Strikingly, we found no statistical evidence for host-virus codivergence in GBV-A, indicating that substitution rates in the GB viruses should not be estimated from host divergence times.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus

The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.

Sequence and Structural Analysis of the 5 ` Noncoding Region of Hepatitis C Virus in Patients With Chronic Infection

Hepatitis C virus (HCV), exhibits considerable genetic diversity, but presents a relatively well conserved 5 ` noncoding region (5 ` NCR) among all genotypes. In this study, the structural features and translational efficiency of the HCV 5 ` NCR sequences were analyzed using the programs RNAfold, RNAshapes and RNApdist and with a bicistronic dual luciferase expression system, respectively. RNA structure prediction software indicated that base substitutions will alter potentially the 5 ` NCR structure. The heterogeneous sequence observed on 5 ` NCR led to important changes in their translation efficiency in different cell culture lines. Interactions of the viral RNA with cellular transacting factors may vary according to the cell type and viral genome polymorphisms that may result in the translational efficiency observed. J. Med. Virol. 81: 1212-1219, 2009. (C) 2009 Wiley-Liss, Inc.

Influence of hepatitis G virus (GB virus C) on the prognosis of HIV-infected women

This study was undertaken to evaluate the prevalence of GB virus C (GBV-C) viraemia and anti-E2 antibody, and to assess the effect of co-infection with GBV-C and HIV during a 10-year follow-up of a cohort of 248 HIV-infected women. Laboratory variables (mean and median CD4 counts, and HIV and GBV-C viral loads) and clinical parameters were investigated. At baseline, 115 women had past exposure to GBV-C: 57 (23%) were GBV-C RNA positive and 58 (23%) were anti-E2 positive. There was no statistical difference between the groups (GBV-C RNA + /anti-E2 -, GBV-C RNA - /anti-E2 + and GBV-C RNA - /anti-E2 -) regarding baseline CD4 counts or HIV viral loads (P = 0.360 and 0.713, respectively). Relative risk of death for the GBV-C RNA + /anti-E2 - group was 63% lower than that for the GBV-C RNA - /anti-E2 - group. Multivariate analysis demonstrated that only HIV loads >= 100,000 copies/mL and AIDS-defining illness during follow-up were associated with shorter survival after AIDS development. It is likely that antiretroviral therapy (ART) use in our cohort blurred a putative protective effect related to the presence of GBV-C RNA.