EHDV-2 Infection Prevalence Varies in Culicoides sonorensis after Feeding on Infected White-Tailed Deer over the Course of Viremia.

Epizootic hemorrhagic disease viruses (EHDVs) are arboviral pathogens of white-tailed deer and other wild and domestic ruminants in North America. Transmitted by various species of Culicoides, EHDVs circulate wherever competent vectors and susceptible ruminant host populations co-exist. The impact of variation in the level and duration of EHDV viremia in white-tailed deer (Odocoileus virginianus) on Culicoides infection prevalence is not well characterized. Here we examined how infection prevalence in a confirmed North American vector of EHDV-2 (Culicoides sonorensis) varies in response to fluctuations in deer viremia. To accomplish this, five white-tailed deer were experimentally infected with EHDV-2 and colonized C. sonorensis were allowed to feed on deer at 3, 5, 7, 10, 12, 14, 18, and 24 days post infection (dpi). Viremia profiles in deer were determined by virus isolation and titration at the same time points. Blood-fed Culicoides were assayed for virus after a 10-day incubation (27 °C) period. We found that increases in deer EHDV blood titers significantly increased both the likelihood that midges would successfully acquire EHDV and the proportion of midges that reached the titer threshold for transmission competence. Unexpectedly, we identified four infected midge samples (three individuals and one pool) after feeding on one deer 18 and 24 dpi, when viremia was no longer detectable by virus isolation. The ability of ruminants with low-titer viremia to serve as a source of EHDV for blood-feeding Culicoides should be explored further to better understand its potential epidemiological significance.

Prognostic factors and outcome of Liposarcoma patients: a retrospective evaluation over 15 years.

BACKGROUND: Soft tissue sarcomas are rare entities with over 50 histological subtypes. Liposarcoma (LS) is the most common neoplasm in this group; it is a complex neoplasm that is divided into different histological subtypes. Different therapy options, such as surgical resection, radiation, and chemotherapy, are available. Depending on the subtype, location, status of the resection margins and metastatic status, different therapy options are used. Therefore, the aim of this study was to determine the prognostic factors influencing the survival of patients affected by LS with consideration for the grading, histological subtype, state of the resection margin, size, location, metastases and local recurrence in a retrospective, single-centre analysis over 15 years. METHODS: We included 133 patients (male/female = 67/66) in this study. We recorded the histologic subtype, grade, TNM classification, localization, biopsy technique, tumour margins, number of operations, complications, radiation and dose, chemotherapy, survival, recrudescence, metastases and follow-up. Survivorship analysis was performed. RESULTS: We detected 56 (43%; 95%-CI 34.6-51.6%) atypical LS cases, 21 (16.2%; 95%-CI 9.8-22.5) dedifferentiated LS cases, 40 (30.8%; 95%-CI 22.8-38.7) myxoid LS cases and 12 (9.2%; 95%-CI 4.3-14.2) pleomorphic LS cases. G1 was the most common grade, which was followed by G3. Negative margins (R0) were detected in 67 cases (53.6%; 95%-CI 44.9-62.3) after surgical resection. Local recurrence was detected in 23.6% of cases. The presence of metastases and dedifferentiated LS subtype as well as negative margins, grade and tumour size are significant prognostic factors of the survival rates (p < 0.015). CONCLUSION: Grading, LS subtype, negative margins after surgery, metastases and tumour size are independently associated with disease-specific survival, and patients with local recurrence had lower survival rates. We hope our investigation may facilitate a further prospective study and clinical decision-making in LS.

European Bluetongue Serotype 8: Disease Threat Assessment for U.S. Sheep.

Bluetongue virus (BTV) is an orbivirus transmitted by biting midges (Culicoides spp.) that can result in moderate to high morbidity and mortality primarily in sheep and white-tailed deer. Although only 5 serotypes of BTV are considered endemic to the United States, as many as 11 incursive serotypes have been detected in livestock and wildlife in the past 16 years. Introductions of serotypes, with unknown virulence and disease risk, are constant threats to US agriculture. One potential incursive serotype of particular concern is the European strain of BTV-8, which was introduced into Northern Europe in 2006 and caused unprecedented livestock disease and mortality during the 2006-2007 vector seasons. To assess disease risk of BTV-8 in a common white-faced American sheep breed, eight Polled Dorset yearlings were experimentally infected and monitored for clinical signs. Viremia and viral tissue distribution were detected and quantified by real-time qRT-PCR. Overall, clinical disease was moderate with no mortality. Viremia reached as high as 9.7 log10 particles/mL and persisted at 5 logs or higher through the end of the study (28 days). Virus distribution in tissues was extensive with the highest mean titers at the peak of viremia (day 8) in the kidney (8.38 log10 particles/mg) and pancreas (8.37 log10 particles/mg). Virus persisted in tissues of some sheep at 8 logs or higher by day 28. Results of this study suggest that should BTV-8 emerge in the United States, clinical disease in this common sheep breed would likely be similar in form, duration, and severity to what is typically observed in severe outbreaks of endemic serotypes, not the extraordinary disease levels seen in Northern Europe. In addition, a majority of exposed sheep would be expected to survive and act as significant BTV-8 reservoirs with high titer viremias for subsequent transmission to other livestock and wildlife populations.

Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro.

A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infected with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) accumulated hsp70 transcripts throughout the 72-hour course of infection in the midgut, hemocytes, and fat body. While a maximal 17- or 15-fold induction of hsp70 was noted in the midgut and hemocytes, respectively, by 72 hours postinfection, the level of hsp70 transcription in the fat body of larvae was greater than two orders of magnitude higher than in mock-infected larvae. These results were largely mirrored in cultures of infected cells, and a potentiation effect was observed in cells that were both heat shocked and infected. In contrast, Spodoptera frugiperda multiple nucleopolyhedrovirus and ultraviolet-inactivated HzSNPV did not stimulate hsp70 transcription in these non-permissive larvae and in cell culture, respectively. Taken together, this report documents baculovirus-mediated upregulation of hsp70 in the host and demonstrates the requirement for productive infection for hsp70 induction in vitro and in vivo.

Determination and analysis of the genome sequence of Spodoptera littoralis multiple nucleopolyhedrovirus.

The Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV), a pathogen of the Egyptian cotton leaf worm S. littoralis, was subjected to sequencing of its entire DNA genome and bioassay analysis comparing its virulence to that of other baculoviruses. The annotated SpliMNPV genome of 137,998 bp was found to harbor 132 open reading frames and 15 homologous repeat regions. Four unique genes not present in SpltMNPV were identified, as were 14 genes that were absent or translocated by comparison. Bioassay analysis of experimentally infected Spodoptera frugiperda revealed an extended killing time for SpliMNPV as compared to S. frugiperda MNPV (SfMNPV), but a level of mortality similar to that caused by infection with SfMNPV and superior to that of Autographa californica MNPV (AcMNPV). Although extensive similarity was observed between the genome structure and predicted translation products of SpliMNPV and Spodoptera litura MNPV (SpltMNPV), genetic distances between isolates of SpliMNPV and SpltMNPV suggest that they are in fact different species of genus Alphabaculovirus.

Gut Transcription in Helicoverpa zea is Dynamically Altered in Response to Baculovirus Infection.

The Helicoverpa zea transcriptome was analyzed 24 h after H. zea larvae fed on artificial diet laced with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Significant differential regulation of 1,139 putative genes (p < 0.05 T-test with Benjamini and Hochberg False Discovery Rate) was detected in the gut epithelial tissue; where 63% of these genes were down-regulated and 37% of genes were up-regulated compared to the mock-infected control. Genes that play important roles in digestive physiology were noted as being generally down-regulated. Among these were aminopeptidases, trypsin-like serine proteases, lipases, esterases and serine proteases. Genes related to the immune response reacted in a complex nature having peptidoglycan binding and viral antigen recognition proteins and antiviral pathway systems down-regulated, whereas antimicrobial peptides and prophenoloxidase were up-regulated. In general, detoxification genes, specifically cytochrome P450 and glutathione S-transferase were down-regulated as a result of infection. This report offers the first comparative transcriptomic study of H. zea compared to HzSNPV infected H. zea and provides further groundwork that will lead to a larger understanding of transcriptional perturbations associated with viral infection and the host response to the viral insult in what is likely the most heavily infected tissue in the insect.

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates.

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.

Baculovirus induced transcripts in hemocytes from the larvae of Heliothis virescens.

Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae.

The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters.

Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 (EICP22) and UL5 (EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4hours post-infection, was localized throughout the nucleus and cytoplasm, and was not present in purified virions. EHV-1 lacking UL4P expression was infectious and displayed cell tropism and pathogenic properties in the mouse model similar to those of parental and revertant viruses. Reporter assays demonstrated that the UL4P has a broad inhibitory function, suggesting a potential role in establishing and/or maintaining DIP-mediated persistent infection.

The IR4 auxiliary regulatory protein expands the in vitro host range of equine herpesvirus 1 and is essential for pathogenesis in the murine model.

IR4, an early regulatory protein of equine herpesvirus 1 (EHV-1), is not a DNA-binding protein, but interacts with the sole immediate-early protein (IEP) to increase both IEP site-specific DNA-binding and IEP-mediated trans-activation of EHV-1 promoters. To investigate the biological properties of IR4 and ascertain whether this regulatory protein is essential for virus growth, bacterial artificial chromosome methods were employed to generate an IR4-null EHV-1. The IR4 gene was dispensable for EHV-1 growth in non-immortalized equine NBL-6 cells, but virus replication was delayed and was reduced by greater than 10-fold. In addition, replication of the IR4 mutant was abrogated in all other cell types tested, including equine ETCC tumor cells and cells of mouse, rabbit, monkey, and human origin. Further, in contrast to the highly pathogenic parent virus, the IR4 deletion mutant failed to cause disease in the CBA mouse as judged by assessing body weight and clinical signs and was unable to replicate in the murine lung. To define the nature of the block in the replication of the IR4-null virus, molecular analyses were carried out in RK-13 rabbits' cells infected with the IR4-deleted virus and revealed that: 1) the synthesis of the sole IEP was not inhibited; 2) the synthesis of early viral proteins examined was either not affected or was delayed to late times; 3) viral DNA replication was inhibited by more than 99.9%; and 4) synthesis of essential late proteins such as glycoprotein D and glycoprotein K was prevented. These findings indicate that the IR4 protein is required for EHV-1 DNA replication in non-permissive cells, and, like its homologues in other alphaherpesviruses, contributes a function required for virus replication in a variety of cell types.

The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein.

The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed.