Inter-laboratory evaluation of the performance parameters of a Lateral Flow Test device for the detection of Bluetongue virus-specific antibodies.

Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities.

Alphaherpesvirus use and misuse of cellular actin and cholesterol.

Two major structural elements of a cell are the cytoskeleton and the lipid membranes. Actin and cholesterol are key components of the cytoskeleton and membranes, respectively, and are involved in a plethora of different cellular processes. This review summarizes and discusses the interaction of alphaherpesviruses with actin and cholesterol during different stages of the replication cycle: virus entry, replication and assembly in the nucleus, and virus egress. Elucidating these interactions not only yields novel insights into the biology of these important pathogens, but may also shed new light on cell biological aspects of actin and cholesterol, and lead to novel avenues in the design of antiviral strategies.

Pseudorabies virus US3-mediated inhibition of apoptosis does not affect infectious virus production.

Preventing apoptosis during the early stages of infection of a host cell is generally thought to result in a higher yield of progeny virus. The US3 protein kinase of pseudorabies virus (PRV) and herpes simplex virus (HSV) is able to protect infected cells from apoptosis, which may be one of the reasons why both US3null PRV and US3null HSV replicate to lower virus titres in several cell types. However, such potential correlation between the higher amount of apoptosis in US3null virus-infected cells and the lower virus titres of US3null virus has not been investigated directly. In the current study, we found that a broad-spectrum caspase-inhibitor efficiently inhibited apoptosis in swine testicle and human laryngeal epidermoid carcinoma cells infected with US3null or wild-type (WT) PRV. However, inhibition of apoptosis did not affect US3null or WT PRV extracellular or cell-associated virus titres, nor did it restore the small plaque phenotype of US3null PRV.

Pseudorabies virus US3- and UL49.5-dependent and -independent downregulation of MHC I cell surface expression in different cell types.

Many herpesviruses interfere with the MHC I antigen-processing pathway in order to limit elimination by cytotoxic T-lymphocytes. For varicelloviruses, the largest subgroup of alphaherpesviruses, two viral proteins have been reported to downregulate MHC I cell surface expression: UL49.5 for BoHV-1, PRV, and EHV-1 and the US3 orthologue for VZV. Here, we report that PRV reduces MHC I cell surface expression during infection in a cell-type-dependent manner. In ST cells, a kinase-active US3 was necessary but not sufficient to downregulate cell surface MHC I expression, whereas US3 was not required in PK-15 cells and porcine alveolar macrophages (PAM). MHC I downregulation was not (PAM, ST) or only partly (PK-15) dependent on UL49.5. In conclusion, we show that the mechanism(s) of PRV-mediated cell surface MHC I downregulation are cell-type-dependent, with variable roles for US3, UL49.5, and additional, yet unidentified early viral proteins.

Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases.

The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1(-/-) and PAK2(-/-) mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs.

The kinase activity of pseudorabies virus US3 is required for modulation of the actin cytoskeleton.

Different viruses exploit the host cytoskeleton to facilitate replication and spread. The conserved US3 protein of the alphaherpesvirus pseudorabies virus induces actin stress fiber disassembly and formation of actin-containing cell projections, which are associated with enhanced intercellular virus spread. Proteins of members of other virus families, notably vaccinia virus F11L protein and human immunodeficiency virus Nef protein, induce actin rearrangements that are very similar to those induced by US3. Interestingly, unlike F11L and Nef, the US3 protein displays serine/threonine kinase activity. Here, we report that the kinase activity of pseudorabies virus US3 is absolutely required for its actin modulating activity. These data show that different viruses have developed independent mechanisms to induce very similar actin rearrangements.

A point mutation in the putative ATP binding site of the pseudorabies virus US3 protein kinase prevents Bad phosphorylation and cell survival following apoptosis induction.

The multifunctional US3 protein kinase is conserved among alphaherpesviruses. Like the herpes simplex virus US3 protein kinase, the pseudorabies virus (PRV) US3 protein confers resistance against apoptosis. In the current report, we introduced a point mutation in the putative ATP binding site of the PRV US3 protein kinase. We found that, in contrast to the wild type PRV US3, the point-mutated PRV US3 does not protect cells from apoptosis induced by PRV infection or staurosporine treatment. In addition, we found that the presence of wild type PRV US3, but not of the point-mutated PRV US3, results in phosphorylation of the pro-apoptotic Bad protein in PRV-infected ST and HEp-2 cells. In PRV-infected ST cells, but not in HEp-2 cells, an additional, US3- and phosphorylation-independent alteration of Bad could be observed. These results suggest that the kinase activity of the US3 protein of PRV is crucial to protect cells from apoptotic cell death during infection, at least partly by leading to phosphorylation of the pro-apoptotic Bad protein.