BSR Cells Research Articles

Immunogenicity evaluation of a bivalent vaccine based on a recombinant rabies virus expressing gB protein of FHV-1 in mice and cats

Feline viral rhinotracheitis (FVR) is caused by the feline herpesvirus-1 (FHV-1), which commonly results in upper respiratory symptoms, and can result in death in the kittens and weak cats. Rabies is an infectious disease with zoonotic characteristics highly relevant to public health and also poses a serious threat to cats. Vaccines are the most effective method to control the spread of both FHV-1 and RABV and have the advantage that they produce long-term specific immune responses. In this study, we constructed a bivalent vaccine against FHV-1 and rabies virus (RABV) simultaneously. The vaccine was constructed by cloning FHV-1 gB into a RABV based vector, and the recombinant RABV (SRV9-FHV-gB) expressing the FHV-1 gB protein was rescued. The growth characteristics of SRV9-FHV-gB were analyzed on NA and BSR cells. To assess the immunogenicity of the vaccine, mice and cats were immunized with SRV9-FHV-gB supplemented with Gel02 adjuvant.The SRV9-FHV-gB exhibited the same growth characteristics as the parent virus SRV9 in both BSR cells and NA cells. The safety of SRV9-FHV-gB was evaluated using 5-day-old and 14-day-old suckling mice. The results showed that mice infected with the SRV9-FHV-gB survived for longer than those in the SRV9 group. Mice immunized with inactivated SRV9-FHV-gB produced high titers of specific antibodies against FHV-1 and neutralizing antibodies against RABV. Cats that received three immunizations with SRV9-FHV-gB also produced neutralizing antibodies against both FHV-1 and RABV. This study represents the first time that a bivalent vaccine targeting FHV-1 and RABV has been constructed, laying the foundations and providing inspiration for the development of other multivalent vaccines.

Purification of Epizootic Hemorrhagic Disease Virus (and Other Orbiviruses) Particles from Infected Mammalian or Insect Cells.

Epizootic hemorrhagic disease virus (EHDV), like other orbiviruses, infects and replicates in mammalian and insect vector cells. Within its ruminant hosts EHDV, like bluetongue virus (BTV), it has mainly been associated with infection of endothelial cells of capillaries as well as leukocyte subsets. Furthermore, EHDV infects and replicates within its biological vector, Culicoides biting midges and Culicoides-derived cells. A wide range of common laboratory cell lines such as BHK, BSR, and Vero cells are susceptible to infection with certain EHDV strains. Cell culture supernatants of infected cells are commonly used for both in vivo and in vitro infection studies. For specific virological or immunological studies, using highly purified virus particles, however, might be beneficial or even required. Here we describe a purification method for EHDV particles, which had been originally developed for certain strains of BTV.

Formulation and Biomedical Activity of Oil-in-Water Nanoemulsion Combining Tinospora smilacina Water Extract and Calophyllum inophyllum Seeds Oil.

Tinospora smilacina is a native plant used in traditional medicine by First Nations peoples in Australia to treat inflammation. In our previous study, an optimised Calophyllum inophyllum seed oil (CSO) nanoemulsion (NE) showed improved biomedical activities such as antimicrobial, antioxidant activity, cell viability and in vitro wound healing efficacy compared to CSO. In this study, a stable NE formulation combining T. smilacina water extract (TSWE) and CSO in a nanoemulsion (CTNE) was prepared to integrate the bioactive compounds in both native plants and improve wound healing efficacy. D-optimal mixture design was used to optimise the physicochemical characteristics of the CTNE, including droplet size and polydispersity index (PDI). Cell viability and in vitro wound healing studies were done in the presence of CTNE, TSWE and CSO against a clone of baby hamster kidney fibroblasts (BHK-21 cell clone BSR-T7/5). The optimised CTNE had a 24 ± 5 nm particle size and 0.21± 0.02 PDI value and was stable after four weeks each at 4 °C and room temperature. According to the results, incorporating TSWE into CTNE improved its antioxidant activity, cell viability, and ability to promote wound healing. The study also revealed that TSWE has >6% higher antioxidant activity than CSO. While CTNE did not significantly impact mammalian cell viability, it exhibited wound-healing properties in the BSR cell line during in vitro testing. These findings suggest that adding TSWE may enhance CTNE's potential as a wound-healing treatment. This is the first study demonstrating NE formulation in which two different plant extracts were used in the aqueous and oil phases with improved biomedical activities.

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.

Efficient Production of Evolutionary Nanoparticle Newcastle Disease Virus

Viruses are routinely isolated from infected cells through freeze–thaw (F–T) cycles or sonication. The aim of this study was to compare different methods for efficient isolation of Newcastle disease virus (NDV) particles from BSR-T7/5 (BSR) cells. The BSR cells were infected with NDV LaSota strain, and the virus particles were isolated via F–T, sonication, sonication followed by F–T, and F–T followed by sonication. The infection and proliferation kinetics of the virus were analyzed by cytopathic observation and monitoring of hemagglutination (HA) titers. The virus isolated by sequential F–T and sonication was amplified through five passages of BSR cells, and then used to infect the HepG2 cells. The viability and apoptosis rates of the infected cells were evaluated by Cell Counting Kit-8 assay and Annexin V-FITC/PI staining respectively. We successfully obtained NDV particles from persistently infected BSR cells through all four methods, which indicated that the LaSota can effectively replicate in BSR cells. However, F–T followed by sonication was optimum in terms of separation effect. The virus particles isolated by this method still exhibited cytolytic activity against HepG2 cells. Thus, our novel method can be applied to NDV production and generation of tumor vaccines.

A Novel and Secure Pseudovirus Reporter System Based Assay for Neutralizing and Enhancing Antibody Assay Against Marburg Virus.

Marburg virus (MARV) is one of the principal members of the filovirus family, which can cause fatal hemorrhagic fever in humans. There are currently no prophylactic and therapeutic drugs on the market, and the high pathogenicity and infectivity of MARV make its research highly dependent on biosafety level 4 conditions, severely hindering the development of vaccines and therapies. Therefore, the development of medicines, such as MARV serological diagnosis, vaccines, and therapeutic antibody drugs, urgently needs a safe, convenient, and biosafety level 2 detection method to measure the neutralizing activity of MARV antibodies. To this end, we report a neutralization assay relying on a Rabies virus (RABV) reverse genetic operating system. We constructed infectious clones carrying the eGFP reporter gene and the full length of the original unmodified MARV GP gene. Based on the critical parameters of phylogenetic analysis, recombinant viruses targeting representative strains in the two major MARV lineages were successfully rescued. These pseudoviruses are safe in mice, and their inability to infect cells after being neutralized by antibodies can be visualized under a fluorescence microscope. We tested the system using the neutralizing antibody MR191. MR191 can significantly block the infection of BSR cells with pseudovirus. We compared it with the traditional lentivirus-type pseudovirus system to verify the system’s credibility and obtained the same results as reported in the literature. In general, we have established a safe and visualized method for evaluating the neutralizing activity of MARV antibodies. Compared with traditional methods, it has the advantages of convenient operation, short cycle, and low cost. It is a candidate method that can replace actual viruses for a neutralization assay.

A new cluster of West Nile virus lineage 1 identified in Spain

<h3>Purpose</h3> West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. In Spain, its active circulation provokes outbreaks in birds, horses and lately in humans. In September 2017, an outbreak affecting raptors, in the province of Málaga (Andalusia), was investigated. One northern goshawk (<i>Accipiter gentilis</i>) died, while two peregrine falcons (<i>Falco peregrinus</i>) recovered. Here we report the isolation and characterization of a new WNV lineage 1 variant from this outbreak. <h3>Methods & Materials</h3> Heart, feathers, brain and kidney samples from the goshawk were collected and firstly analysed by real-time RT-PCR for WNV (Jiménez-Clavero <i>et al</i>., 2006, https://doi.org/doi:10.1177/104063870601800505). Virus isolation was performed in BSR and Vero cells. The whole genome of the isolate was sequenced by Sanger method. Phylogenetic analyses were performed including a wide range of representatives of: 1) all described WNV lineages, and 2) the different clusters of WNV lineage 1. <h3>Results</h3> All samples from the goshawk were positive for WNV lineage 1 by RT-PCR. Virus isolation was successful in heart and brain samples. A full genome sequence of 10,919 nt was obtained for the WNV isolated from the brain (named Spain/2017/NG-b). Phylogenetic analyses confirmed that Spain/2017/NG-b belonged to WNV clade 1a. Surprisingly, this isolate was placed neither in the cluster 2 of this clade, which comprises most of the recent European and Mediterranean strains (including the preceding Spanish ones), nor in other closest clusters. When analysing the polyprotein, amino acid substitutions that define WNV clusters 1-4 were not found in Spain/2017/NG-b isolate (May <i>et al.</i>, 2011, https://doi.org/10.1128/jvi.01963-10). Moreover, it presented 12 unique residues compared to cluster 2 viruses. <h3>Conclusion</h3> These results strongly support that Spain/2017/NG-b represents a new, independent introduction of WNV into the Western Mediterranean since it showed no close relationship to other strains from this region. Therefore, we propose to associate this variant to a new cluster 7 within WNV clade 1a. Finally, a WNV outbreak occurred in equids in December 2017, nearby the raptors´ outbreak above mentioned, suggests the ability of this new variant to infect and cause disease also in horses.

Extended characterisation of five archival tick-borne viruses provides insights for virus discovery in Australian ticks

BackgroundA subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks.MethodsWe performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry.ResultsOur sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus.ConclusionsGenetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.Graphical

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.

Evaluation of Multiplicity of Infection (MOI) and Harvesting Time on the Production of CVS-11 Strain of Rabies Virus in BSR Cell Line

Evaluation of Multiplicity of Infection (MOI) and Harvesting Time on the Production of CVS-11 Strain of Rabies Virus in BSR Cell Line

Immune responses in mice and pigs after oral vaccination with rabies virus vectored Nipah disease vaccines

Nipah virus (NiV) is a re-emerging zoonotic pathogen that causes high mortality in humans and pigs. Oral immunization in free-roaming animals is one of the most practical approaches to prevent NiV pandemics. We previously generated a recombinant rabies viruses (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, rERAG333E, which contains a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) and serves as an oral vaccine for dog rabies. In this study, we generated two recombinant RABVs, rERAG333E/NiVG and rERAG333E/NiVF, expressing the NiV Malaysian strain attachment glycoprotein (NiV-G) or fusion glycoprotein (NiV-F) gene based on the rERAG333E vector platform. Both rERAG333E/NiVG and rERAG333E/NiVF displayed growth properties similar to those of rERAG333E and caused marked syncytia formation after co-infection in BSR cell culture. Adult and suckling mice intracerebrally inoculated with the recombinant RABVs showed NiV-G and NiV-F expression did not increase the virulence of rERAG333E. Oral vaccination with rERAG333E/NiVG either singularly or combined with rERAG333E/NiVF induced significant NiV neutralizing antibody against NiV and RABV, and IgG to NiV-G or NiV-F in mice and pigs. rERAG333E/NiVG and rERAG333E/NiVF thus appeared to be suitable candidates for further oral vaccines for potential animal targets in endemic areas of NiV disease and rabies.

PCR-based reverse genetics strategy for bluetongue virus recovery

BackgroundBluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription.ResultIn this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others.ConclusionIn this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.

Cholesterol 25-hydroxylase suppresses rabies virus infection by inhibiting viral entry.

Cholesterol-25-hydroxylase (CH25H) is a reticulum-associated membrane protein that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC). Recent studies have revealed that CH25H is an interferon-stimulated gene (ISG) that suppresses infection by several viruses. In the present study, we found that overexpression of both human and murine CH25H inhibited rabies virus (RABV) infection in HEK-293T (293T) cells. In contrast, silencing of CH25H enhanced RABV replication in 293T cells, and a catalytic mutant of CH25H lost its ability to inhibit RABV infection. Treatment with the oxysterol 25-hydroxycholesterol (25HC), the product of CH25H, dramatically decreased RABV replication in 293T, BSR and N2a cells by inhibiting viral membrane penetration. These data provide insights into the antiviral function of CH25H against RABV infection, which can potentially be used as a therapeutic agent for rabies.

Dermaseptins as potential antirabies compounds

Over the last 20 years, natural peptides playing a key role in defense mechanisms and innate immunity have been isolated from unicellular organisms. Amphibian skin secretes dermaseptins, 24–34 amino acids in length that have a wide antimicrobial spectrum incorporating yeast, fungi, protozoa, bacteria and enveloped viruses. The anti-rabies virus (RABV) activity of dermaseptins S3 (30aa) and S4 (28aa) from Phyllomedusa sauvagei has been investigated, and further dissected its molecular basis by comparing punctual mutation or deletion of S4 analogues. The results showed that: (1) S4 is more active than S3 against RABV infection, 89% versus 38% inhibition at 7.5 μM; (2) the 5 NH2-aa of S4 are crucial for its inhibitory potential (S46–28 lost any inhibition) but the COOH terminus stabilizes the inhibitory potential (S41–16 showed only 23% inhibition at 7.5 μM); (3) there is a correlation between viral inhibition and dermaseptin cytotoxicity, which remains however moderated for BSR cells (≤12% at 10 μM). A single mutation in position 4 (S4M4K) slightly reduced cytotoxicity while keeping its antiviral activity, 97% at 7.5 μM. S4 and S4M4K showed an antiviral activity in vitro when provided 1 h after infection. In vivo experiments in mice by intramuscular injection of non-toxic doses of dermaseptin S4M4K 1 h post-infection by a lethal dose of RABV at the same site allowed more than 50% improvement in mice survival. This study highlights the potential interest of dermaseptins as non-expansive alternatives to rabies immunoglobulins for the treatment of rabies that continues to claim about 60,000 human lives per year worldwide, almost exclusively in developing countries.

Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity.

ABSTRACTAmong the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus. BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

Genetically modified rabies virus-vectored Ebola virus disease vaccines are safe and induce efficacious immune responses in mice and dogs

Ebola viruses (EBOVs) are zoonotic pathogens that cause EBOV disease (EVD) with high case fatality in humans. Currently, EVD vaccines are still under development in several countries. Here, we generated two recombinant rabies viruses (RABVs), rERAG333E/ZGP and rERAG333E/SGP, expressing the Zaire EBOV glycoprotein (ZGP) or Sudan EBOV glycoprotein (SGP) gene based on a modified ERA vaccine strain (rERAG333E) vector platform. The recombinant RABVs retained growth properties similar to those of the vector virus in BSR cell culture and efficiently expressed ZGP or SGP. After intracerebral (i.c.) inoculation with rERAG333E/ZGP or rERAG333E/SGP, all adult mice showed no signs of disease or weight loss and suckling mice maintained similar survivorship curve as those mice inoculated with control vector rERAG333E, demonstrating that ZGP or SGP expression did not increase the virulence of the vector. Mouse immunization studies showed that vaccination with rERAG333E/ZGP and rERAG333E/SGP induced Zaire or Sudan EBOV neutralizing antibody (VNA) responses and IgG, IgG2a responses to ZGP or SGP, suggesting their potential as oral or inactivated bivalent vaccines against rabies and EVD. Most importantly, all dogs immunized orally with rERAG333E/ZGP developed long-lasting ZEBOV and RABV VNA responses with or without previous rabies vaccine immunization history. Live rERAG333E with EBOV GP thus appear to have the potential to be oral vaccines for free-roaming animals in endemic areas of EVD and rabies, and may serve as inactivated vaccines for use in humans.

Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus

In an effort to simplify and expand the utility of African horse sickness virus (AHSV) reverse genetics, different plasmid-based reverse genetics systems were developed. Plasmids containing cDNAs corresponding to each of the full-length double-stranded RNA genome segments of AHSV-4 under control of a T7 RNA polymerase promoter were co-transfected in cells expressing T7 RNA polymerase, and infectious AHSV-4 was recovered. This reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. Subsequently, a T7 RNA polymerase expression cassette was incorporated into one of the AHSV-4 rescue plasmids. This modified 5-plasmid set enabled virus recovery in BSR or L929 cells, thus offering the possibility to generate AHSV-4 in any cell line. Moreover, mutant and cross-serotype reassortant viruses were recovered. These plasmid DNA-based reverse genetics systems thus offer new possibilities for investigating AHSV biology and development of designer AHSV vaccine strains.

African horse sickness virus infects BSR cells through macropinocytosis

Cellular pathways involved in cell entry by African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, have not yet been determined. Here, we show that acidic pH is required for productive infection of BSR cells by AHSV-4, suggesting that the virus is likely internalized by an endocytic pathway. We subsequently analyzed the major endocytic routes using specific inhibitors and determined the consequences for AHSV-4 entry into BSR cells. The results indicated that virus entry is dynamin dependent, but clathrin- and lipid raft/caveolae-mediated endocytic pathways were not used by AHSV-4 to enter and infect BSR cells. Instead, binding of AHSV-4 to BSR cells stimulated uptake of a macropinocytosis-specific cargo and inhibition of Na+/H+ exchangers, actin polymerization and cellular GTPases and kinases involved in macropinocytosis significantly inhibited AHSV-4 infection. Altogether, the data suggest that AHSV-4 infects BSR cells by utilizing macropinocytosis as the primary entry pathway.

Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4.

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4.

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells).

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional ‘types’ have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.