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Porcine reproductive and respiratory syndrome viruses (PRRSV-1 and -2) are the causative agents of one of the most important infectious diseases affecting the global pig industry. Previous studies, largely focused on PRRSV-2, have shown that non-structural protein-1α (NSP1α) and NSP1β modulate host cell responses; however, the underlying molecular mechanisms remain to be fully elucidated. Therefore, we aimed to identify novel PRRSV-1 NSP1–host protein interactions to improve our knowledge of NSP1-mediated immunomodulation. NSP1α and NSP1β from a representative western European PRRSV-1 subtype 1 field strain (215-06) were used to screen a cDNA library generated from porcine alveolar macrophages (PAMs), the primary target cell of PRRSV, using the yeast-2-hybrid system. This identified 60 putative binding partners for NSP1α and 115 putative binding partners for NSP1β. Of those taken forward for further investigation, 3 interactions with NSP1α and 27 with NSP1β were confirmed. These proteins are involved in the immune response, ubiquitination, nuclear transport, or protein expression. Increasing the stringency of the system revealed NSP1α interacts more strongly with PIAS1 than PIAS2, whereas NSP1β interacts more weakly with TAB3 and CPSF4. Our study has increased our knowledge of the PRRSV-1 NSP1α and NSP1β interactomes, further investigation of which could provide detailed insight into PRRSV immunomodulation and aid vaccine development.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) causes huge economic losses to the European pig industry. PRRSV-1 is divided into 3 subtypes and exhibits considerable antigenic heterogeneity. Due to its high mutation rate, PRRSV-1 is constantly evolving, and highly virulent, particularly subtype 3 strains, are continually emerging. The mechanism(s) underlying PRRSV-1 virulence have not been fully elucidated. In vivo studies have implicated replication kinetics, cell tropism and an enhanced pro-inflammatory cytokine response as potential contributing factors. However, few strains have been directly compared and differences in in vivo study design have hindered comparison, thus limiting our understanding of PRRSV-1 virulence. To address this knowledge gap, we sought to develop a reverse genetics and ex vivo model system, to attempt to identify correlates of PRRSV-1 virulence and attenuation in vitro. Herein we describe the use of primary porcine bone marrow-derived macrophages (BMDM) to investigate the growth kinetics and induced cytokine profiles of the highly virulent SU1-Bel strain, the low virulence 215-06 strain and the attenuated Olot/91 strain. We show that infection of BMDM with virulent PRRSV-1 strains induced higher expression of IL-6 and IL-8 and lower expression of TNF-α when compared with the attenuated strain. In addition, BMDM infected with SU1-Bel secreted significantly more IFN-α than those infected with PRRSV-1 strains of lower virulence. Interestingly, despite inducing less IFN-α than SU1-Bel, Olot/91 induced much higher levels of expression of several interferon-stimulated genes (ISGs), suggesting that Olot/91 may be less able to counteract type I IFN signaling which may contribute to its attenuated phenotype.

Foot-and-mouth disease is an economically devastating disease of livestock caused by foot-and-mouth disease virus (FMDV). Vaccination is the most effective control measure in place to limit the spread of the disease; however, the success of vaccination campaigns is hampered by the antigenic diversity of FMDV and the rapid rate at which new strains emerge that escape pre-existing immunity. FMDV has seven distinct serotypes, and within each serotype are multiple strains that often induce little cross-protective immunity. The diversity of FMDV is a consequence of the high error rate of the RNA-dependent RNA polymerase, accompanied by extensive recombination between genomes during co-infection. Since multiple serotypes and strains co-circulate in regions where FMDV is endemic, co-infection is common, providing the conditions for recombination, and also for other events such as trans-encapsidation in which the genome of one virus is packaged into the capsid of the co-infecting virus. Here, we demonstrate that the co-infection of cells with two FMDVs of different serotypes results in trans-encapsidation of both viral genomes. Crucially, this facilitates the infection of new cells in the presence of neutralizing antibodies that recognize the capsid that is encoded by the packaged genome.

Foot-and-mouth disease (FMD) is endemic in large parts of sub-Saharan Africa, Asia and South America, where outbreaks in cloven-hooved livestock threaten food security and have severe economic impacts. Vaccination in endemic regions remains the most effective control strategy. Current FMD vaccines are produced from chemically inactivated foot-and-mouth disease virus (FMDV) grown in suspension cultures of baby hamster kidney 21 cells (BHK-21). Strain diversity means vaccines produced from one subtype may not fully protect against circulating disparate subtypes, necessitating the development of new vaccine strains that “antigenically match”. However, some viruses have proven difficult to adapt to cell culture, slowing the manufacturing process, reducing vaccine yield and limiting the availability of effective vaccines, as well as potentiating the selection of undesired antigenic changes. To circumvent the need to cell culture adapt FMDV, we have used a systematic approach to develop recombinant suspension BHK-21 that stably express the key FMDV receptor integrin αvβ6. We show that αvβ6 expression is retained at consistently high levels as a mixed cell population and as a clonal cell line. Following exposure to field strains of FMDV, these recombinant BHK-21 facilitated higher virus yields compared to both parental and control BHK-21, whilst demonstrating comparable growth kinetics. The presented data supports the application of these recombinant αvβ6-expressing BHK-21 in future FMD vaccine production.

Abstract Childhood onset clinical syndromes involving intellectual disability and dysmorphic features, such as polydactyly, suggest common developmental pathways link seemingly unrelated phenotypes. We identified a consanguineous family of Saudi origin with varying complex features including intellectual disability, speech delay, facial dysmorphism and polydactyly. Combining, microarray based comparative genomic hybridisation (CGH) to identify regions of homozygosity, with exome sequencing, led to the identification of homozygous mutations in five candidate genes (RSPH6A, ANKK1, AMOTL1, ALKBH8, TRAPPC6A), all of which appear to be pathogenic as predicted by Proven, SIFT and PolyPhen2 and segregate perfectly with the disease phenotype. We therefore looked for differences in expression levels of each protein in HEK293 cells, expressing either the wild-type or mutant full-length cDNA construct. Unexpectedly, wild-type TRAPPC6A appeared to be unstable, but addition of the proteasome inhibitor MG132 stabilised its expression. Mutations have previously been reported in several members of the TRAPP complex of proteins, including TRAPPC2, TRAPPC9 and TRAPPC11, resulting in disorders involving skeletal abnormalities, intellectual disability, speech impairment and developmental delay. TRAPPC6A joins a growing list of proteins belonging to the TRAPP complex, implicated in clinical syndromes with neurodevelopmental abnormalities.

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is an economically devastating disease affecting several important livestock species. FMDV is antigenically diverse and exists as seven serotypes comprised of many strains which are poorly cross-neutralised by antibodies induced by infection or vaccination. Co-infection and recombination are important drivers of antigenic diversity, especially in regions where several serotypes co-circulate at high prevalence, and therefore experimental systems to study these events in vitro would be beneficial. Here we have utilised recombinant FMDVs containing an HA or a FLAG epitope tag within the VP1 capsid protein to investigate the products of co-infection in vitro. Co-infection with viruses from the same and from different serotypes was demonstrated by immunofluorescence microscopy and flow cytometry using anti-tag antibodies. FLAG-tagged VP1 and HA-tagged VP1 could be co-immunoprecipitated from co-infected cells, suggesting that newly synthesised capsids may contain VP1 proteins from both co-infecting viruses. Furthermore, we provide the first demonstration of trans-encapsidation of an FMDV genome into capsids comprised of proteins encoded by a co-infecting heterologous virus. This system provides a useful tool for investigating co-infection dynamics in vitro, particularly between closely related strains, and has the advantage that it does not depend upon the availability of strain-specific FMDV antibodies.

Type I interferons (IFNs) are produced by most cells in response to virus infection and stimulate a program of anti-viral gene expression in neighboring cells to suppress virus replication. Type III IFNs have similar properties, however their effects are limited to epithelial cells at mucosal surfaces due to restricted expression of the type III IFN receptor. Rotavirus (RV) replicates in intestinal epithelial cells that respond predominantly to type III IFNs, and it has been shown that type III rather than type I IFNs are important for controlling RV infections in vivo. The RV NSP1 protein antagonizes the host type I IFN response by targeting IRF-3, IRF-5, IRF-7, or β-TrCP for proteasome-mediated degradation in a strain-specific manner. Here we provide the first demonstration that NSP1 proteins from several human and animal RV strains antagonize type III as well as type I IFN induction. We also show that NSP1 is a potent inhibitor of IRF-1, a previously undescribed property of NSP1 which is conserved among human and animal RVs. Interestingly, all NSP1 proteins were substantially more effective inhibitors of IRF-1 than either IRF-3 or IRF-7 which has significance for evasion of basal anti-viral immunity and type III IFN induction in the intestinal epithelium.