African swine fever virus (ASFV) is a major threat for pig health and meat production in many countries. The development and commercialization of vaccine candidates are complicated by efficacy and safety concerns. Improved vaccine design requires further studies to identify factors that regulate immune responses to vaccines leading to protective immunity against a virulent challenge. In a previous study, we reported that infection with the moderately virulent ASFV field strain Estonia 2014 was less severe in specific pathogen-free (SPF) pigs than in conventional farm pigs, which differ in their gut microbiome and their basal immune activation status. As shown previously using intramuscular infection, SPF pigs were more resilient to oronasal infection with the ASFV Estonia 2014 strain compared to farm pigs, which showed increased fever and clinical signs. All SPF and farm pigs nevertheless survived the infection and remained viremic for approximately 4 months. When all animals had no detectable viremia, both groups were rechallenged with the virulent ASFV Armenia 2008 strain. SPF pigs were fully protected against disease and showed little or no viremia upon re-challenge. In contrast, farm pigs developed high viremia, high proinflammatory cytokine responses, severe clinical signs, and 40% (2 of 5 pigs) reached humane endpoints. Our findings suggest that limited prior immune exposure to other pathogens and/or the microbiome composition of SPF pigs promotes resilience to infection with a moderately virulent strain such as Estonia 2014, and importantly promotes the development of a strong protective immune response against a second challenge with a virulent ASFV strain. In conclusion, testing safety and efficacy of live attenuated vaccine candidates should take into account the specific hygiene conditions and the associated changes of general immune status of pigs in clinical trials.

African swine fever virus (ASFV) is the etiological agent of African swine fever, a highly contagious hemorrhagic disease affecting both wild boars and domestic pigs with lethality rates up to 100%. Until now, the most effective measure to prevent an outbreak of ASFV was early detection. In this situation, whole genome sequencing (WGS) allows the gathering of detailed information about the identity and epidemiology of the virus. However, due to the large genome size and complex genome ends, WGS is challenging. Current WGS workflows require either elaborate enrichment methods or are based on tiled PCR approaches, which are susceptible to genetic differences between ASFV strains. To overcome this, we developed a novel approach for WGS of ASFV, using the Phi29 DNA polymerase-based multiple displacement amplification in combination with only seven primers. Furthermore, we applied an alkaline-based DNA denaturation step to significantly increase the number of viral reads, which resolves the near-full genome of ASFV. This novel isothermal WGS approach can be used in authorized laboratories for the genomic epidemiological analysis of ASFV outbreaks caused by different genotypes.

African swine fever virus (ASFV) has been spreading through Europe, Asia, and the Caribbean after its introduction in Georgia in 2007 and, due to its particularly high mortality rate, poses a continuous threat to the pig industry. The golden standard to trace back the ASFV is whole genome sequencing, but it is a cost and time-intensive methodology. A more efficient way of tracing the virus is to amplify only specific genomic regions relevant for genotyping. This is mainly accomplished by amplifying single amplicons by PCR followed by Sanger sequencing. To reduce costs and processivity time, we evaluated a multiplex PCR based on the four primer sets routinely used for ASFV genotyping (B646L, E183L, B602L, and intergenic I73R-I329L), which was followed by Nanopore ligation-based amplicon sequencing. We show that with this protocol, we can genotype ASFV DNA originating from different biological matrices and correctly classify multiple genotypes and strains using a single PCR reaction. Further optimization of this method can be accomplished by adding or swapping the primer sets used for amplification based on the needs of a specific country or region, making it a versatile tool that can speed up the processing time and lower the costs of genotyping during ASFV outbreaks.

Peste des petits ruminants (PPR) is an acute disease of small ruminants caused by a morbillivirus. Clinical observation of the disease in the field revealed that several species of small ruminants are affected to varying degrees. This difference in disease-related effects could depend either on the host or on the virulence of the virus strain. A previous study highlighted the difference in virulence between two strains of PPRV used to infect Saanen goats. For this breed, PPRV Morocco 2008 strain (MA08) was highly virulent while PPRV Côte d’Ivoire 1989 (IC89) strain induced mild disease. Experimental studies generally based on healthy and young animals do not permit exploration of the natural variability of the host susceptibility to PPRV. Therefore, building on the previous study on Saanen goats, the current study focussed on this breed of goat and used commercially available animals with an unknown history of infection with other pathogens. Results confirmed the previous disease pattern for PPRV IC89 and MA08 strains. Viral RNA detection, macroscopic and histological lesions were stronger for the highly virulent MA08 strain. We show here for the first time that viral RNA can be detected in the tissues of vaccinated animals. Viral RNA was also detected for the first time in serum samples, which is in agreement with the role of circulating immune cells in transporting the virus into host target organs. Thus, this study provides insight into the pathogenesis of strains of different virulence of PPRV and will help to better understand the onset of the disease.

African Swine Fever virus (ASFV) is a large double-enveloped DNA virus of the Asfarviridae family that causes a lethal hemorrhagic disease in domestic pigs and wild boars. Since 2007, a highly virulent genotype II strain has emerged and spread in Europe and South-East Asia, where millions of animals succumbed to the disease. Field- and laboratory-attenuated strains of ASFV cause highly variable clinical disease severity and survival, and mechanisms remain unclear. We hypothesized that the immunological and hygienic status of pigs is a determinant of ASF disease course. Here we compared the immunological profile at baseline and in response to ASFV infection in specific pathogen-free (SPF) and farm-raised Large White domestic pigs. At steady state, SPF pigs showed lower white blood cell counts and a lower basal inflammatory and antiviral transcriptomic profile compared to farm pigs associated with profound differences in gut microbiome composition. After inoculation with a highly virulent ASFV genotype II strain (Armenia 2008), severe clinical signs, viremia and pro-inflammatory cytokines appeared sooner in SPF pigs, indicating a reduced capacity to control early virus replication. In contrast, during infection with an attenuated field isolate (Estonia 2014), SPF pigs presented a milder and shorter clinical disease with full recovery, whereas farm pigs presented severe protracted disease with 50% lethality. Interestingly, farm pigs showed higher production of inflammatory cytokines, whereas SPF pigs produced more anti-inflammatory IL-1ra early after infection, and presented a stronger expansion of leukocytes in the recovery phase. Altogether, our data indicate that the hygiene-dependent innate immune status has a double-edge sword impact on immune responses in ASF pathogenesis. While the higher baseline innate immune activity helps the host in reducing initial virus replication, it promotes immunopathological cytokine responses, and delays lymphocyte proliferation after infection with an attenuated strain. Such effects should be considered for live vaccine development and vigilance.

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications. AUTHOR SUMMARY This work identifies a novel function of lung-resident MSCs during virus-induced acute lung injury. These findings contribute to the understanding of host response and lung repair mechanisms during a highly prevalent clinical situation and may have therapeutic implications.