Comparative evaluation of disease dynamics in wild boar and domestic pigs experimentally inoculated intranasally with the European highly virulent African swine fever virus genotype II strain "Armenia 2007".

Since the reintroduction of African swine fever virus (ASFV) in Europe in 2007 and its subsequent spread to Asia, wild boar has played a crucial role in maintaining and disseminating the virus. There are significant gaps in the knowledge regarding infection dynamics and disease pathogenesis in domestic pigs and wild boar, particularly at the early infection stage. We aimed to compare domestic pigs and wild boar infected intranasally to mimic natural infection with one of the original highly virulent genotype II ASFV isolates (Armenia 2007). The study involved euthanising three domestic pigs and three wild boar on days 1, 2, 3, and 5 post-infection, while four domestic pigs and four wild boar were monitored until they reached a humane endpoint. The parameters assessed included clinical signs, macroscopic lesions, viremia levels, tissue viral load, and virus shedding in nasal and rectal swabs from day 1 post-infection. Compared with domestic pigs, wild boar were more susceptible to ASFV, with a shorter incubation period and earlier onset of clinical signs. While wild boar reached a humane endpoint earlier than domestic pigs did, the macroscopic lesions were comparatively less severe. In addition, wild boar had earlier viremia, and the virus was also detected earlier in tissues. The medial retropharyngeal lymph nodes were identified as key portals for ASFV infection in both subspecies. No viral genome was detected in nasal or rectal swabs until shortly before reaching the humane endpoint in both domestic pigs and wild boar, suggesting limited virus shedding in acute infections.

Molecular Epidemiology Questions Transmission Pathways Identified During the Year 2000 Outbreak of Classical Swine Fever in the UK.

The last outbreak of classical swine fever (CSF) in the UK occurred in 2000. A total of 16 domestic pig holdings in the East Anglia region were confirmed as infected over a 3-month period. Obtaining viral genome sequences has since become easier and more cost-effective and has accordingly been applied to trace viral transmission events for a variety of viruses. The rate of genetic evolution varies for different viruses and is influenced by different transmission events, which will vary according to the epidemiology of an outbreak. To examine if genetic changes over the course of any future CSF outbreak would occur to supplement epidemiological investigations and help to track virus movements, the E2 gene and full genome of the virus present in archived tonsil samples from 14 of these infected premises were sequenced. Insufficient changes occurred in the full E2 gene to discriminate between the viruses from the different premises. In contrast, between 5 and 14 nucleotide changes were detected between the genome sequence of the virus from the presumed index case and the sequences from the other 13 infected premises. Phylogenetic analysis of these full CSFV genome sequences identified clusters of closely related viruses that allowed to corroborate some of the transmission pathways inferred by epidemiological investigations at the time. However, other sequences were more distinct and raised questions about the virus transmission routes previously implicated. We are thus confident that in future outbreaks, real-time monitoring of the outbreak via full genome sequencing will be beneficial.

Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions.

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Evaluation of Lesions and Viral Antigen Distribution in Domestic Pigs Inoculated Intranasally with African Swine Fever Virus Ken05/Tk1 (Genotype X).

The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.

Inactivation of African Swine Fever Virus by reagents commonly used in containment laboratories.

Rapid and effective virus inactivation is an essential step for safe diagnostic testing and for research and vaccine development using infectious viruses. We characterised the reduction of African Swine Fever Virus (ASFV) infectivity using Virkon™ S (Lanxess) 1% w/v disinfectant, FACS™ Lysing buffer (BD), and AVL™ buffer (Qiagen), using porcine cell culture. No virus was detected following a 30 s 20:1 v/v mixing ratio of Virkon™ S 1% with high titre ASFV, supporting its effective use as a laboratory surface disinfectant. FACS™ Lysing and AVL™ buffers also inactivated ASFV, permitting safe removal of treated infected samples from high containment facilities.

Substitution of warthog NF-κB motifs into RELA of domestic pigs is not sufficient to confer resilience to African swine fever virus.

African swine fever virus (ASFV) causes a lethal, haemorrhagic disease in domestic swine that threatens pig production across the globe. Unlike domestic pigs, warthogs, which are wildlife hosts of the virus, do not succumb to the lethal effects of infection. There are three amino acid differences between the sequence of the warthog and domestic pig RELA protein; a subunit of the NF-κB transcription factor that plays a key role in regulating the immune response to infections. Domestic pigs with all 3 or 2 of the amino acids from the warthog RELA orthologue have been generated by gene editing. To assess if these variations confer resilience to ASF we established an intranasal challenge model with a moderately virulent ASFV. No difference in clinical, virological or pathological parameters were observed in domestic pigs with the 2 amino acid substitution. Domestic pigs with all 3 amino acids found in warthog RELA were not resilient to ASF but a delay in onset of clinical signs and less viral DNA in blood samples and nasal secretions was observed in some animals. Inclusion of these and additional warthog genetic traits into domestic pigs may be one way to assist in combating the devastating impact of ASFV.

Phylogenetic analysis of infectious pancreatic necrosis virus in Ireland reveals the spread of a virulent genogroup 5 subtype previously associated with imports.

Infectious pancreatic necrosis is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality and disease-management costs. Significant outbreaks of the disease occurred in farmed Atlantic salmon in Ireland between 2003 and 2007, associated with imported ova and smolts. As the virus was known to occur in the country since the development of aquaculture in the 1980s, this study examined archived samples to determine whether these older isolates were associated with virulent forms. The study showed that two genotypes of IPNV were present in the 1990s, genotype 3 and genotype 5. A more virulent subtype of the virus first appeared in 2003 associated with clinical outbreaks of IPN, and this subtype is now the most prevalent form of IPNV found in the country. The data also indicated that IPNV in Ireland is more closely related to Scottish and continental European isolates than to Norwegian, Chilean and Australasian genogroup 5 isolates.