SARS CoV-2 infections in animals, two years into the pandemic.

In December 2019, several cases of pneumonia caused by a novel coronavirus, later identified as SARS-CoV-2, were detected in the Chinese city of Wuhan. Due to its rapid worldwide spread, on 11 March 2020 the World Health Organization declared a pandemic state. Since this new virus is genetically similar to the coronaviruses of bats, SARS-CoV-2 was hypothesized to have a zoonotic origin. Within a year of the appearance of SARS-CoV-2, several cases of infection were also reported in animals, suggesting human-to-animal and animal-to-animal transmission among mammals. Natural infection has been found in companion animals as well as captive animals such as lions, tigers, and gorillas. Among farm animals, so far, minks have been found to be susceptible to SARS-CoV-2 infection, whereas not all the relevant studies agree on the susceptibility of pigs. Experimental infections have documented the susceptibility to SARS-CoV-2 of further animal species, including mice, hamsters, cats, dogs, ferrets, raccoon dogs, cattle, and non-human primates. Experimental infections have proven crucial for clarifying the role of animals in transmission and developing models for viral pathogenesis and immunotherapy. On the whole, this review aims to update and critically revise the current information on natural and experimental SARS-CoV-2 infections in animals.

Shelf life of diluted human interferon-α for veterinary clinical use.

BACKGROUND: Oral, low-dose, human interferon (IFN)-α treatments have shown efficacy in different models of viral and autoimmune diseases. Human IFN-α is commonly used at a daily dose between 1 and 10 IU/kg body weight. Registered products matching these doses at convenient concentrations are lacking. AIM: The stability of sterile, diluted, human leukocyte IFN-α was investigated under cold-chain and room temperature conditions. RESULTS: Diluted IFN-α displayed moderate decay at room temperature (20-23 °C), which should restrict the shelf life to 3-4 days after compounding. On the contrary, a substantial stability was demonstrated over 6 months under cold-chain conditions. Such a stability of IFN-α was correlated with the maintenance of Class I MHC-modulating properties. CONCLUSION: A shelf life of six months can be envisaged for sterile, diluted preparations of human IFN-α under cold-chain conditions, and batch size should be adjusted in accordance with this property.

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine.

UNLABELLED: Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. IMPORTANCE: Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs.

A simple method for measuring porcine circovirus 2 whole virion particles and standardizing vaccine formulation.

Porcine Circovirus 2 (PCV2) is involved in porcine circovirus-associated disease, that causes great economic losses to the livestock industry worldwide. Vaccination against PCV2 proved to be very effective in reducing disease occurrence and it is currently performed on a large scale. Starting from a previous model concerning Foot-and Mouth Disease Virus antigens, we developed a rapid and simple method to quantify PCV2 whole virion particles in inactivated vaccines. This procedure, based on sucrose gradient analysis and fluorometric evaluation of viral genomic content, allows for a better standardization of the antigen payload in vaccine batches. It also provides a valid indication of virion integrity. Most important, such a method can be applied to whole virion vaccines regardless of the production procedures, thus enabling meaningful comparisons on a common basis. In a future batch consistency approach to PCV2 vaccine manufacture, our procedure represents a valuable tool to improve in-process controls and to guarantee conformity of the final product with passmarks for approval. This might have important repercussions in terms of reduced usage of animals for vaccine batch release, in the framework of the current 3Rs policy.

The autumn low milk yield syndrome in Brown Swiss cows in continental climates: hypotheses and facts.

Extensive research has been conducted globally on the impact of heat stress (HS) on animal health and milk production in dairy cows. In this article, we examine the possible reasons for the decrease in milk production in Brown Swiss (BS) cows during the autumn season, known as the autumn low milk yield syndrome (ALMYS). This condition has been extensively studied in high-yielding Holstein Friesian (HF) cattle and has also been observed in BS cows with a daily milk yield of around 30 kg. Our hypothesis is that the drop in milk yield and the increased prevalence of mastitis in autumn, as found in our recent studies, may be a long-term consequence of summer HS. We re-evaluate our previous findings in light of the possible manifestation of an HS-related form of ALMYS in BS cows. As milk yield, mastitis spread, and reproductive function of cows are interrelated and have seasonal dependence, we examine the consistency of our hypothesis with existing data. The significant drop in milk yield in BS cows in autumn (by 2.0-3.2 kg), as well as the threshold of milk yield decrease (temperature-humidity index of 70.7), may point in favour of the manifestation of ALMYS in BS cows, similar to HF cows. Only the percentage effect of seasonal factor (59.4%; p < 0.05) on milk yield of BS cows was significant. HS-related ALMYS provides a robust conceptual framework for diverse sets of productive and animal health data in BS cows, similar to observations in high-yielding HF cattle. However, the limitations associated with the lack of additional data (e.g. immunological indicators) suggest the need for further research to confirm ALMYS in BS breed.

Different Immune Control of Gram-Positive and Gram-Negative Mammary Infections in Dairy Cows.

In the dairy industry, bovine mastitis represents a major concern due to substantial production losses and costs related to therapies and early culling. The mechanisms of susceptibility and effective response to intra-mammary infections are still poorly understood. Therefore, we investigated innate immunity in acellular bovine skim milk through cytofluorimetric analyses of bacterial killing activity against both Gram-positive and Gram-negative pathogens. Freshly cultured E. coli and S. aureus strains were incubated with colostrum and milk samples at different lactation time points from two groups of cows, purportedly representing mastitis-resistant and mastitis-susceptible breeds; bacterial cells were analyzed for vitality by flow cytometry following incorporation of vital dyes. N-acetyl-ß-D-glucosaminidase (NAGase) activity was also investigated in milk and colostrum samples. Our findings revealed that colostrum and milk bacterial killing activity was greater against S. aureus compared to E. coli., with this activity correlated with milk NAGase levels. Furthermore, both killing of S. aureus and NAGase activity were negatively correlated to the elapsed time of lactation. Interestingly, samples from the allegedly mastitis-resistant breed displayed higher bacterial killing and NAGase activities. Our study suggests that diverse control mechanisms are exerted against Gram-positive and Gram-negative pathogens in the mammary glands of cows, probably beyond those already described in the literature.

Short communication: Lymphoproliferative response to lipopolysaccharide and incidence of infections in periparturient dairy cows.

This preliminary study aimed at assessing whether the in vitro proliferation of peripheral blood mononuclear cells in response to lipopolysaccharide permits individual characterization of periparturient dairy cows, and whether this parameter may be associated with incidence of infections and with some of the single nucleotide polymorphisms located on the toll-like receptor 4 (TLR4) gene. Based on the average response of peripheral blood mononuclear cells to lipopolysaccharide over 7 time points during the transition period, 31 cows were categorized as low (LO), medium (MED), and high (HI) responders. This categorization identified 7 HI, 19 MED, and 5 LO cows, respectively. Genomic DNA was genotyped for P-226 C>G and E3+2021 C>T TLR4 single nucleotide polymorphisms. Monitoring of the health status revealed that 8 of the 31 cows suffered from clinical mastitis, metritis, or interdigital dermatitis during the first 60d in milk. The association study pointed out that none of the HI cows and all of the LO cows developed an infection; cows with the CCGT haplotype remained healthy and none of them belonged to the LO responder category.

Evaluation of a robotic system for the recovery of peripheral blood mononuclear cells.

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.

The Swine IFN System in Viral Infections: Major Advances and Translational Prospects.

Interferons (IFNs) are a family of cytokines that play a pivotal role in orchestrating the innate immune response during viral infections, thus representing the first line of defense in the host. After binding to their respective receptors, they are able to elicit a plethora of biological activities, by initiating signaling cascades which lead to the transcription of genes involved in antiviral, anti-inflammatory, immunomodulatory and antitumoral effector mechanisms. In hindsight, it is not surprising that viruses have evolved multiple IFN escape strategies toward efficient replication in the host. Hence, in order to achieve insight into preventive and treatment strategies, it is essential to explore the mechanisms underlying the IFN response to viral infections and the constraints thereof. Accordingly, this review is focused on three RNA and three DNA viruses of major importance in the swine farming sector, aiming to provide essential data as to how the IFN system modulates the antiviral immune response, and is affected by diverse, virus-driven, immune escape mechanisms.

The Autumn Low Milk Yield Syndrome in High Genetic Merit Dairy Cattle: The Possible Role of a Dysregulated Innate Immune Response.

The analysis of milk yield data shows that high genetic merit dairy cows do not express their full production potential in autumn. Therefore, we focused on metabolic stress and inflammatory response in the dry and peripartum periods as possible causes thereof. It was our understanding that some cows could not cope with the stress imposed by their physiological and productive status by means of adequate adaptation strategies. Accordingly, this study highlights the noxious factors with a potential to affect cows in the above transition period: hot summer climate, adverse genetic traits, poor coping with unfavorable environmental conditions, outright production diseases and consequences thereof. In particular, the detrimental effects in the dry period of overcrowding, photoperiod change and heat stress on mammary gland development and milk production are highlighted in the context of the autumn low milk yield syndrome. The latter could be largely accounted for by a "memory" effect on the innate immune system induced in summer by diverse stressors after dry-off, according to strong circumstantial and indirect experimental evidence. The "memory" effect is based on distinct epigenetic changes of innate immunity genes, as already shown in cases of bovine mastitis. Following a primary stimulation, the innate immune system would be able to achieve a state known as "trained immunity", a sort of "education" which modifies the response to the same or similar stressors upon a subsequent exposure. In our scenario, the "education" of the innate immune system would induce a major shift in the metabolism of inflammatory cells following their reprogramming. This would entail a higher basal consumption of glucose, in competition with the need for the synthesis of milk. Also, there is strong evidence that the inflammatory response generated in the dry period leads to a notable reduction of dry matter intake after calving, and to a reduced efficiency of oxidative phosphorylation in mitochondria. On the whole, an effective control of the stressors in the dry period is badly needed for better disease control and optimal production levels in dairy cattle.

Therapeutic and Prophylactic Use of Oral, Low-Dose IFNs in Species of Veterinary Interest: Back to the Future.

Cytokines are important molecules that orchestrate the immune response. Given their role, cytokines have been explored as drugs in immunotherapy in the fight against different pathological conditions such as bacterial and viral infections, autoimmune diseases, transplantation and cancer. One of the problems related to their administration consists in the definition of the correct dose to avoid severe side effects. In the 70s and 80s different studies demonstrated the efficacy of cytokines in veterinary medicine, but soon the investigations were abandoned in favor of more profitable drugs such as antibiotics. Recently, the World Health Organization has deeply discouraged the use of antibiotics in order to reduce the spread of multi-drug resistant microorganisms. In this respect, the use of cytokines to prevent or ameliorate infectious diseases has been highlighted, and several studies show the potential of their use in therapy and prophylaxis also in the veterinary field. In this review we aim to review the principles of cytokine treatments, mainly IFNs, and to update the experiences encountered in animals.

Reappraisal of PRRS Immune Control Strategies: The Way Forward.

The control of porcine reproductive and respiratory syndrome (PRRS) is still a major issue worldwide in the pig farming sector. Despite extensive research efforts and the practical experience gained so far, the syndrome still severely affects farmed pigs worldwide and challenges established beliefs in veterinary virology and immunology. The clinical and economic repercussions of PRRS are based on concomitant, additive features of the virus pathogenicity, host susceptibility, and the influence of environmental, microbial, and non-microbial stressors. This makes a case for integrated, multi-disciplinary research efforts, in which the three types of contributing factors are critically evaluated toward the development of successful disease control strategies. These efforts could be significantly eased by the definition of reliable markers of disease risk and virus pathogenicity. As for the host's susceptibility to PRRSV infection and disease onset, the roles of both the innate and adaptive immune responses are still ill-defined. In particular, the overt discrepancy between passive and active immunity and the uncertain role of adaptive immunity vis-à-vis established PRRSV infection should prompt the scientific community to develop novel research schemes, in which apparently divergent and contradictory findings could be reconciled and eventually brought into a satisfactory conceptual framework.

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Dataset of immune responses induced in swine by an inactivated Porcine Circovirus 2b vaccine.

A whole virus, inactivated, Porcine Circovirus 2b (PCV2b) vaccine was submitted to a quantal assay of potency, as explained in detail in our companion paper [1]. To this purpose, twenty, 45-day old piglets, checked for maternally-derived antibody (MDA), were allocated to four groups of 5 animals each; these were vaccinated with 800/266/88/0 nanograms, respectively, of an inactivated PCV2b strain, consisting of two distinct virion populations. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. In the presence of a clear dose-dependent protection in terms of viremia, no such effect was observed in terms of weight gain after challenge. The 800 and 266-ng payloads were associated with neutralizing antibody titers above the MDA levels in oral fluids. Higher levels of viremia in control and 88-ng groups [1] coincided with a higher Natural Killer activity of tracheobronchial lymph node cells from PCV2-infected pigs. The PCV2 ORF2-specific ELISPOT assay for IFN-g- secreting cells showed very few (2-4) ORF2-specific cells/105 peripheral blood mononuclear cells beyond the basal levels under our experimental conditions (non-significant differences among groups). Also, no significant differences were observed in the degree of lymphoid tissue hyperplasia among the different groups.

Protective immunity in swine induced by Porcine Circovirus 2b inactivated vaccines with different antigen payload.

Porcine Circovirus 2 (PCV2) vaccines are poorly standardized in terms of antigen payload and correlates of protection. Therefore, twenty, 45-day old piglets were divided into four groups of 5 animals each and vaccinated with 800 / 266 / 88 / 0 nanograms, respectively, of an inactivated PCV2b strain formulated in the same oil adjuvant. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. No clinical signs were observed in the pigs under study. Viremia was observed after challenge in all the control pigs, as well as in 3 pigs of the 266 and 88-ng groups (one and two, respectively). No pigs of the 800-ng group developed viremia. On the basis of post challenge viremia, the PCV2b vaccine under study had a titer of 11 Protective Doses (PD) 50 %, and 1 PD50 amounted to 74 ng of PCV2b Ag. Neutralizing and ELISA Ab titers showed no obvious correlation with protection in the single animals, even though the 800-ng group developed a significantly higher mean Ab response. All the pigs with a PCV2-specific, IFN-gamma response at 3 weeks after vaccination in whole blood samples were protected against viremia. In lymphoid tissues (mainly tonsils and ileum) the presence of sparse reactive histiocytes and multinucleated giant cells was the only PCV2-associated feature and, by immunohistochemistry, only 3 out of 20 subjects showed a low viral load.

Non-Assembled ORF2 Capsid Protein of Porcine Circovirus 2b Does Not Confer Protective Immunity.

Porcine Circovirus 2 (PCV2) vaccines are based on either inactivated whole virion, or recombinant ORF2 capsid protein assembled into Virus-like Particles (VLPs). No data are available about the immunizing properties of free, non-assembled capsid protein. To investigate this issue, ORF2 of a reference PCV2b strain was expressed in a Baculovirus-based expression system without assembly into VLPs. The free purified protein was formulated into an oil vaccine at three distinct Ag payloads: 10.8/3.6/1.2 micrograms/dose. Each dose was injected intramuscularly into five, 37-day old piglets, carefully matched for maternally-derived antibody. Five control piglets were injected with sterile PBS in oil adjuvant. Twenty-eight days later, all the pigs were challenged intranasally with 105.3 TCID50 of PCV2b strain DV6503. After challenge infection, all the pigs remained in good clinical conditions. The recombinant vaccine did not induce significant antibody and PCV2-specific IFN-γ responses. ELISPOT and lymphocyte proliferation data confirmed poor induction of cell-mediated immunity. In terms of PCV2 viremia, there was no significant difference between vaccinated and control animals. The histological data indicated the absence of a detectable viral load and of PCVAD lesions in both vaccinated and control animals, as well as of histiocytes and multi-nucleated giant cells. We conclude that free, non-assembled ORF2 capsid protein does not induce protective immunity.

Basic information for the development of a toxicity assay in inactivated bacterial vaccines.

This study dealt with the toxicity of inactivated bacteria intended for veterinary autogenous vaccines toward a suitable control assay. Two in vitro methods were used. The [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] (MTT) test, based on the metabolic reaction of a tetrazolium salt in vital cells, was adopted on the basis of previous positive results. The Interleukin (IL)-1 beta release assay on monocyte-derived pig macrophages was carried out for comparative purposes, to evaluate the possible role of the inflammatory response. MTT and IL-1 beta responses showed a significant correlation (P < 0.05) at defined test dilutions of bacterial antigens, whereas no correlation was demonstrated using MTT responses normalized on bacterial cell concentration. Furthermore, the toxic effects shown in the MTT test were positively correlated to the extracellular protein content. On the whole, the above results could be a useful basis for the development of a toxicity assay on inactivated bacterial vaccines. Also, our data point at bacterial autolysis as a major component underlying toxicity.

Immune Response in Young Thoroughbred Racehorses under Training.

Training has a great impact on the physiology of an athlete and, like all stressful stimuli, can trigger an innate immune response and inflammation, which is part of a wider coping strategy of the host to restore homeostasis. The Thoroughbred racehorse is a valid animal model to investigate these changes thanks to its homogeneous training and highly selected genetic background. The aim of this study was to investigate modifications of the innate immune response and inflammation in young untrained Thoroughbred racehorses during the first training season through haematological and molecular investigations. Twenty-nine Thoroughbred racehorses were followed during their incremental 3-month sprint exercise schedule. Blood collection was performed at time 0 (T0; before starting the intense training period), 30 days after T0 (T30), and 90 days after T0 (T90). Haematological parameters (red and white blood cells, haemoglobin, and platelets) were evaluated and haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), and red cells width distribution + standard deviation (RDW-SD) were calculated. Moreover, via RT-qPCR, we investigated the expression of, Interleukin 1ß (IL-1ß), Interleukin 4 (IL-4) Interleukin 6 (IL-6), Interleukin 2 (IL-2), Interleukin 3 (IL-3), Interleukin 5 (IL-5) Interleukin 8 (IL-8), Trasformig Growth Factor ß and α (TGF-ß), Tumor necrosis factor α (TNF-α), and Interferon γ (IFN-γ)genes. Main corpuscular volume (MCV) showed a significant (p = 0.008) increase at T90. Main corpuscular haemoglobin (MCH) and haemoglobin concentration (MCHC) values were significantly augmented at both T30 (p < 0.001) and T90 (p < 0.001). Basophils were significant increased at T30 (p = 0.02) and eosinophils were significantly increased at T90 (p = 0.03). Significant differences in gene expression were found for all the genes under study, with the exception of IFN-γ and TNF-α. In particular, IL-2 (T30, p = 0.011; T90, p = 0.015), IL-4 (T30, p = 0.009; T90, p < 0.001), and IL-8 (T30, p < 0.001; T90, p < 0.001) genes were significantly upregulated at both T30 and T90 with respect to T0, TGF-ß was intensely downregulated at T30 (p < 0.001), IL-5 gene expression was significantly decreased at T90 (p = 0.001), while IL-1ß (p = 0.005) and IL-3 (p = 0.001) expression was strongly augmented at the same time. This study highlighted long-term adjustments of O2 transport capability that can be reasonably traced back to exercise adaptation. Moreover, the observed changes of granulocyte numbers and functions and inflammatory cytokine gene expression confirm a major role of the innate immune system in the response to the complex of stressful stimuli experienced during the training period.

Modulation of Type I Interferon System by African Swine Fever Virus.

African Swine Fever Virus (ASFV) has tropism for macrophages, which seems to play a crucial role in disease pathogenesis and viral dissemination. Previous studies showed that ASFV developed mechanisms to evade type I interferon (IFN) responses. Hence, we analyzed the ability of ASFV strains of diverse virulence to modulate IFN-ß and IFN-α responses. Porcine monocyte-derived macrophages un-activated (moMΦ) or activated with IFN-α (moMΦ + FN-α) were infected with virulent (22653/14) or attenuated (NH/P68) ASFV strains, and expressions of IFN-ß and of 17 IFN-α subtypes genes were monitored over time. ASFV strains of diverse virulence induced different panels of IFN genes: infection of moMΦ with either strains caused statistically significant up-regulation of IFN-α3, -α7/11, whereas only attenuated NH/P68 determined statistically significant up-regulation of IFN-α10, -α12, -α13, -α15, -α17, and IFN-ß. Infection of activated moMΦ with either strains resulted in up-regulation of IFN-ß and many IFN-α subtypes, but statistical significance was found only for IFN-α1, -α10, -α15, -α16, -α17 in response to NH/P68-infection only. These data revealed differences in type I IFNs expression patterns, with differences between strains of diverse virulence. In addition, virulent 22653/14 ASFV seems to have developed mechanisms to suppress the induction of several type I IFN genes.

In vitro Cytokine Responses to Virulent PRRS Virus Strains.

Porcine reproductive and respiratory syndrome (PRRS) affects farmed swine causing heavy direct and indirect losses. The infections sustained by PRRS viruses (PRRSV-1 and PRRSV-2) may give rise to severe clinical cases. This highlights the issue of PRRSV pathogenicity and relevant markers thereof. Since PRRSV strains can be discriminated in terms of immunotypes, we aimed to detect possible correlates of virulence in vitro based on the profile of innate immune responses induced by strains of diverse virulence. To this purpose, 10 field PRRSV isolates were investigated in assays of innate immune response to detect possible features associated with virulence. Tumor necrosis factor-α, interleukin (IL)-1beta, IL-8, IL-10, and caspase-1 were measured in cultures of PRRSV-treated peripheral blood mononuclear cells of PRRS-naive pigs, unable to support PRRSV replication. Two reference PRRSV strains (highly pathogenic and attenuated, respectively), were included in the screening. The PRRSV strains isolated from field cases were shown to vary widely in terms of inflammatory cytokine responses in vitro, which were substantially lacking with some strains including the reference, highly pathogenic one. In particular, neither the field PRRSV isolates nor the reference highly pathogenic strain gave rise to an IL-1beta response, which was consistently induced by the attenuated strain, only. This pattern of response was reversed in an inflammatory environment, in which the attenuated strain reduced the ongoing IL-1beta response. Results indicate that some pathogenic PRRSV strains can prevent a primary inflammatory response of PBMCs, associated with reduced permissiveness of mature macrophages for PRRSV replication in later phases.