Enhanced infectivity of bovine viral diarrhoea virus (BVDV) in arginase-producing bovine monocyte-derived macrophages.

Macrophages are important cells of the innate immunity that play a major role in Bovine Viral Diarrhea Virus (BVDV) pathogenesis. Macrophages are not a homogenous population; they exist in different phenotypes, typically divided into two main categories: classically (pro-inflammatory) and alternatively activated (anti-inflammatory) or M1 and M2, respectively. The role of bovine macrophage phenotypes on BVDV infection is still unclear. This study characterized the interaction between BVDV, and monocyte-derived macrophages (Mo-Mφ) collected from healthy cattle and polarized to an M1 or M2 state by using LPS, INF-γ, IL-4 or azithromycin. Arginase activity quantitation was utilized as a marker of the M2 Mo-Mφ spectrum. There was a significant association between arginase activity and the replication rate of BVDV strains of different genotypes and biotypes. Inhibition of arginase activity also reduced BVDV infectivity. Calves treated with azithromycin induced Mo-Mφ of the M2 state produced high levels of arginase. Interestingly, azithromycin administered in vivo increased the susceptibility of macrophages to BVDV infection ex vivo. Mo-Mφ from pregnant dams and calves produced higher arginase levels than those from non-pregnant adult animals. The increased infection of arginase-producing alternatively activated bovine macrophages with BVDV supports the need to delve into a possible leading role of M2 macrophages in establishing the immune-suppressive state during BVDV convalescence.

Serological screening of SARS-CoV-2 infection in companion animals of Buenos Aires suburbs.

The Coronavirus Disease 2019 (COVID-19) is a zoonotic disease caused by the pandemic virus SARS-CoV-2. Domestic and wild animals are susceptible to infection and are potential reservoirs for virus variants. To date, there is no information about the exposure of companion animals in Buenos Aires Suburbs, the area with the largest population in Argentina where the highest number of COVID-19 human cases occurred during the first infection wave. Here we developed a multi-species indirect ELISA to measure antibodies reactive to the SARS-CoV-2 receptor-binding domain (RBD) from several vertebrates constituting the class Mammalia, making it a valuable tool for field serosurveillance. The ELISA cut-off value was estimated by sera from dogs, cats, cattle, and pigs sampled before 2019 (n = 170), considering a 98% percentile and a grey zone to completely exclude any false positive result. Specificity was confirmed by measuring levels of neutralizing antibodies against canine coronavirus, the avidity of specific antibodies, and their capacity to impede the binding of a recombinant RBD protein to VERO cells in an In-Cell ELISA. Sera from 464 cats and dogs sampled in 2020 and 2021 ("pandemic" samples) were assessed using the RBD-ELISA. Information on COVID-19 disease in the household and the animals' lifestyles was collected. In Buenos Aires Suburbs cats were infected at a higher proportion than dogs, seroprevalence was 7.1 and 1.68%, respectively. Confirmed COVID-19 in the caregivers and outdoor lifestyle were statistically associated with seropositivity in cats. The risk of cats getting infected living indoors in COVID-19-negative households was null. The susceptibility of mammals to SARS-CoV-2, the possibility of transmission between animals themselves and humans, together with the free-roaming lifestyle typical of Buenos Aires suburban companion animals, urge pursuing responsible animal care and avoiding human interaction with animals during the disease course. The multi-species RBD-ELISA we developed can be used as a tool for serosurveillance of SARS-CoV-2 infection in mammalians (domestic and wild), guiding further targeted virological analyses to encounter susceptible species, interspecies transmission, and potential virus reservoirs in our region.

In-vivo Activity of IFN-λ and IFN-α Against Bovine-Viral-Diarrhea Virus in a Mouse Model.

Bovine-viral-diarrhea virus (BVDV) can cause significant economic losses in livestock. The disease is controlled with vaccination and bovines are susceptible until vaccine immunity develops and may remain vulnerable if a persistently infected animal is left on the farm; therefore, an antiviral agent that reduces virus infectivity can be a useful tool in control programs. Although many compounds with promising in-vitro efficacy have been identified, the lack of laboratory-animal models limited their potential for further clinical development. Recently, we described the activity of type I and III interferons, IFN-α and IFN-λ respectively, against several BVDV strains in-vitro. In this study, we analyzed the in-vivo efficacy of both IFNs using a BALB/c-mouse model. Mice infected with two type-2 BVDV field strains developed a viremia with different kinetics, depending on the infecting strain's virulence, that persisted for 56 days post-infection (dpi). Mice infected with the low-virulence strain elicited high systemic TNF-α levels at 2 dpi. IFNs were first applied subcutaneously 1 day before or after infection. The two IFNs reduced viremia with different kinetics, depending on whether either one was applied before or after infection. In a second experiment, we increased the number of applications of both IFNs. All the treatments reduced viremia compared to untreated mice. The application of IFN-λ pre- and post-infection reduced viremia over time. This study is the first proof of the concept of the antiviral potency of IFN-λ against BVDV in-vivo, thus encouraging further trails for a potential use of this cytokine in cattle.

Bovine Interferon Lambda Is a Potent Antiviral Against SARS-CoV-2 Infection in vitro.

Interferon lambda (IFN-λ) is an antiviral naturally produced in response to viral infections, with activity on cells of epithelial origin and located in the mucosal surfaces. This localized activity results in reduced toxicity compared to type I IFNs, whose receptors are ubiquitously expressed. IFN-λ has been effective in the therapy of respiratory viral infections, playing a crucial role in potentiating adaptive immune responses that initiate at mucosal surfaces. Human IFN-λ has polymorphisms that may cause differences in the interaction with the specific receptor in the human population. Interestingly, bovine IFN-λ3 has an in silico-predicted higher affinity for the human receptor than its human counterparts, with high identity with different human IFN-λ variants, making it a suitable antiviral therapeutic candidate for human health. Here, we demonstrate that a recombinant bovine IFN-λ (rbIFN-λ) produced in HEK-293 cells is effective in preventing SARS-CoV-2 infection of VERO cells, with an inhibitory concentration 50% (IC50) between 30 and 50 times lower than that of human type I IFN tested here (α2b and ß1a). We also demonstrated the absence of toxicity of rbIFN-λ in human PBMCs and the lack of proinflammatory activity on these cells. Altogether, our results show that rbIFN-λ is as an effective antiviral potentially suitable for COVID-19 therapy. Among other potential applications, rbIFN-λ could be useful to preclude virus dispersion to the lungs and/or to reduce transmission from infected people. Moreover, and due to the non-specific activity of this IFN, it can be potentially effective against other respiratory viruses that may be circulating together with SARS-CoV-2.

Interferon lambda protects cattle against bovine viral diarrhea virus infection.

Interferon lambda (IFN-λ) plays an important role in inducing an antiviral state in mucosal surfaces and has been used as an effective biotherapeutic against several viral diseases. Here we performed a proof of concept study on the activity of a biologically active recombinant bovine IFN-λ (rIFN-λ) produced in eukaryotic cells against Bovine Viral Diarrhea Virus (BVDV) in cattle. We first confirmed the lack of toxicity of different concentrations of rIFN-λ in bovine peripheral blood cells and the safety of its subcutaneous application in calves in doses up to 12 IU/kg. The antiviral activity of the rIFN-λ against BVDV was assessed in calves that were inoculated with 6 IU/kg of rIFN-λ (n = 4) or mock-treated (n = 2) two days before and after challenge with a BVDV type-2 non-cytopathic strain. Mock-treated animals developed respiratory disease, shedded the virus from 4 to 7 days post-infection (dpi) and had viremia between 4 and 14 dpi. Conversely, calves treated with rIFN-λ did not develop clinical symptoms. The virus was not found in nasal secretions or sera. Only one animal had a positive viral RNA detection in serum at 7 dpi. All infected animals treated with rIFN-λ increased systemic type-I IFNs levels at 4 dpi. The antiviral treatment induced an earlier onset of the anti-BVDV neutralizing antibodies. Altogether, these results constitute the proof-of-principle of bovine IFN-λ as an antiviral biotherapeutic to protect cattle against the clinical disease caused by BVDV.

In Vitro and In Vivo Characterization of a Typical and a High Pathogenic Bovine Viral Diarrhea Virus Type II Strains.

Non-cytopathic (ncp) type 2 bovine viral diarrhea virus (BVDV-2) is widely prevalent in Argentina causing high mortality rates in cattle herds. In this study, we characterized an Argentinean ncp BVDV-2 field isolate (98-124) compared to a high-virulence reference strain (NY-93), using in silico analysis, in vitro assays, and in vivo infections of colostrum-deprived calves (CDC) to compare pathogenic characters and virulence. In vitro infection of bovine peripheral blood mononuclear cells (PBMC) with BVDV 98-124 induced necrosis shortly after infection while NY-93 strain increased the apoptotic rate in infected cells. Experimental infection of CDC (n = 4 each) with these strains caused an enteric syndrome. High pyrexia was detected in both groups. Viremia and shedding were more prolonged in the CDC infected with the NY-93 strain. In addition, NY-93 infection elicited a severe lymphopenia that lasted for 14 days, whereas 98-124 strain reduced the leukocyte counts for 5 days. All infected animals had a diminished lymphoproliferation activity in response to a mitogen. Neutralizing and anti-NS3 antibodies were detected 3 weeks after infection in all infected calves. Virulence was associated with a more severe clinical score, prolonged immune-suppression, and a greater window for transmission. Studies of apoptosis/necrosis performed after in vitro PBMC infection also revealed differences between both strains that might be correlated to the in vivo pathogenesis. Our results identified 98-124 as a low-virulence strain.

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity.

Low-cost high-throughput methods applicable to any virus strain are required for screening antiviral compounds against multiple field strains. Colorimetric cell-viability assays are used for this purpose as long as the viruses are cytopathic (CP) in cell culture. However, bovine viral diarrhoea virus (BVDV) strains circulating in the field are mostly non-cytopathic (NCP). An In Cell-ELISA aimed to measure viral infectivity by detecting a conserved protein produced during viral replication (non-structural protein 3, "NS3") was developed. The ELISA is performed without harvesting the cells, directly on the 96-wells culture plate. NS3 In Cell-ELISA was tested for its ability to assess BVDV-specific antiviral activity of recombinant bovine type I and III IFNs. Results correlated to those measured by qRT-PCR and virus titration. NS3 In Cell-ELISA was also efficient in estimating the IC50 of two compounds with different antiviral activity. Estimation of the 50% inhibition dose of each IFN using six BVDV strains of different biotype and genotype showed that CP strains were more susceptible to both IFNs than NCP, while type 2 NCP viruses were more sensitive to IFN-I. The In Cell-ELISA format using a detector antibody against a conserved non-structural protein can be potentially applied to accurately measure infectivity of any viral strain.