Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different in vitro and in vivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced in vitro and in vivo by the decrease of proinflammatory cytokines.

SARS-CoV-2 in Captive Nonhuman Primates, Spain, 2020-2023.

We conducted a serologic and molecular study to assess exposure of captive nonhuman primates (NHPs) to SARS-CoV-2 in Spain during the 2020-2023 COVID-19 pandemic. We found limited exposure of NHPs to SARS-CoV-2. Biosafety measures must be strictly maintained to avoid SARS-CoV-2 reverse-zoonotic transmission in the human-NHP interface.

Advances and gaps in SARS-CoV-2 infection models.

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age.

Extended Viral Shedding of MERS-CoV Clade B Virus in Llamas Compared with African Clade C Strain.

Middle East respiratory syndrome coronavirus (MERS-CoV) clade B viruses are found in camelids and humans in the Middle East, but clade C viruses are not. We provide experimental evidence for extended shedding of MERS-CoV clade B viruses in llamas, which might explain why they outcompete clade C strains in the Arabian Peninsula.

Type I and III IFNs produced by the nasal epithelia and dimmed inflammation are features of alpacas resolving MERS-CoV infection.

While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.

Porcine circovirus 3 (PCV-3) variability: Is it in the virus or in the classification criteria?

The continuous discovery of new viruses during the last decades has increased the need for new classification approaches and rules. Currently, the International Committee on Taxonomy of Viruses classifies viruses up to the species level. However, because of the higher variability of most of these infectious agents, a below-species categorization is often required for proper epidemiological investigations. Unfortunately, variable criteria are typically proposed by different research groups, leading to misleading and poorly reproducible results. This scenario occurred for the recently identified Porcine circovirus 3. Although genotype definition standards had been defined by a group of experts in the field, recent articles have been published introducing new genotypes, whose classification rules are not reported. We therefore would like to stress the usefulness of defining and maintaining a common language to allow proper results comparison among groups. We consider the consensus opinion of a heterogeneous expert team as the most valuable approach. Nevertheless, if other approaches are proposed, the disclosure of the criteria and the comparison with previous literature should be deemed mandatory to allow effective results reproducibility, interpretation and sharing.

First detection of porcine circovirus 4 (PCV-4) in Europe.

Porcine circovirus 4 (PCV-4) is a novel virus recently discovered (2019) in domestic pigs from China, although several studies have proven its circulation since 2008. Later, PCV-4 was also detected in wild boar populations from China and domestic pigs from South Korea and Thailand. Currently, Asia is so far the only continent where this novel virus has been reported; few studies carried out in South America and Europe failed in the attempt to detect it. The objective of this Comment is to communicate the first detection of PCV-4 in Europe, specifically in wild boar and domestic pigs from Mid-South-Western Spain. A retrospective study was carried out on wild boar and domestic pigs, both extensively (Iberian breed) and intensively raised, from Spain and Italy, sampled between 1998 and 2022. PCV-4 genome detection was attempted using different conventional or quantitative real time PCR (qPCR) protocols and some positive results were confirmed through Sanger sequencing. A total of 57 out of 166 (34.3%) Spanish wild boar and 9 out of 223 (4%) Iberian pigs (both geographically located in the Mid-South-Western Spain) were qPCR positive, while the rest of tested animals from North-Eastern Spain and Italy were negative. Partial sequences of Rep or Cap genes of selected samples confirmed the presence of PCV-4. The relatively high prevalence in wild boar and the low one in Iberian pigs from the same areas suggests intra- and interspecific transmission, being the wild boar a potential viral reservoir. The epidemiological and clinical importance of these findings are currently unknown, but guarantees further research on this novel virus.

Review on the methodology to assess respiratory tract lesions in pigs and their production impact.

Porcine respiratory disease is one of the most important health problems in pig production worldwide. Cranioventral pulmonary consolidation (CVPC) and pleurisy are the two most common lesions in the respiratory tract of slaughtered pigs. The present review paper discusses pathogens involved in the lesions, lesion prevalence, scoring systems, advantages and disadvantages of slaughterhouse examination, and the impact of CVPC and pleurisy on performance, carcass, and meat quality. Cranioventral pulmonary consolidation and pleurisy in slaughter pigs are characteristic for infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae, respectively, although other pathogens may cause similar lesions and/or be involved in their development. The overall prevalence of CVPC and pleurisy in slaughter pigs are still high, being the prevalence of CVPC generally higher than that of chronic pleurisy. The advantages and disadvantages of slaughterhouse examination are discussed in relation to practical aspects, the assessment of lesions, the number and representativeness of the examined animals and the interpretation and value of the results for the stakeholders. The main scoring methods for CVPC and pleurisy are shortly reviewed. In general, scoring methods can be applied rapidly and easily, although significant variation due to abattoir and observer remains. Artificial intelligence-based technologies that automatically score lesions and facilitate processing of data may aid solving these problems. Cranioventral pulmonary consolidation and pleurisy have a major negative impact on pig performance, and the effects increase the extension of the lesions and/or presence of multiple lesions. The performance losses caused by these lesions, however, vary significantly between studies and farms, possibly due to differences in study population and used methodology. Both lesions also have a negative impact on different carcass and meat quality parameters, leading to increased risk for poor processing and storage of the carcasses. Monitoring lung lesions of slaughter pigs should be optimized and implemented routinely; however, it is recommended to complement this information with farm data and laboratory results for specific pathogens.

Detection of SARS-CoV-2 in a cat owned by a COVID-19-affected patient in Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is considered a zoonotic pathogen mainly transmitted human to human. Few reports indicate that pets may be exposed to the virus. The present report describes a cat suffering from severe respiratory distress and thrombocytopenia living with a family with several members affected by COVID-19. Clinical signs of the cat prompted humanitarian euthanasia and a detailed postmortem investigation to assess whether a COVID-19-like disease was causing the condition. Necropsy results showed the animal suffered from feline hypertrophic cardiomyopathy and severe pulmonary edema and thrombosis. SARS-CoV-2 RNA was only detected in nasal swab, nasal turbinates, and mesenteric lymph node, but no evidence of histopathological lesions compatible with a viral infection were detected. The cat seroconverted against SARS-CoV-2, further evidencing a productive infection in this animal. We conclude that the animal had a subclinical SARS-CoV-2 infection concomitant to an unrelated cardiomyopathy that led to euthanasia.

Rapid SARS-CoV-2 Inactivation in a Simulated Hospital Room Using a Mobile and Autonomous Robot Emitting Ultraviolet-C Light.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has made mask-wearing, physical distancing, hygiene, and disinfection complementary measures to control virus transmission. Especially for health facilities, we evaluated the efficacy of an UV-C autonomous robot to inactivate SARS-CoV-2 desiccated on potentially contaminated surfaces. ASSUM (autonomous sanitary sterilization ultraviolet machine) robot was used in an experimental box simulating a hospital intensive care unit room. Desiccated SARS-CoV-2 samples were exposed to UV-C in 2 independent runs of 5, 12, and 20 minutes. Residual virus was eluted from surfaces and viral titration was carried out in Vero E6 cells. ASSUM inactivated SARS-CoV-2 by ≥ 99.91% to ≥ 99.99% titer reduction with 12 minutes or longer of UV-C exposure and onwards and a minimum distance of 100cm between the device and the SARS-CoV-2 desiccated samples. This study demonstrates that ASSUM UV-C device is able to inactivate SARS-CoV-2 within a few minutes.

Evaluation of alpaca tracheal explants as an ex vivo model for the study of Middle East respiratory syndrome coronavirus (MERS-CoV) infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to public health. Here, we established an ex vivo alpaca tracheal explant (ATE) model using an air-liquid interface culture system to gain insights into MERS-CoV infection in the camelid lower respiratory tract. ATE can be infected by MERS-CoV, being 103 TCID50/mL the minimum viral dosage required to establish a productive infection. IFNs and antiviral ISGs were not induced in ATE cultures in response to MERS-CoV infection, strongly suggesting that ISGs expression observed in vivo is rather a consequence of the IFN induction occurring in the nasal mucosa of camelids.

Middle East respiratory syndrome coronavirus infection in camelids.

Middle East respiratory syndrome coronavirus (MERS-CoV) is the cause of a severe respiratory disease with a high case fatality rate in humans. Since its emergence in mid-2012, 2578 laboratory-confirmed cases in 27 countries have been reported by the World Health Organization, leading to 888 known deaths due to the disease and related complications. Dromedary camels are considered the major reservoir host for this virus leading to zoonotic infection in humans. Dromedary camels, llamas, and alpacas are susceptible to MERS-CoV, developing a mild-to-moderate upper respiratory tract infection characterized by epithelial hyperplasia as well as infiltration of neutrophils, lymphocytes, and some macrophages within epithelium, lamina propria, in association with abundant viral antigen. The very mild lesions in the lower respiratory tract of these camelids correlate with absence of overt illness following MERS-CoV infection. Unfortunately, there is no approved antiviral treatment or vaccine for MERS-CoV infection in humans. Thus, there is an urgent need to develop intervention strategies in camelids, such as vaccination, to minimize virus spillover to humans. Therefore, the development of camelid models of MERS-CoV infection is key not only to assess vaccine prototypes but also to understand the biologic mechanisms by which the infection can be naturally controlled in these reservoir species. This review summarizes information on virus-induced pathological changes, pathogenesis, viral epidemiology, and control strategies in camelids, as the intermediate hosts and primary source of MERS-CoV infection in humans.

Chronological brain lesions after SARS-CoV-2 infection in hACE2-transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease, but it can also affect other organs including the central nervous system. Several animal models have been developed to address different key questions related to Coronavirus Disease 2019 (COVID-19). Wild-type mice are minimally susceptible to certain SARS-CoV-2 lineages (beta and gamma variants), whereas hACE2-transgenic mice succumb to SARS-CoV-2 and develop a fatal neurological disease. In this article, we aimed to chronologically characterize SARS-CoV-2 neuroinvasion and neuropathology. Necropsies were performed at different time points, and the brain and olfactory mucosa were processed for histopathological analysis. SARS-CoV-2 virological assays including immunohistochemistry were performed along with a panel of antibodies to assess neuroinflammation. At 6 to 7 days post inoculation (dpi), brain lesions were characterized by nonsuppurative meningoencephalitis and diffuse astrogliosis and microgliosis. Vasculitis and thrombosis were also present and associated with occasional microhemorrhages and spongiosis. Moreover, there was vacuolar degeneration of virus-infected neurons. At 2 dpi, SARS-CoV-2 immunolabeling was only found in the olfactory mucosa, but at 4 dpi intraneuronal virus immunolabeling had already reached most of the brain areas. Maximal distribution of the virus was observed throughout the brain at 6 to 7 dpi except for the cerebellum, which was mostly spared. Our results suggest an early entry of the virus through the olfactory mucosa and a rapid interneuronal spread of the virus leading to acute encephalitis and neuronal damage in this mouse model.

Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs.

Streptococcus suis is one of the most important bacterial swine pathogens affecting post-weaned piglets, causing mainly meningitis, arthritis and sudden death. It not only results in severe economic losses but also raises concerns over animal welfare and antimicrobial resistance and remains an important zoonotic agent in some countries. The definition and diagnosis of S. suis-associated diseases can be complex. Should S. suis be considered a primary or secondary pathogen? The situation is further complicated when referring to respiratory disease, since the pathogen has historically been considered as a secondary pathogen within the porcine respiratory disease complex (PRDC). Is S. suis a respiratory or strictly systemic pathogen? S. suis is a normal inhabitant of the upper respiratory tract, and the presence of potentially virulent strains alone does not guarantee the appearance of clinical signs. Within this unclear context, it has been largely proposed that co-infection with some viral and bacterial pathogens can significantly influence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field. In this review, we critically addressed studies reporting an epidemiological link (mixed infections or presence of more than one pathogen at the same time), as well as in vitro and in vivo studies of co-infection of S. suis with other pathogens and discussed their limitations and possibilities for improvement and proposed recommendations for future studies.

Piglet innate immune response to Streptococcus suis colonization is modulated by the virulence of the strain.

Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of piglets by S. suis is very common and occurs early in life. The clinical outcome of infection is influenced by the virulence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohistochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inoculation. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites, with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response in piglets that appears to be related to the virulence potential of the colonizing strain.

Assessment of the in vitro growing dynamics and kinetics of the non-pathogenic J and pathogenic 11 and 232 Mycoplasma hyopneumoniae strains.

Information on the in vitro growth of pathogenic and non-pathogenic Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains' daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC®25934™) type strain and the pathogenic 11 (ATCC®25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.

Exploratory field study on the effect of Porcine circovirus 2 (PCV2) sow vaccination on serological, virological and reproductive parameters in a PCV2 subclinically infected sow herd.

BACKGROUND: This study sought to evaluate the effect of sow vaccination against Porcine circovirus 2 (PCV2) on reproductive parameters during two consecutive reproductive cycles. The study was performed in a PCV2 subclinical infected breeding herd (PCV2 circulation but absence of major reproductive problems). Ninety-four pregnant sows were primo-immunized with a commercial PCV2 vaccine and ninety-seven were injected with phosphate-buffered saline at 6 and 3 weeks before the first studied farrowing, and then boosted at 2 weeks before the second one. Blood samples were taken throughout the study to assess PCV2 DNA load and antibodies. At farrowing, main reproductive parameters and piglet vitality index were registered. In addition, in those litters with more than three mummified or stillborn piglets, microscopic examination and PCV2 antigen detection in foetal myocardium was done. RESULTS: Vaccinated sows showed significantly higher antibody levels compared to the non-vaccinated counterparts. PCV2 DNA was only detected at farrowing in 2 (4.2%) non-vaccinated sows. Vaccinated sows had 1.3 more live-born piglets per litter at the second cycle than non-vaccinated counterparts. Piglets from vaccinated sows had significantly higher (+ 12.7%) vitality score than the ones born from non-vaccinated sows. No PCV2 compatible lesions neither PCV2 antigen were detected in the tested foetal hearts. CONCLUSIONS: The present study represents a first attempt to demonstrate that PCV2 sow vaccination may have a positive influence on prolificacy and vitality of the offspring in a subclinical infected breeding herd. However, since reproductive outcomes at farm level may be affected by a number of factors, further studies would be needed to confirm this association.

Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus.

Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs. To determine whether other animals are potential reservoirs, we inoculated MERS-CoV into llamas, pigs, sheep, and horses and collected nasal and rectal swab samples at various times. The presence of MERS-CoV in the nose of pigs and llamas was confirmed by PCR, titration of infectious virus, immunohistochemistry, and in situ hybridization; seroconversion was detected in animals of both species. Conversely, in sheep and horses, virus-specific antibodies did not develop and no evidence of viral replication in the upper respiratory tract was found. These results prove the susceptibility of llamas and pigs to MERS-CoV infection. Thus, the possibility of MERS-CoV circulation in animals other than dromedaries, such as llamas and pigs, is not negligible.

ICTV Virus Taxonomy Profile: Circoviridae.

The family Circoviridae comprises viruses with small, circular, single-stranded DNA (ssDNA) genomes, including the smallest known animal viruses. Members of this family are classified into two genera, Circovirus and Cyclovirus, which are distinguished by the position of the origin of replication relative to the coding regions and the length of the intergenic regions. Within each genus, the species demarcation threshold is 80 % genome-wide nucleotide sequence identity. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Circoviridae, which is available at www.ictv.global/report/circoviridae.

Differential Expression of the Middle East Respiratory Syndrome Coronavirus Receptor in the Upper Respiratory Tracts of Humans and Dromedary Camels.

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor--dipeptidyl peptidase 4 (DPP4)--is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission.