Two DNA vaccines protect against severe disease and pathology due to SARS-CoV-2 in Syrian hamsters.

The SARS-CoV-2 pandemic is an ongoing threat to global health, and wide-scale vaccination is an efficient method to reduce morbidity and mortality. We designed and evaluated two DNA plasmid vaccines, based on the pIDV-II system, expressing the SARS-CoV-2 spike gene, with or without an immunogenic peptide, in mice, and in a Syrian hamster model of infection. Both vaccines demonstrated robust immunogenicity in BALB/c and C57BL/6 mice. Additionally, the shedding of infectious virus and the viral burden in the lungs was reduced in immunized hamsters. Moreover, high-titers of neutralizing antibodies with activity against multiple SARS-CoV-2 variants were generated in immunized animals. Vaccination also protected animals from weight loss during infection. Additionally, both vaccines were effective at reducing both pulmonary and extrapulmonary pathology in vaccinated animals. These data show the potential of a DNA vaccine for SARS-CoV-2 and suggest further investigation in large animal and human studies could be pursued.

Treatment with Ad5-Porcine Interferon-α Attenuates Ebolavirus Disease in Pigs.

Under experimental conditions, pigs infected with Ebola Virus (EBOV) develop disease and can readily transmit the virus to non-human primates or pigs. In the event of accidental or intentional EBOV infection of domestic pigs, complex and time-consuming safe depopulation and carcass disposal are expected. Delaying or preventing transmission through a reduction in viral shedding is an absolute necessity to limit the spread of the virus. In this study, we tested whether porcine interferon-α or λ3 (porIFNα or porIFNλ3) delivered by a replication-defective human type 5 adenovirus vector (Ad5-porIFNα or Ad5-porIFNλ3) could limit EBOV replication and shedding in domestic pigs. Our results show that pigs pre-treated with Ad5-porIFNα did not develop measurable clinical signs, did not shed virus RNA, and displayed strongly reduced viral RNA load in tissues. A microarray analysis of peripheral blood mononuclear cells indicated that Ad5-porIFNα treatment led to clear upregulation in immune and inflammatory responses probably involved in protection against disease. Our results indicate that administration of Ad5-porIFNα can potentially be used to limit the spread of EBOV in pigs.

Characterization of Reston virus infection in ferrets.

Among the five currently recognized type viruses within the genus Ebolavirus, Reston virus (RESTV) is not known to cause disease in humans, although asymptomatic infections have been confirmed in the past. Intriguingly, despite the absence of pathogenicity in humans, RESTV is highly lethal to nonhuman primates and has been isolated from domestic pigs co-infected with other viruses in the Philippines and China. Whether infection in these animals can support the eventual emergence of a human-pathogenic RESTV remains unclear and requires further investigation. Unfortunately, there is currently no lethal small animal model available to investigate RESTV pathogenicity or pan-ebolavirus therapeutics. Here we show that wild type RESTV is uniformly lethal in ferrets. In this study, ferrets were challenged with 1260 TCID50 of wild type RESTV either intramuscularly or intranasally and monitored for clinical signs, survival, virus replication, alteration in serum biochemistry and blood cell counts. Irrespective of the route of challenge, viremia occurred in all ferrets on day 5 post-infection, and all animals succumbed to infection between days 9 and 11. Additionally, several similarities were observed between this model and the other ferret models of filovirus infection, including substantial decreases in lymphocyte and platelet counts and abnormalities in serum biochemistry indicating hepatic injury. The ferret model represents the first uniformly lethal model for RESTV infection, and it will undoubtedly prove useful for evaluating virus pathogenicity as well as pan-ebolavirus countermeasures.

Detection of genome, antigen, and antibodies in oral fluids from pigs infected with foot-and-mouth disease virus.

Virus nucleic acids and antibody response to pathogens can be measured using swine oral fluids (OFs). Detection of foot-and-mouth disease virus (FMDV) genome in swine OFs has previously been demonstrated. Virus isolation and viral antigen detection are additional confirmatory assays for diagnosing FMDV, but these methods have not been evaluated using swine OF. The objectives of this study were to further validate the molecular detection of FMDV in oral fluids, evaluate antigen detection and FMDV isolation from swine OFs, and develop an assay for isotypic anti-FMDV antibody detection in OFs. Ribonucleic acid (RNA) from FMDV was detected in OFs from experimentally infected pigs by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) from 1 day post-infection (dpi) to 21 dpi. Foot-and-mouth disease virus (FMDV) was isolated from OFs at 1 to 5 dpi. Additionally, FMDV antigens were detected in OFs from 1 to 6 dpi using a lateral flow immunochromatographic strip test (LFIST), which is a rapid pen-side test, and from 2 to 3 dpi using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). Furthermore, FMDV-specific immunoglobulin A (IgA) was detected in OFs using an isotype-specific indirect ELISA starting at dpi 14. These results further demonstrated the potential use of oral fluids for detecting FMDV genome, live virus, and viral antigens, as well as for quantifying mucosal IgA antibody response.

Chez les porcs les acides nucléiques viraux et la production d'anticorps contre des agents pathogènes peuvent être mesurés en utilisant les fluides oraux (FO). La détection du génome du virus de la fièvre aphteuse (VFA) dans les FO de porcs a été démontrée précédemment. L'isolement viral et la détection d'antigène viral sont des épreuves de confirmation supplémentaires pour diagnostiquer la présence du VFA, mais ces méthodes n'ont pas été évaluées en utilisant des FO porcins. Les objectifs de la présente étude étaient de valider un peu plus la détection moléculaire du VFA dans les FO, d'évaluer la détection d'antigènes et l'isolement du VFA à partir de FO porcins, et de développer une épreuve pour la détection d'anticorps isotypiques anti-VFA dans les FO. L'ARN du VFA fut détecté dans les FO de porcs infectés expérimentalement par réaction quantitative en temps réel d'amplification en chaine par la polymérase utilisant la transcriptase réverse à partir du jour 1 post-infection (PI) jusqu'au jour 21 PI. Le VFA fut isolé à partir des FO aux jours 1 à 5 PI. De plus, les antigènes du VFA ont été détectés dans les FO des jours 1 à 6 PI en utilisant une épreuve sur bandelette d'immunochromatographie par flot latéral, un test rapide pouvant être réalisé à la ferme, ainsi que de 2 à 3 j PI en utilisant une épreuve immuno-enzymatique (ELISA) double-sandwich. Également, à partir du jour 14 PI des immunoglobulines A (IgA) spécifiques au VFA ont été détectées dans les FO au moyen d'une épreuve ELISA indirecte spécifique pour les isotypes. Ces résultats démontrent d'une manière additionnelle le potentiel d'utilisation des FO pour détecter le génome du VFA, du virus vivant, et des antigènes viraux, de même que pour quantifier la production d'IgA par les muqueuses.(Traduit par Docteur Serge Messier).

Localization of annexins A1 and A2 in the respiratory tract of healthy calves and those experimentally infected with Mannheimia haemolytica.

Annexins A1 and A2 are proteins known to function in the stress response, dampening inflammatory responses and mediating fibrinolysis. We found, in healthy cattle recently arrived to a feedlot, that lower levels of these proteins correlated with later development of pneumonia. Here we determine the localization of annexin A1 and A2 proteins in the respiratory tract and in leukocytes, in healthy calves and those with Mannheimia haemolytica pneumonia. In healthy calves, immunohistochemistry revealed cytoplasmic expression of annexin A1 in the surface epithelium of large airways, tracheobronchial glands and goblet cells, to a lesser degree in small airways, but not in alveolar epithelium. Immunocytochemistry labeled annexin A1 in the cytoplasm of neutrophils from blood and bronchoalveolar lavage fluid, while minimal surface expression was detected by flow cytometry in monocytes, macrophages and lymphocytes. Annexin A2 expression was detected in surface epithelium of small airways, some mucosal lymphocytes, and endothelium, with weak expression in large airways, tracheobronchial glands and alveolar septa. For both proteins, the level of expression was similar in tissues collected five days after intrabronchial challenge with M. haemolytica compared to that from sham-inoculated calves. Annexins A1 and A2 were both detected in leukocytes around foci of coagulative necrosis, and in necrotic cells in the center of these foci, as well as in areas outlined above. Thus, annexins A1 and A2 are proteins produced by airway epithelial cells that may prevent inflammation in the healthy lung and be relevant to development of pneumonia in stressed cattle.

Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves.

Strategies to control bovine respiratory disease depend on accurate classification of disease risk. An objective method to refine the risk classification of beef calves could be economically beneficial, improve welfare by preventing unexpected disease occurrences, refine and reduce the use of antibiotics in beef production, and facilitate alternative methods of disease control. The objective of this study was to identify proteins in bronchoalveolar lavage fluid (BALF) of stressed healthy calves that predict later disease outcome, serve as biomarkers of susceptibility to pneumonia, and play a role in pathogenesis. BALF was collected from 162 healthy beef calves 1-2 days after weaning and transportation. Difference in gel electrophoresis (DIGE) and mass spectrometry were used to compare proteins in samples from 7 calves that later developed respiratory disease compared to 7 calves that remained healthy. Calves that later developed pneumonia had significantly lower levels of annexin A1, annexin A2, peroxiredoxin I, calcyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3. Differences in annexin levels were partially confirmed by western blot analysis. Thus, lower levels of annexins A1 and A2 are potential biomarkers of increased susceptibility to pneumonia in recently weaned and transported feedlot cattle. Since annexins are regulated by glucocorticoids, this finding may reflect individual differences in the stress response that predispose to pneumonia. These findings also have implications in pathogenesis. Annexins A1 and A2 are known to prevent neutrophil influx and fibrin deposition respectively, and may thus act to minimize the harmful effects of the inflammatory response during development of pneumonia.