The efficacy of intravenous application of the Mycobacterium Cell Wall Fraction (AMPLIMUNE) in the treatment of subclinical mastitis caused by S. Aureus.

The aim of the study was to investigate the effect of the immunostimulant Mycobacterium Cell Wall Fraction (MCWF) on the treatment of S. aureus SCM by intravenous application. The study included 45 HF dairy cows in 2nd and 3rd month after parturition divided into three groups (n = 15 per group): the MC + group - cows with S. aureus SCM treated with MCWF; the MC- group - cows with S. aureus SCM, with no treatment; and the C group - the control group of healthy cow with no treatment. Samples were collected 0th (I sample), 7th (II), and 14th day (III) from the day of SCM diagnosis and on day 21st (IV). A greater influx of leukocytes was confirmed into milk after 7 days after MCWF treatment in MC + group, which was followed by increase of WBC and LYM in blood. These results support the hypothesis of effective action of MCWF, and in quarters with lower-grade infection, bacteriological cure was achieved. The MC- group had a statistically higher concentration of TBARS and CAT activity in milk, while MC + group had lower blood serum LDH activity, which indicates a positive effect of the MCWF application and a lower exposure of the tissue to lipide peroxidation and inflammation caused by S. aureus. The application of MCWF would give new possibilities in the prevention and therapy of mammary gland diseases without fear of the presence of residues and the emergence of bacterial resistance. In future studies, the effects of local and systemic application of MCWF in the treatment of S. aureus SCM should be compared.

Genealogy of an in-vivo passaged isolate of western Canadian bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a primary respiratory pathogen in calves. Clinical infection with this pathogen has been experimentally modelled to assess vaccine efficacy using a field isolate (Asquith) of BRSV that has been sequentially passaged in vivo in neonatal calves to maintain virulence. The objective of this retrospective cumulative analysis of passages over approximately 20 years was to determine if there have been any changes in the viral genome of this isolate because of this process. Sequence analyses indicated that the Asquith isolate placed genetically in a clade comprising US and some European isolates and a recently described Chinese BRSV isolate (DQ). Furthermore, there were rare changes in bases over time in the N, G, and F gene segments examined when comparing among different passages ranging from 1996 to 2019. These results indicated the absence of significant mutations in the absence of significant adaptive immunological pressure.

Le virus respiratoire syncitial bovin (BRSV) est un agent pathogène respiratoire primaire chez les veaux. Une infection clinique avec cet agent pathogène a été expérimentalement modélisée pour évaluer l'efficacité vaccinale en utilisant un isolat de champ (Asquith) de BRSV qui a été passé séquentiellement in vivo chez des veaux nouveau-nés pour maintenir sa virulence. L'objectif de cette analyse rétrospective cumulative des passages sur une période d'approximativement 20 ans était de déterminer s'il y avait eu des changements dans le génome viral de cet isolat à cause de ce processus. L'analyse des séquences indiquaient que l'isolat Asquith se positionnait génétiquement dans un clade comprenant des isolats américains et quelques isolats européens et un isolat chinois de BRSV récemment décrit (DQ). Également, il y avait de rares changements de bases dans le temps dans les segments de gènes N, G et F examinés lors de la comparaison parmi les différents passages allant de 1996 à 2019. Ces résultats indiquent l'absence de mutation significative en absence de pression immunologique adaptative significative.(Traduit par Docteur Serge Messier).

Efficacy of Souroubea-Platanus Dietary Supplement Containing Triterpenes in Beagle Dogs Using a Thunderstorm Noise-Induced Model of Fear and Anxiety.

A novel botanical dietary supplement, formulated as a chewable tablet containing a defined mixture of Souroubea spp. vine and Platanus spp. Bark, was tested as a canine anxiolytic for thunderstorm noise-induced stress (noise aversion). The tablet contained five highly stable triterpenes and delivered 10 mg of the active ingredient betulinic acid (BA) for an intended 1 mg/kg dose in a 10 kg dog. BA in tablets was stable for 30 months in storage at 23 °C. Efficacy of the tablets in reducing anxiety in dogs was assessed in a blinded, placebo-controlled study by recording changes in blood cortisol levels and measures of behavioral activity in response to recorded intermittent thunder. Sixty beagles were assigned into groups receiving: placebo, 0.5×, 1×, 2×, and 4× dose, or the positive control (diazepam), for five days. Reduction in anxiety measures was partially dose-dependent and the 1× dose was effective in reducing inactivity time (p = 0.0111) or increased activity time (p = 0.0299) compared with placebo, indicating a decrease in anxiety response. Cortisol measures also showed a dose-dependent reduction in cortisol in dogs treated with the test tablet.

Influenza A Virus in Swine: Epidemiology, Challenges and Vaccination Strategies.

Influenza A viruses cause acute respiratory infections in swine that result in significant economic losses for global pig production. Currently, three different subtypes of influenza A viruses of swine (IAV-S) co-circulate worldwide: H1N1, H3N2, and H1N2. However, the origin, genetic background and antigenic properties of those IAV-S vary considerably from region to region. Pigs could also have a role in the adaptation of avian influenza A viruses to humans and other mammalian hosts, either as intermediate hosts in which avian influenza viruses may adapt to humans, or as a "mixing vessel" in which influenza viruses from various origins may reassort, generating novel progeny viruses capable of replicating and spreading among humans. These potential roles highlight the importance of controlling influenza A viruses in pigs. Vaccination is currently the main tool to control IAV-S. Vaccines containing whole inactivated virus (WIV) with adjuvant have been traditionally used to generate highly specific antibodies against hemagglutinin (HA), the main antigenic protein. WIV vaccines are safe and protect against antigenically identical or very similar strains in the absence of maternally derived antibodies (MDAs). Yet, their efficacy is reduced against heterologous strains, or in presence of MDAs. Moreover, vaccine-associated enhanced respiratory disease (VAERD) has been described in pigs vaccinated with WIV vaccines and challenged with heterologous strains in the US. This, together with the increasingly complex epidemiology of SIVs, illustrates the need to explore new vaccination technologies and strategies. Currently, there are two different non-inactivated vaccines commercialized for swine in the US: an RNA vector vaccine expressing the HA of a H3N2 cluster IV, and a bivalent modified live vaccine (MLV) containing H1N2 γ-clade and H3N2 cluster IV. In addition, recombinant-protein vaccines, DNA vector vaccines and alternative attenuation technologies are being explored, but none of these new technologies has yet reached the market. The aim of this article is to provide a thorough review of the current epidemiological scenario of IAV-S, the challenges faced in the control of IAV-S infection and the tools being explored to overcome those challenges.

Effects of the Administration of a Non-specific Immune Stimulant Around Transportation on Health and Performance of Jersey and Jersey-Cross Heifer Calves During the Rearing Period: Randomized Clinical Trial.

Our objective was to evaluate the effects of a non-specific immune stimulant (IS) administered around transportation on health scores (HS), average daily gain (ADG), disease treatment and mortality of Jersey and Jersey-cross calves during the rearing period. Newborn calves (4 d ± 1) were randomly allocated to receive either 1 mL of saline (CON; n = 438), 1 mL of IS before transport (BTIS; n = 431), or 1 mL of IS immediately after transport (ATIS; n = 436). Calves were health scored weekly for 3 weeks after transport. The data were analyzed using multivariable linear mixed models and multivariable logistic regression models. Kaplan-Meier survival analysis was performed for time to event analysis. Treatment, birth weight, breed, site of birth, serum total solids, dam parity, season of enrollment, and metaphylaxis were offered to models. Differences in respiratory and fecal HS, and ADG between treatment groups were not statistically significant. A total of 196 (15.0%) calves were treated at least once for any disease and 52 calves were treated multiple times. The proportion of calves treated for respiratory disease and/or diarrhea were 14.4, 14.4, and 16.2% for BTIS, ATIS and CON groups, respectively. Although the differences in the likelihood of treatment for both respiratory disease and/or diarrhea during the first 9 weeks of life was not statistically different between groups, we observed that more calves in the control group received disease treatments around 15 days of age compared with calves that received IS. The likelihood of treatment for respiratory diseases alone during the first 30 days of life was smaller in the calves that received IS before transportation when compared to the control group. Only 18 (1.4%) calves died within the study period. The calf mortality likelihood was not statistically different between study groups; however, fewer calves in the IS groups died when compared to CON. In conclusion, the use of IS around transportation did not influence weekly HS, ADG, and the number of disease treatments during the rearing period, but administering IS before transportation resulted in fewer treatments of respiratory diseases during the first 30 days post-transport and marginally lower mortality rates during the rearing period.

Imepitoin Shows Benzodiazepine-Like Effects in Models of Anxiety.

Imepitoin is a low affinity partial agonist for the benzodiazepine binding site of γ-aminobutyric acid (GABAA) receptors, and is currently used as an antiepileptic in dogs. Here we tested imepitoin for anxiolytic properties. In an in vitro model, imepitoin was capable of preventing the effect of corticotrophin releasing factor (CRF) on locus coeruleus neurons without suppressing the basal activity of these cells, an activity which is suggestive for an anti-stress effect of imepitoin. In addition, we applied a battery of standard rodent preclinical tests for anxiety behavior including elevated plus mazes in mice and rats, light-dark-box in mice and rats, social interaction test in rats, or the Vogel conflict test in rats. In all models, the observed profile of imepitoin appeared similar to benzodiazepines and typical for anxiolytic drugs. We also observed anxiolytic activity in dogs in a provoked open field sound-induced fear model, where reactions to noises were elicited by a sound recording of thunderstorms. Imepitoin caused an increase in locomotion measured in distance traveled and an ameliorating effect on cortisol levels in response to thunderstorm noises. For comparison, dexmedetomidine caused a decrease in locomotion and had no effect on cortisol. In all animal models the doses needed for an anxiolytic effect were not associated with sedation. In rodents, there was at least a factor of 10 between anxiolytic doses and doses with mild signs of sedation. In summary, imepitoin showed similar anxiolytic activities as benzodiazepines but without producing the known adverse reactions of benzodiazepines such as sedation.

Safety evaluation of a new anxiolytic product containing botanicals Souroubea spp. and Platanus spp. in dogs.

Separation anxiety and noise aversion are common behavioral problems in dogs. They elicit fear responses such as cowering, seeking out the owner, and attempting to escape. This can result in property damage, injury to the dog, and disruption of the owner-pet bond, possibly leading to pet abandonment or euthanasia. A novel botanical anxiolytic product was evaluated for safety in dogs as the target animal species. Its intended use is for the treatment and prevention of anxiety and noise aversion in dogs. It contains a defined mixture of Souroubea spp. vine and Platanus spp. bark, delivering the active principle, betulinic acid, at a recommended dose of 1 mg/kg body weight (BW). In the current target animal safety study, 16 healthy male beagle dogs were administered either a placebo or the newly formulated botanical tablets at 0.5×, 2.5×, or 5× the recommended dose (1 mg/kg BW) over 28 d. The dogs were monitored for occurrence of any systemic or local adverse events. In the investigation presented here, there were no clinically significant adverse effects following treatment, as determined by clinical observations, physical examinations, BW, hematology, clinical biochemistry, and urinalysis. Pharmacokinetic analysis demonstrated that the concentration of betulinic acid in serum was below 0.020 µg/mL in treated animals. Under the conditions of these studies, the formulated blend of S. sympetala and P. occidentalis, when administered up to 5× the intended dose for 28 consecutive d, showed no adverse effects on the health of dogs.

L'anxiété de séparation et une aversion au bruit sont des problèmes de comportement fréquents chez les chiens. Elles élicitent des réponses de peur telles que des tremblements, la recherche du propriétaire, et une tentative de fuite. Elles peuvent résulter en des dommages à la propriété, des blessures au chien, et un bris du lien propriétaire-animal, pouvant potentiellement mener à l'abandon de l'animal ou l'euthanasie. Un nouveau produit anxiolytique botanique a été évalué pour sa sécurité chez les chiens, l'espèce animale cible. Son utilisation visée est pour le traitement et la prévention de l'anxiété et de l'aversion au bruit chez les chiens. Le produit contient un mélange défini de vigne de Souroubea spp. et d'écorce de Platanus spp., fournissant le principe actif, l'acide bétulinique, à un dosage recommandé de 1 mg/kg de poids corporel (PC). Dans l'étude de sécurité chez l'espèce animale cible, 16 chiens mâles de race beagle en santé ont reçu soit un placebo ou les nouvelles tablettes botaniques à 0,5×, 2,5×, ou 5× la dose recommandée (1 mg/kg PC) pendant 28 jours. Les chiens ont été observés pour l'apparition de manifestions adverses systémiques ou locales. Dans l'étude présentée ici, il n'y eut aucun effet clinique adverse significatif suivant le traitement, tel que déterminé par les observations cliniques, les examens physiques, le PC, et les résultats des analyses hématologiques, de biochimie clinique et urinaires. L'analyse pharmacocinétique a démontré que la concentration d'acide bétulinique dans le sérum était moins de 0,020 µg/mL chez les animaux traités. Dans les conditions des présentes études, le mélange de S. sympetala et de P. occidentalis, lorsqu'administré jusqu'à 5× le dosage prévu pendant 28 jours consécutifs, n'a démontré aucun effet adverse sur la santé des chiens.(Traduit par Docteur Serge Messier).

Neutrophil phagocytosis and respiratory burst activity of dairy cows during the transition period and early lactation.

INTRODUCTION: Hormonal and metabolic changes, as well as energy imbalance, can affect health, production and reproductive performance of dairy cows. In the present study, we evaluated phagocytosis and respiratory burst neutrophil activity during the transition period and early lactation and compared it with biochemical and hematological parameters in dairy cows. METHODOLOGY: Simmental cows (n = 21) were enrolled in the study. Whole blood samples were collected weekly from 3 weeks pre- calving until 6 weeks post calving. Basic metabolic and blood parameters were assessed by routine laboratory analyses, while neutrophil functions were analyzed by commercial test kits. RESULTS: Optimal neutrophil response was observed pre and post calving. The highest value was recorded in the 6th week after calving (89.54 ± 7.61%) and being significantly higher (p < 0.01) as compared to values recorded at two and one week before and one week after calving. The percentage of activated neutrophils was high during the entire study period: from 70.80 ± 5.22% at the beginning of the study to 89.54 ± 7.61% at the end of the study. During the study period, production of Reactive Oxidative Species by neutrophils was positively correlated with ß-hydroxybutyrat and non-esterified fatty acids values (0.454** and 0.423**, respectively) and calcium levels (0.164* and 0.212**, respectively). CONCLUSIONS: The most prominent changes in all parameters had no influence on phagocytic and respiratory burst activity of neutrophils. Neutrophil function is preserved at the optimal level during the transition period and early lactation in Simmental cows.

New Botanical Anxiolytics for Use in Companion Animals and Humans.

As part of our ongoing research into botanical therapies for anxiety disorders, the neotropical vine Souroubea sympetala was chosen for study as a phytochemical discovery strategy focusing on rare Central American plant families. When orally administered to male Sprague-Dawley rats, the crude plant extract, its ethyl acetate fraction, supercritical carbon dioxide fraction, or its isolated triterpenes reduced anxiety and/or fear-related behavior in standardized behavioral models. Pharmacological studies showed that the extracts acted at the benzodiazepine GABAA receptor and reduced corticosterone levels. A preparation containing Souroubea fortified with a second triterpene containing plant, Platanus occidentalis, was shown to be safe in a 28-day feeding trial with beagles at 5 times the intended dose. Subsequent trials with beagles in a thunderstorm model of noise aversion showed that the material reduced anxiety behaviors and cortisol levels in dogs. The formulation has been released for the companion animal market in Canada and the USA under the Trademark "Zentrol." Ongoing research is exploring the use of the material in treatment of anxiety and post-traumatic stress in humans.

An eight-segment swine influenza virus harboring H1 and H3 hemagglutinins is attenuated and protective against H1N1 and H3N2 subtypes in pigs.

Swine influenza virus (SIV) infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method of controlling SIV is through vaccination. The killed SIV vaccines currently in use must be closely matched to the challenge virus, and their protective efficacy is limited. Live attenuated influenza vaccines (LAIV) provide strong, long-lived cell-mediated and humoral immunity against different influenza virus subtypes with no need for antigen matching. Here we report the generation of a new potential LAIV, an eight-segment SIV harboring two different SIV hemagglutinins (HAs), H1 and H3, in the genetic background of H1N1 SIV. This mutant SIV was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1) SIV. While this H1-H3 chimeric SIV, when propagated in vitro in the presence of exogenous neuraminidase, showed kinetics and growth properties similar to those of the parental wild-type virus, in vivo it was highly attenuated in pigs, demonstrating a great potential for serving as a dual LAIV. Furthermore, vaccination with the H1-H3 virus elicited robust immune responses, which conferred complete protection against infections with both H1 and H3 SIV subtypes in pigs.

Pandemic H1N1 influenza virus-like particles are immunogenic and provide protective immunity to pigs.

The outbreak of the 2009 influenza pandemic underscored the important role of swine in influenza virus evolution and the emergence of novel viruses with pandemic potential. Vaccination is the most common practice to control swine influenza in swine industry. Influenza virus-like particle (VLP) vaccines are an alternative approach and have been demonstrated to be immunogenic and confer protection against influenza virus challenge in chickens, mice and ferrets. In this study, we generated VLPs consisting of HA, NA and M1 proteins derived from pandemic virus A/California/04/2009 in insect cells. The immunogenicity and efficacy following vaccination of VLPs were evaluated in swine. Our data showed that vaccination using VLPs elicited robust levels of serum IgG, mucosal IgA, and viral neutralizing antibodies against A/Sw/Manitoba/MAFRI32/2009 H1N1. Following challenge with pandemic H1N1 2009, vaccinated pigs were protected, displaying reduced lung lesions, virus shedding and inhibition of virus replication in the lungs compared to non-vaccinated control pigs. Thus, VLPs can serve as a promising vaccination strategy to control influenza in swine.

Regulation of influenza A virus induced CXCL-10 gene expression requires PI3K/Akt pathway and IRF3 transcription factor.

Influenza A virus infects human airway epithelial cells, and induces the CXC chemokine gamma interferon (IFN-γ)-inducible protein CXCL-10/IP-10 production. To understand the regulation of CXCL-10, we investigated the role of PI3K/AKT pathway in regulating virus induced CXCL-10 production. Previously we have shown that wild type (WT) influenza A virus infection activates PI3K/AKT pathway, whereas PR8-SH3-mf-1 mutant virus is unable to activate this pathway. Here we report that WT influenza A virus infection induced CXCL-10 production in A549 cells. PR8-SH3-mf-1 mutant virus infection led to reduced level of CXCL-10 mRNA transcription and protein expression. To define the transcriptional regulation factors that are important in this process, we performed studies using several mutant CXCL-10 promoter-luciferase constructs. Mutation of either of four Forkhead binding sites and two NF-κB response elements in CXCL-10 promoter did not alter promoter activity induced by WT virus. However, mutation of ISRE binding site markedly reduced luciferase activity. Our data suggested that PI3K/AKT pathway contributes to influenza A virus induced CXCL-10 production. This process is involved in binding of IRF3 to the ISRE binding site in CXCL-10 promoter region.

An elastase-dependent attenuated heterologous swine influenza virus protects against pandemic H1N1 2009 influenza challenge in swine.

Influenza virus infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method to control influenza is through vaccination. However, currently used killed influenza virus vaccines must be closely matched to the challenge virus. The ability of an elastase-dependent live attenuated influenza A virus was evaluated to protect pigs against the pandemic H1N1 2009 influenza virus. Pigs vaccinated intranasally or intratracheally with the elastase-dependent swine influenza virus (SIV) vaccine had significantly reduced macroscopic and microscopic lung lesions and lower viral loads in the lung and in nasal swabs. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs. In addition, low levels of cross-neutralizing antibodies to H1N1 2009 were elicited prior to challenge by the swine adapted H1N1 avian strain vaccine.

Immunogenicity and protective efficacy of an elastase-dependent live attenuated swine influenza virus vaccine administered intranasally in pigs.

Influenza A virus is an important respiratory pathogen of swine that causes significant morbidity and economic impact on the swine industry. Vaccination is the first choice for prevention and control of influenza infections. Live attenuated influenza vaccines (LAIV) are approved for use in humans and horses and their application provides broad protective immunity, however no LAIV against swine influenza virus (SIV) exists in the market. Previously we reported that an elastase-dependent mutant SIV A/Sw/Sk-R345V (R345V) derived from A/Sw/Saskatchewan/18789/02 (H1N1) (SIV/Sk02) is highly attenuated in pigs. Two intratracheal administrations of R345V induced strong cell-mediated and humoral immune responses and provided a high degree of protection to antigenically different SIV infection in pigs. Here we evaluated the immunogenicity and the protective efficacy of R345V against SIV infection by intranasal administration, the more practical route for vaccination of pigs in the field. Our data showed that intranasally administered R345V live vaccine is capable of inducing strong antigen-specific IFN-γ response from local tracheo-bronchial lymphocytes and antibody responses in serum and respiratory mucosa after two applications. Intranasal vaccination of R345V provided pigs with complete protection not only from parental wild type virus infection, but also from homologous antigenic variant A/Sw/Indiana/1726/88 (H1N1) infection. Moreover, intranasal administration of R345V conferred partial protection from heterologous subtypic H3N2 SIV infection in pigs. Thus, R345V elastase-dependent mutant SIV can serve as a live vaccine against antigenically different swine influenza viruses in pigs.

The PI3K/Akt pathway inhibits influenza A virus-induced Bax-mediated apoptosis by negatively regulating the JNK pathway via ASK1.

It has previously been reported that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. In addition, it has been shown that the mutant influenza A virus PR8-SH3-mf-1, which is unable to activate the PI3K/Akt pathway, is more pro-apoptotic than the wild-type (WT) virus. However, the molecular pathways involved in regulating this process remain unknown. Here, it is reported that, although both WT and PR8-SH3-mf-1 viruses induced apoptosis, the PR8-SH3-mf-1 virus consistently showed greater potential to induce mitochondrial membrane disruption, cytochrome c release, and translocation and conformational change of Bax than the WT virus. Furthermore, the PR8-SH3-mf-1 virus was unable to phosphorylate apoptosis signal-regulating kinase 1 (ASK1) but induced higher levels of c-jun N-terminal kinase (JNK) phosphorylation than the WT virus. Blocking JNK activity could inhibit virus-induced Bax activation and apoptosis. These results reveal that, during influenza A virus infection, the PI3K/Akt pathway negatively regulates the JNK pathway via ASK1, thereby inhibiting JNK-dependent, Bax-mediated apoptosis.

Elastase-dependent live attenuated swine influenza A viruses are immunogenic and confer protection against swine influenza A virus infection in pigs.

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.

Reverse genetics-generated elastase-dependent swine influenza viruses are attenuated in pigs.

Influenza A virus causes significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. It has been shown that conversion of the haemagglutinin (HA) cleavage site from a trypsin-sensitive motif to an elastase-sensitive motif resulted in attenuated viruses in mouse models. However, application of this attenuation approach in a natural host has not been achieved yet. Here, we report that using reverse genetics, we generated two mutant SIVs derived from strain A/SW/SK/18789/02 (H1N1). Mutant A/SW/SK-R345V carries a mutation from arginine to valine at aa 345 of HA. Similarly, mutant A/SW/SK-R345A encodes alanine instead of arginine at aa 345 of HA. Our data showed that both mutants are solely dependent on neutrophil elastase cleavage in tissue culture. These tissue culture-grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics to the wild-type virus. In addition, SIV mutants were able to maintain their genetic information after multiple passaging on MDCK cells. Furthermore, mutant SIVs were highly attenuated in pigs. Thus, these mutants may have the potential to serve as live attenuated vaccines.