Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

Mechanosensitive channel gating by delipidation.

The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-ß-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.

Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.

Structural Investigation on How Guest Loading of Poly(2-oxazoline)-Based Micelles Affects the Interaction with Simulated Intestinal Fluids.

Drug loading of polymer micelles can have a profound effect on their particle size and morphology as well as their physicochemical properties. In turn, this influences performance in biological environments. For oral delivery of drugs, the intestinal environment is key, and consequently, a thorough structural understanding of what happens at this material-biology interface is required to understand in vivo performance and tailor improved delivery vehicles. In this study, we address this interface in vitro through a detailed structural characterization of the colloidal assemblies of polymeric micelles based on poly(2-oxazolines) with three different guest loadings with the natural product curcumin (17-52 wt %) in fed-state simulated intestinal fluids (FeSSIF). For this, we employ NMR spectroscopy, in particular, 1H NMR, 1H-1H-NOESY, and 1H DOSY experiments complemented by quantum chemical calculations and cryo-TEM measurements. Through this mixture of methods, we identified curcumin-taurocholate interactions as central interaction patterns alongside interactions with the polymer and lipids. Furthermore, curcumin molecules can be exchanged between polymer micelles and bile colloids, an important prerequisite for their uptake. Finally, increased loading of the polymer micelles with curcumin resulted in a larger number of vesicles as taurocholate─through coordination with Cur─is less available to form nanoparticles with the lipids. The loading-dependent behavior found in this study deviates from previous work on a different drug substance highlighting the need for further studies including different drug molecules and polymer types to improve the understanding of events on the molecular level.

The nucleotide excision repair (NER) system of Helicobacter pylori: role in mutation prevention and chromosomal import patterns after natural transformation.

BACKGROUND: Extensive genetic diversity and rapid allelic diversification are characteristics of the human gastric pathogen Helicobacter pylori, and are believed to contribute to its ability to cause chronic infections. Both a high mutation rate and frequent imports of short fragments of exogenous DNA during mixed infections play important roles in generating this allelic diversity. In this study, we used a genetic approach to investigate the roles of nucleotide excision repair (NER) pathway components in H. pylori mutation and recombination. RESULTS: Inactivation of any of the four uvr genes strongly increased the susceptibility of H. pylori to DNA damage by ultraviolet light. Inactivation of uvrA and uvrB significantly decreased mutation frequencies whereas only the uvrA deficient mutant exhibited a significant decrease of the recombination frequency after natural transformation. A uvrC mutant did not show significant changes in mutation or recombination rates; however, inactivation of uvrC promoted the incorporation of significantly longer fragments of donor DNA (2.2-fold increase) into the recipient chromosome. A deletion of uvrD induced a hyper-recombinational phenotype. CONCLUSIONS: Our data suggest that the NER system has multiple functions in the genetic diversification of H. pylori, by contributing to its high mutation rate, and by controlling the incorporation of imported DNA fragments after natural transformation.

Efficacy of a Modified Live Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) Vaccine against Experimental Infection with PRRSV AUT15-33 in Weaned Piglets.

In this study, the efficacy of the commercial modified live PRRSV-1 vaccine "Ingelvac PRRSFLEX® EU" was assessed in weaned piglets experimentally infected with PRRSV strain AUT15-33. Seventy-four weaned piglets were allocated to five groups. Vaccinated (groups 1, 2, and 5) and non-vaccinated piglets (groups 3 and 4), infected with either a low dose (103 TCID50/dose; groups 2 and 4) or a high dose (105 TCID50/dose; groups 1 and 3) of the virus, were compared regarding clinical signs, average daily weight gain (ADG), lung lesions, viral load in serum, oral swabs, and tissue samples. In comparison to vaccinated animals, coughing increased notably in the second week after challenge in non-vaccinated piglets. During the same time period, vaccinated, high-dose-infected piglets showed significantly higher ADG (p < 0.05) than non-vaccinated, high-dose-infected animals. All infected piglets reached approximately the same viremia levels, but vaccinated animals showed both a significantly reduced viral load in oral fluid (p < 0.05) and tissue samples and significantly reduced lung lesions (p < 0.05). In conclusion, vaccination was able to increase ADG, reduce the amount of viral shedding via oral fluids, and reduce the severity of lung lesions and the viral load in tissue samples under experimental conditions.

Concentration and composition dependent aggregation of Pluronic- and Poly-(2-oxazolin)-Efavirenz formulations in biorelevant media.

Many drugs and drug candidates are poorly water-soluble. Intestinal fluids play an important role in their solubilization. However, the interactions of intestinal fluids with polymer excipients, drugs and their formulations are not fully understood. Here, diffusion ordered spectroscopy (DOSY) and nuclear Overhauser effect spectroscopy (NOESY), complemented by cryo-TEM were employed to address this. Efavirenz (EFV) as model drug, the triblock copolymers Pluronic® F-127 (PF127) and poly(2-oxazoline) based pMeOx-b-pPrOzi-b-pMeOx (pOx/pOzi) and their respective formulations were studied in simulated fed-state intestinal fluid (FeSSIF). For the individual polymers, the bile interfering nature of PF127 was confirmed and pure pOx/pOzi was newly classified as non-interfering. A different and more complex behaviour was however observed if EFV was involved. PF127/EFV formulations in FeSSIF showed concentration dependent aggregation with separate colloids at low formulation concentrations, a merging of individual particles at the solubility limit of EFV in FeSSIF and joint aggregates above this concentration. In the case of pOx/pOzi/EFV formulations, coincident diffusion coefficients for pOx/pOzi, lipids and EFV indicate joint aggregates across the studied concentration range. This demonstrates that separate evaluation of polymers and drugs in biorelevant media is not sufficient and their mixtures need to be studied to learn about concentration and composition dependent behaviour.

Effects of three commercial vaccines against porcine parvovirus 1 in pregnant gilts.

Porcine parvovirosis is a common and important cause of reproductive failure in naïve dams. Even though vaccination is generally effective at preventing disease occurrence, the homology between the vaccine and challenge strains has been recently suggested to play a role in protection. Therefore, the purpose of this study was to evaluate and compare the efficacy of three currently available commercial vaccines against porcine parvovirus genotype 1 (PPV1) in an experimental model using pregnant gilts. Seventy-seven PPV1-negative gilts were included in the trial and randomly allocated to four groups. In group 1, gilts received two doses, three weeks apart, of a PPV1 subunit vaccine (ReproCyc® ParvoFLEX). Following the same scheme, gilts from group 2 received two doses of a PPV1 bivalent vaccine (ERYSENG® PARVO). In group 3, gilts received two doses, four weeks apart, of a PPV1 octavalent vaccine (Porcilis® Ery + Parvo + Lepto). Lastly, gilts from group 4 were left untreated and were used as challenge controls. All gilts were artificially inseminated three weeks after completion of vaccination. Pregnant animals were subsequently challenged around 40 days of gestation with a heterologous PPV1 strain. Foetuses were harvested at around day 90 of gestation and evaluated for their macroscopic appearance (i.e., normal, mummified, or autolytic). Along the study, safety parameters after vaccination, antibody responses against PPV1 and viremia in gilts were also measured. All the foetuses in the challenge control group were mummified, which validated the challenge model, whereas the three evaluated vaccines protected the progeny against PPV1 by preventing the appearance of clinical manifestations associated to parvovirosis. Remarkably, the PPV1 subunit vaccine induced an earlier seroconversion of gilts and was the only vaccine that could prevent viremia after challenge. This vaccine also achieved the largest average litter size accompanied with a high average proportion of clinically healthy foetuses.

In Situ Hybridization of PRRSV-1 Combined with Digital Image Analysis in Lung Tissues of Pigs Challenged with PRRSV-1.

Betaarterivirus suid 1 and 2 are the causative agents of porcine reproductive and respiratory syndrome (PRRS), which is one of the most significant diseases of the swine industry, causing significant economic losses in the main pig producing countries. Here, we report the development of a novel, RNA-based in situ hybridization technique (RNAscope) to detect PRRS virus (PRRSV) RNA in lung tissues of experimentally infected animals. The technique was applied to lung tissues of 20 piglets, which had been inoculated with a wild-type, highly pathogenic PRRSV-1 strain. To determine the RNAscope's applicability as a semi-quantitative method, we analysed the association between the proportion of the virus-infected cells measured with an image analysis software (QuPath) and the outcome of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed in parallel. The results of the quantitative approach of these two molecular biological methods show significant association (pseudo R2 = 0.3894, p = 0.004). This is the first time RNAscope assay has been implemented for the detection of PRRSV-1 in experimental animals.

Binding of a Pocket Factor to Hepatitis B Virus Capsids Changes the Rotamer Conformation of Phenylalanine 97.

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.

Gilt Vaccination with a Mixed Administration of a PRRS MLV and a PPV1 Subunit Vaccine Protects against Heterologous PRRSV1 Infection and Prevents Detrimental Effects on Piglet Performance.

The efficacy of the combined administration of a porcine reproductive and respiratory syndrome (PRRS) modified live virus (MLV) vaccine and a porcine parvovirus 1 (PPV1) subunit vaccine in gilts was addressed in two experiments. Experiment A aimed to establish a 4-week onset of immunity (OOI). Gilts were randomly distributed in three treatment groups: non-vaccinated control animals (group 1), animals vaccinated with the combined vaccine (group 2), and a third group that consisted of animals vaccinated with the PRRS MLV vaccine alone (group 3). Four weeks after the first vaccination, gilts were challenged with a heterologous PRRS virus 1 (PRRSV1) and euthanized three weeks after. Besides this, experiment B pursued a 17-week duration of immunity (DOI). In this case, gilts were distributed in the same treatment groups, but for the third group, which consisted of non-vaccinated, non-challenged animals were used instead. For the DOI assessment, gilts were artificially inseminated 4 weeks after the first vaccination, challenged at day 90 of gestation, and followed up, together with their offspring, until day 20 post-farrowing. Serology and viremia post-challenge were determined in gilts from both experiments, while farrowing and piglet performance were only evaluated in experiment B. Overall, the combined vaccine helped to protect gilts from viremia post-challenge and, consequently, to prevent PRRS clinical symptoms and diminish the proportion of piglets infected congenitally or early in life. The combined vaccine also elicited a significant improvement in piglet survival rate and growth performance until weaning. The present results reveal efficacy and lack of interference of the mixed use of the tested vaccines against PRRSV1 infection, with at least 4-week OOI and 17-week DOI.

Field evaluation of the safety and compatibility of a combined vaccine against porcine parvovirus 1 and porcine reproductive and respiratory syndrome virus in breeding animals.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus 1 (PPV1) are two common causes of reproductive failure. ReproCyc® ParvoFLEX is a novel subunit vaccine based on the protective viral protein (VP) 2 of PPV1 that has been recently licensed in the European (EU) market, whereas ReproCyc® PRRS EU is a porcine reproductive and respiratory syndrome (PRRS) modified live virus (MLV) vaccine authorized in 2015. The present work sought to evaluate the safety and compatibility of the combined administration of the abovementioned vaccines in target animals under the context of a field PRRSV (experiment A) and PPV1 (experiment B) infection. To achieve this objective, safety and lack of vaccines' antigen interference were established according to the absence of significant differences between the combined vaccinated animals (PPRSV+PPV1) and the single vaccinated animals against PRRSV or PPV1. In both experiments, gilts and sows were evaluated for local and systemic reactions after vaccination as well as for reproductive and productive performance. In addition, tissues from abortions, mummified fetuses and stillborn piglets were analyzed for the presence of PRRSV and PPV1. Lastly, serology and viremia were determined in experiment B. RESULTS: No relevant differences in terms of safety, reproductive and productive performance between the single vaccinated and the combined vaccinated animals in either experiment were observed. CONCLUSIONS: ReproCyc® PRRS EU mixed with ReproCyc® ParvoFLEX can be used as a safe method of protection against the detrimental effects of PRRSV and PPV1 infections in breeding female pigs in one single injection. The present results also open up opportunities to tackle reproductive problems as a whole by combining control programs against swine reproductive pathogens.

Evaluation of PRRSv specific, maternally derived and induced immune response in Ingelvac PRRSFLEX EU vaccinated piglets in the presence of maternally transferred immunity.

In this study, we analyzed PRRS virus (PRRSv) specific lymphocyte function in piglets vaccinated with Ingelvac PRRSFLEX EU® at two and three weeks of age in the presence of homologous maternal immunity. Complete analysis of maternal immunity to PRRSv was evaluated postpartum, as well as passive transfer of antibodies and T cells to the piglet through colostrum intake and before and after challenge with a heterologous PRRSv at ten weeks of age. Maternal-derived antibodies were detected in piglets but declined quickly after weaning. However, vaccinated animals restored PRRSv-specific antibody levels by anamnestic response to vaccination. Cell analysis in colostrum and milk revealed presence of PRRSv-specific immune cells at suckling with higher concentrations found in colostrum than in milk. In addition, colostrum and milk contained PRRSv-specific IgA and IgG that may contribute to protection of newborn piglets. Despite the presence of PRRSv-specific Peripheral Blood Mononuclear cells (PBMCs) in colostrum and milk, no PRRSv-specific cells could be detected from blood of the piglets at one or two weeks of life. Nevertheless, cellular immunity was detectable in pre-challenged piglets up to 7 weeks after vaccination while the non-vaccinated control group showed no interferon (IFN) γ response to PRRSv stimulation. After challenge, all piglets developed a PRRSv-specific IFNγ-response, which was more robust at significantly higher levels in vaccinated animals compared to the primary response to PRRSv in non-vaccinated animals. Cytokine analysis in the lung lumen showed a reduction of pro-inflammatory responses to PRRSv challenge in vaccinated animals, especially reduced interferon (IFN) α levels. In conclusion, vaccination of maternally positive piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX EU induced a humoral and cellular immune response to PRRSv and provided protection against virulent, heterologous PRRSv challenge.

Gain and loss of multiple genes during the evolution of Helicobacter pylori.

Sequence diversity and gene content distinguish most isolates of Helicobacter pylori. Even greater sequence differences differentiate distinct populations of H. pylori from different continents, but it was not clear whether these populations also differ in gene content. To address this question, we tested 56 globally representative strains of H. pylori and four strains of Helicobacter acinonychis with whole genome microarrays. Of the weighted average of 1,531 genes present in the two sequenced genomes, 25% are absent in at least one strain of H. pylori and 21% were absent or variable in H. acinonychis. We extrapolate that the core genome present in all isolates of H. pylori contains 1,111 genes. Variable genes tend to be small and possess unusual GC content; many of them have probably been imported by horizontal gene transfer. Phylogenetic trees based on the microarray data differ from those based on sequences of seven genes from the core genome. These discrepancies are due to homoplasies resulting from independent gene loss by deletion or recombination in multiple strains, which distort phylogenetic patterns. The patterns of these discrepancies versus population structure allow a reconstruction of the timing of the acquisition of variable genes within this species. Variable genes that are located within the cag pathogenicity island were apparently first acquired en bloc after speciation. In contrast, most other variable genes are of unknown function or encode restriction/modification enzymes, transposases, or outer membrane proteins. These seem to have been acquired prior to speciation of H. pylori and were subsequently lost by convergent evolution within individual strains. Thus, the use of microarrays can reveal patterns of gene gain or loss when examined within a phylogenetic context that is based on sequences of core genes.

Initial vaccination and revaccination with Type I PRRS 94881 MLV reduces viral load and infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes respiratory distress in pigs, reproductive failure in breeding-age gilts and sows, and can have devastating economic consequences in domestic herds. Several PRRS vaccines are available commercially. This study compared the effectiveness of single-vaccination and revaccination schedules using the PRRS 94881 Type I modified live virus (MLV) vaccine ReproCyc® PRRS EU with no vaccination (challenge control) in protecting against a PRRS virus (PRRSV) challenge in non-pregnant gilts. RESULTS: Data were available from 48 gilts across three groups: a challenge control group (n = 16), which received no vaccination; a revaccination group (n = 16), which received ReproCyc® PRRS EU on Days 0 and 56; and a single vaccination group (n = 16), which received ReproCyc® PRRS EU on Day 56. All gilts were PRRSV RNA-negative (based on reverse transcription and quantitative polymerase chain reaction [RT-qPCR]) and PRRSV seronegative (based on enzyme-linked immunosorbent assay [ELISA]) at Day 0. All gilts were challenged with PRRSV strain 190136 on Day 91.Viral RNA loads in both vaccination groups were significantly reduced compared with the challenge control group on Days 98 (P < 0.0001) and 101 (P < 0.0001), indicating that vaccinated gilts were better able to respond to challenge than unvaccinated gilts. At all timepoints following challenge, mean viral RNA load and the percentage of PRRSV RNA-positive gilts were numerically higher in the single-vaccination group than in the revaccination group; these differences were statistically significant on Day 101 (P = 0.0434). Furthermore, viremia levels after challenge were significantly lower in the revaccination group than in the single-vaccination group based on median area under the curve (AUC) values for viral RNA load from Day 91 to Day 112, suggesting that revaccinated gilts had better protection from viral infection than gilts who received a single vaccination. Protection from viremia did not correlate with the proportion of seropositive gilts on Day 91. In the single-vaccination group, 94% of pigs were seropositive on Day 91 compared with 56% in the revaccination group. Vaccination was well tolerated and no safety concerns were identified. CONCLUSIONS: Both single-vaccination and revaccination with ReproCyc® PRRS EU were effective in reducing PRRSV viremia post-challenge. These findings have important implications for herd management as both the single-vaccination and revaccination schedules protect against PRRSV challenge, with revaccination appearing to provide better protection from viremia than single vaccination.

Long duration of immunity against a type 1 heterologous PRRS virus challenge in pigs immunised with a novel PRRS MLV vaccine: a randomised controlled study.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread in commercial pig farms worldwide, and has a significant cost to the swine industry. Herd owners need a vaccine that will confer long-lasting immunity to prevent PRRSV infection and transmission. The studies described here evaluated duration of immunity conferred by a European-derived PRRS (isolate 94,881) modified live virus (MLV) vaccine, Ingelvac PRRSFLEX® EU, at 20, 24, and 26 weeks post-vaccination. Primary endpoints were the assessment of gross and histological lung lesions and viral RNA load in lung tissue 10 days following heterologous PRRSV challenge. Secondary endpoints included clinical observations, average daily weight gain (ADWG) and viral RNA load in serum 10 days post-challenge. Three blinded, vaccination-challenge efficacy studies were performed using separate cohorts of pigs (n = 56 per study). Pigs received either Ingelvac PRRSFLEX® EU (Group 1) or placebo (Groups 2 and 3). Groups 1 and 2 were subsequently challenged with heterologous European PRRSV isolate 205,817 at 20, 24 or 26 weeks post-vaccination. RESULTS: Mean gross lung lesion scores were significantly lower in Group 1 than in Group 2 at 24 and 26 weeks (p <  0.0001), but not at 20 weeks (p = 0.299). Significantly lower mean histological lung lesion scores were observed in Group 1 versus Group 2 at 20 (p = 0.0065), 24 (p <  0.0001) and 26 weeks (p <  0.0001). Mean viral RNA load in lung tissue was significantly lower in Group 1 than in Group 2 (p <  0.0001) at 20 (p <  0.0001), 24 (p <  0.0001) and 26 weeks (p <  0.0001). Cumulative viral RNA loads in serum during days 1-10 post-challenge were significantly lower in Group 1 than in Group 2 (p <  0.0001) in all studies. A significant increase in ADWG was observed in Group 1 compared with Group 2 at 20 weeks (p = 0.0027) and 24 weeks (p = 0.0004), but not at 26 weeks (p = 0.1041). There were no significant differences in clinical signs post-challenge in any study. CONCLUSION: These results suggest that Ingelvac PRRSFLEX® EU confers long-term immunity to European heterologous PRRSV, which is maintained up to 26 weeks after vaccination, corresponding to the expected lifespan of commercial pigs.

Safety and early onset of immunity with a novel European porcine reproductive and respiratory syndrome virus vaccine in young piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) can be difficult to manage in commercial settings. A novel type I PRRSV vaccinal strain (94881) was evaluated for safety and efficacy/onset of immunity (OOI) in piglets. In 2 experiments, groups of piglets were vaccinated intramuscularly (IM) at approximately 14 d of age with a maximum-range commercial dose, an overdose, or a placebo in experiment 1 and either a minimum-range commercial dose or a placebo in experiment 2. The piglets in experiment 1 were evaluated for local and systemic reactions from days -2 through 14 after vaccination. The piglets in experiment 2 were challenged with a virulent heterologous type I PRRSV isolate 14 d after vaccination and observed once daily for general health from days -1 through 12 after vaccination and once daily for clinical signs associated with challenge from days 13 through 24 after vaccination. The average daily weight gain (ADWG) and the results of serologic and viremia testing were evaluated in experiments 1 and 2. Lung lesion scores and results of testing for PRRSV in lung tissue were evaluated in experiment 2. In experiment 1 the vaccine was shown to be safe, as there were no relevant differences between the vaccinated piglets and the piglets given a placebo. In experiment 2 the vaccine's efficacy, with an OOI of 14 d after vaccination, was established, as the vaccinated and challenged piglets exhibited significantly lower lung lesion scores, viremia, viral load in lung tissue, and total clinical sign scores, along with a significantly greater ADWG, compared with the placebo-vaccinated and challenged piglets.

La gestion du virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être difficile dans un environnement de production commerciale. Une nouvelle souche vaccinale du VSRRP de type 1 (94881) a été évaluée d'un point de vue sécurité et efficacité/début de l'immunité (DDI) chez des porcelets. Dans deux expériences, des groupes de porcelets ont été vaccinés par voie intramusculaire (IM) à l'âge d'environ 14 j avec une dose commerciale maximale, une surdose, ou un placebo dans l'expérience 1 et une dose commerciale minimale ou un placebo dans l'expérience 2. Les porcelets dans l'expérience 1 furent évalués pour des réactions locale et systémique à compter du jour −2 jusqu'au jour 14 post-vaccination. Les porcelets dans l'expérience 2 furent soumis à une infection défi avec un isolat virulent hétérologue de VSRRP de type 1 14 j après la vaccination et observés une fois par jour pour leur état de santé général du jour −1 jusqu'au jour 12 après la vaccination et une fois par jour pour des signes cliniques associés avec l'infection du jour 13 au jour 24 après l'infection. Le gain moyen quotidien (GMQ) et les résultats des analyses sérologiques et de virémie ont été évalués dans les expériences 1 et 2. Les pointages de lésions pulmonaires et les résultats de détection du VSRRP dans le tissu pulmonaire ont été évalués dans l'expérience 2. Dans l'expérience 1, le vaccin s'est montré sécuritaire étant donné qu'il n'y avait aucune différence significative entre les porcelets vaccinés et les porcelets ayant reçu un placebo. Dans l'expérience 2, l'efficacité du vaccin, avec un DDI de 14 j après la vaccination, a été établie, étant donné que les porcelets vaccinés, et soumis à une infection défi avaient des valeurs significativement moins élevées de pointage de lésions pulmonaires, de virémie, de charge virale dans le tissu pulmonaire, et des pointages de signes cliniques totaux, avec un GMQ significativement plus élevé, comparativement au porcs vaccinés avec un placebo et soumis à une infection défi.(Traduit par Docteur Serge Messier).

Vaccination of piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX® EU provides protection against heterologous field challenge in the face of homologous maternally derived antibodies.

BACKGROUND: Due to difficulties in eradicating porcine reproductive and respiratory syndrome (PRRS) linked to biosecurity challenges, transmission of the virus and the lack of efficient DIVA vaccines, successful control of PRRS requires a combination of strict management measures and vaccination of both sows and piglets. The present study aimed to assess the efficacy of a recently developed MLV vaccine (Ingelvac PRRSFLEX® EU) in piglets at 2 and 3-weeks of age in the presence of homologous maternally derived antibodies as the dams were vaccinated with the same vaccine strain (ReproCyc® PRRS EU). METHODS: The study was carried out on a Hungarian farrow to finish farm naturally infected with PRRSv. The study was designed as a blind, placebo controlled side by side trial. ORF5 sequence similarity of the vaccine strain and the resident field strain was 87.8 %. PRRS specific real-time quantitative PCR was performed from serum samples to measure both the viral load and the frequency of virus positive animals. RESULTS: At the time of the natural infection observed in the control group at 10-12 weeks of age, the number of viraemic animals did not increase significantly in the vaccinated group. To understand the infection dynamics, positive PCR samples with low Ct values were sequenced (ORF5) and the data analysis indicated the circulation of wild type virus in both groups, however wild type virus was only found in non-vaccinated animals. CONCLUSIONS: Our data indicate that piglets vaccinated at as early as 2 weeks of age with Ingelvac PRRSFLEX® EU were protected both in terms of proportion of viraemic animals and viraemia levels. It has to be highlighted that these results were achieved in piglets with high levels of homologous maternally derived antibodies (MDA) at the time of vaccination.

Assessment of safety and reproductive performance after vaccination with a modified live-virus PRRS genotype 1 vaccine in pregnant sows at various stages of gestation.

The objective of the present study was to assess safety and efficacy of a new modified live-virus porcine reproductive and respiratory syndrome (PRRS) genotype 1 vaccine in pregnant sows at various stages of gestation under field conditions. A total of 505 sows and gilts were allocated to two treatment groups and maintained in separate facilities. Animals of group 1 were vaccinated with a commercial modified live genotype 1 PRRSV vaccine (control product, CP), while animals of group 2 were immunized with a new modified live genotype 1 PRRSV vaccine (investigational veterinary product, IVP) (ReproCyc® PRRS EU, Boehringer Ingelheim Vetmedica GmbH). Injection site reactions were noted to be significantly less frequent in the IVP group compared to the CP group for pain (p=0.039), redness (p=0.030), heat (p=0.016) and swelling (p=0.002). The mean total number of piglets alive at weaning did not differ significantly between both study groups (10.6 vs. 11.0, p=0.375). However, pre-weaning mortality was significantly higher (p=0.005) in piglets from the CP group (14.1% vs. 10.9%). Analyses of reproductive performance data for both groups did not result in statistically significant differences between CP group and IVP group for number of piglets alive (12.7 and 12.6, respectively), healthy live (11.9 and 11.8), weak (0.7 and 0.5), stillborn (1.0 and 0.8) and mummified piglets (0.3 and 0.2) per litter. No differences were detected between both groups for piglet birth weights, while body weights at weaning (7.2kg vs. 6.6kg, p=0.026) and average daily gain (0.2445kg vs. 0.2211kg, p=0.037) were significantly higher in piglets from the IVP group. In conclusion, the administration of a single dose of ReproCyc® PRRS EU to sows and gilts at various stages of gestation confirmed non-inferiority to a commercial PRRS vaccine regarding safety and efficacy parameters under field conditions.

Safety and efficacy of a novel European vaccine for porcine reproductive and respiratory virus in bred gilts.

Porcine reproductive and respiratory syndrome virus (PRRSV) can be devastating to commercial breeding operations. The objective of this study was to evaluate a novel European PRRSV vaccinal strain for safety and efficacy in bred gilts. In 2 experiments, 110 gilts were vaccinated intramuscularly and the vaccine was evaluated for safety and efficacy. Gilts in Experiment 1 were evaluated for local and systemic reactions and gilts in both experiments were observed for clinical signs of disease through farrow. In both experiments, piglet clinical observations, piglet average daily weight gain (ADWG), gilt serology [determined by enzyme-linked immunosorbent assay (ELISA)], gilt and piglet viremia [determined by quantitative real-time polymerase chain reaction (qPCR)], as well as piglet lung lesion scores and PRRS virus in lung tissue (qPCR) were determined. The vaccine was shown to be safe as there were no significant differences among groups in either experiment. Efficacy was established in Experiment 2 as both vaccinated groups were associated with desirable significant differences in percentage of gilts with abnormal clinical findings; gilt viral load post-challenge [day 125, day of farrowing (DOF), and DOF + 13]; percentages of alive, healthy live, weak live, and mummified piglets per litter at farrowing and weaning; percentage of piglets per gilt that were positive for viremia; percentage of piglets per gilt with clinical disease; and piglet viral load on DOF. It was concluded that a vaccine formulated from the PRRSV modified live virus (MLV) strain 94881 is a safe and effective method of protection against the detrimental effects of virulent PRRSV infection in breeding female pigs.

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être dévastateur pour les opérations de reproduction commerciales. L'objectif de la présente étude était d'évaluer l'innocuité et l'efficacité d'une nouvelle souche vaccinale européenne du VSRRP chez des cochettes saillies. Lors de deux expériences, 110 cochettes ont été vaccinées par voie intramusculaire afin d'évaluer l'innocuité et l'efficacité du vaccin. Les cochettes de l'Expérience 1 ont été évaluées pour la présence de réactions locales et systémiques et les cochettes dans les deux expériences ont été observées pour vérifier la présence de signes cliniques de maladie jusqu'au moment de la mise-bas. Lors des deux expériences on nota les observations cliniques des porcelets, le gain de poids quotidien moyen des porcelets (GMQ), les titres sérologiques des truies [déterminés par épreuve immuno-enzymatique (ELISA)], la virémie chez les cochettes et les porcelets [déterminées par réaction d'amplification en chaine par la polymérase quantitative (qACP)], ainsi que par les pointages de lésions pulmonaires des porcelets et la quantité de virus SRRP dans le tissu pulmonaire (qACP). Le vaccin s'est avéré sécuritaire et il n'y avait pas de différence significative entre les groupes dans les deux expériences. L'efficacité a été établie lors de l'Expérience 2 alors que les deux groupes vaccinés ont été associés à des différences significatives souhaitées dans le pourcentage de cochettes avec des trouvailles cliniques anormales; dans la charge virale post-challenge par cochette [jour 125, jour de la mise-bas (JMB), et JMB + 13]; pourcentages de porcelets vivants, vivants en santé, vivants faibles, et momifiés par portée au moment de la mise-bas et au sevrage; pourcentages de porcelets par truie qui étaient positifs pour une virémie; pourcentage de porcelets par cochette avec une maladie clinique; et charge virale des porcelets au JMB. Il a été conclu qu'un vaccin formulé à partir de la souche 94881 du VSRRP vivant modifié est une méthode sécuritaire et efficace de protection contre les effets néfastes d'une infection par le VSRRP chez des porcs femelles de reproduction.(Traduit par Docteur Serge Messier).