Effect of vaccination route (intradermal vs. intramuscular) against porcine reproductive and respiratory syndrome using a modified live vaccine on systemic and mucosal immune response and virus transmission in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a viral disease with worldwide distribution and an enormous economic impact. To control PRRS virus (PRRSV) infection, modified live vaccines (MLVs) are widely used in the field, mainly administered via an intramuscular (IM) route. Currently, some MLVs are authorized for intradermal (ID) administration, which has many practical and welfare advantages. The objectives of the study were to compare the immune responses (systemic in blood and mucosal in lungs) and vaccine efficacy in preventing challenge strain transmission after IM or needle-free ID immunization of piglets with an MLV against PRRSV-1 (MLV1). METHODS: Groups of sixteen 5-week-old specific pathogen-free piglets were vaccinated with Porcilis PRRS® (MSD) either by an IM (V+ IM) or ID route (V+ ID) using an IDAL®3G device or kept unvaccinated (V-). Four weeks after vaccination, in each group, 8 out of the 16 piglets were challenged intranasally with a PRRSV-1 field strain, and one day later, the inoculated pigs were mingled by direct contact with the remaining 8 sentinel noninoculated pigs to evaluate PRRSV transmission. Thus, after the challenge, each group (V+ IM, V+ ID or V-) included 8 inoculated and 8 contact piglets. During the postvaccination and postchallenge phases, PRRSV replication (RT-PCR), PRRSV-specific antibodies (ELISA IgG and IgA, virus neutralization tests) and cell-mediated immunity (ELISPOT Interferon gamma) were monitored in blood and bronchoalveolar lavages (BALs). RESULTS: Postvaccination, vaccine viremia was lower in V+ ID pigs than in V+ IM pigs, whereas the cell-mediated immune response was detected earlier in the V+ ID group at 2 weeks postvaccination. In the BAL fluid, a very low mucosal immune response (humoral and cellular) was detected. Postchallenge, the vaccine efficacy was similar in inoculated animals with partial control of PRRSV viremia in V+ ID and V+ IM animals. In vaccinated sentinel pigs, vaccination drastically reduced PRRSV transmission with similar estimated transmission rates and latency durations for the V+ IM and V+ ID groups. CONCLUSIONS: Our results show that the tested MLV1 induced a faster cell-mediated immune response after ID immunization two weeks after vaccination but was equally efficacious after IM or ID immunization towards a challenge four weeks later. Considering the practical and welfare benefits of ID vaccination, these data further support the use of this route for PRRS MLVs.

Dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype 1 PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates primarily in pulmonary alveolar macrophages (PAMs) and the resulting lung damage is influenced by strain virulence. To better understand the pathogenesis of PRRSV infection, we performed a longitudinal study of the PAM population and lung cytokines in specific pathogen-free pigs infected either with the highly pathogenic Lena strain or with the low pathogenic Finistere strain in comparison to uninfected pigs. Bronchoalveolar lavage fluid (BALF) and blood were collected to follow viral, cellular and cytokine changes in lung with respect to clinical signs and systemic events. Compared to Finistere-infected pigs, Lena-infected pigs exhibited more severe clinical signs and 10- to 100-fold higher viral loads in BALF and blood. Similarly, they showed an earlier drop in BALF cell viability and phagocytic activity along with a decrease in the macrophage count. From 8 to 15 days post-infection (dpi), monocytes increased both in BALF and blood from Lena-infected pigs. BALF and blood showed contrasting cytokine patterns, with low increase of IFN-α and TNF-α levels and high increase for IL-1α and IL-8 in BALF after Lena-infection. In contrast, in the blood, the increase was marked for IFN-α and TNF-α but limited for IL-1ß and IL-8. Down-regulation of PAM functions combined with inflammatory cytokine and monocyte recruitment may promote lung pathogenesis and virus replication in PRRSV infections with the highly pathogenic Lena strain. In contrast, the low pathogenic Finistere strain showed prolonged viral replication in lung, possibly related to the weak IFN-γ response.

Exploring type I interferon pathway: virulent vs. attenuated strain of African swine fever virus revealing a novel function carried by MGF505-4R.

African swine fever virus represents a significant reemerging threat to livestock populations, as its incidence and geographic distribution have surged over the past decade in Europe, Asia, and Caribbean, resulting in substantial socio-economic burdens and adverse effects on animal health and welfare. In a previous report, we described the protective properties of our newly thermo-attenuated strain (ASFV-989) in pigs against an experimental infection of its parental Georgia 2007/1 virulent strain. In this new study, our objective was to characterize the molecular mechanisms underlying the attenuation of ASFV-989. We first compared the activation of type I interferon pathway in response to ASFV-989 and Georgia 2007/1 infections, employing both in vivo and in vitro models. Expression of IFN-α was significantly increased in porcine alveolar macrophages infected with ASFV-989 while pigs infected with Georgia 2007/1 showed higher IFN-α than those infected by ASFV-989. We also used a medium-throughput transcriptomic approach to study the expression of viral genes by both strains, and identified several patterns of gene expression. Subsequently, we investigated whether proteins encoded by the eight genes deleted in ASFV-989 contribute to the modulation of the type I interferon signaling pathway. Using different strategies, we showed that MGF505-4R interfered with the induction of IFN-α/ß pathway, likely through interaction with TRAF3. Altogether, our data reveal key differences between ASFV-989 and Georgia 2007/1 in their ability to control IFN-α/ß signaling and provide molecular mechanisms underlying the role of MGF505-4R as a virulence factor.

CP7_E2alf oral vaccination confers partial protection against early classical swine fever virus challenge and interferes with pathogeny-related cytokine responses.

The conventional C-strain vaccine induces early protection against classical swine fever (CSF), but infected animals cannot be distinguished from vaccinated animals. The CP7_E2alf marker vaccine, a pestivirus chimera, could be a suitable substitute for C-strain vaccine to control CSF outbreaks. In this study, single oral applications of CP7_E2alf and C-strain vaccines were compared for their efficacy to induce protection against a CSF virus (CSFV) challenge with the moderately virulent Bas-Rhin isolate, in pigs as early as two days post-immunization. This work emphasizes the powerful potential of CP7_E2alf vaccine administered orally by a rapid onset of partial protection similar to that induced by the C-strain vaccine. Furthermore, our results revealed that both vaccinations attenuated the effects induced by CSFV on production of the pig major acute phase protein (PigMAP), IFN-α, IL-12, IL-10, and TGF-ß1 cytokines. By this interference, several cytokines that may play a role in the pathogeny induced by moderately virulent CSFV strains were revealed. New hypotheses concerning the role of each of these cytokines in CSFV pathogeny are discussed. Our results also show that oral vaccination with either vaccine (CP7_E2alf or C-strain) enhanced CSFV-specific IgG2 production, compared to infection alone. Interestingly, despite the similar antibody profiles displayed by both vaccines post-challenge, the production of CSFV-specific IgG1 and neutralizing antibodies without challenge was lower with CP7_E2alf vaccination than with C-strain vaccination, suggesting a slight difference in the balance of adaptive immune responses between these vaccines.

Oronasal or Intramuscular Immunization with a Thermo-Attenuated ASFV Strain Provides Full Clinical Protection against Georgia 2007/1 Challenge.

African swine fever (ASF) is a contagious viral disease of suids that induces high mortality in domestic pigs and wild boars. Given the current spread of ASF, the development of a vaccine is a priority. During an attempt to inactivate the Georgia 2007/1 strain via heat treatment, we fortuitously generated an attenuated strain called ASFV-989. Compared to Georgia, the ASFV-989 strain genome has a deletion of 7458 nucleotides located in the 5'-end encoding region of MGF 505/360, which allowed for developing a DIVA PCR system. In vitro, in porcine alveolar macrophages, the replication kinetics of the ASFV-989 and Georgia strains were identical. In vivo, specific-pathogen-free (SPF) pigs inoculated with the ASFV-989 strain, either intramuscularly or oronasally, exhibited transient hyperthermia and slightly decreased growth performance. Animals immunized with the ASFV-989 strain showed viremia 100 to 1000 times lower than those inoculated with the Georgia strain and developed a rapid antibody and cell-mediated response. In ASFV-989-immunized pigs challenged 2 or 4 weeks later with the Georgia strain, no symptoms were recorded and no viremia for the challenge strain was detected. These results show that the ASFV-989 strain is a promising non-GMO vaccine candidate that is usable either intramuscularly or oronasally.

Concomitant Swine Influenza A Virus Infection Alters PRRSV1 MLV Viremia in Piglets but Does Not Interfere with Vaccine Protection in Experimental Conditions.

Modified-live vaccines (MLVs) against porcine reproductive and respiratory syndrome viruses (PRRSVs) are usually administrated to piglets at weaning when swine influenza A virus (swIAV) infections frequently occur. SwIAV infection induces a strong interferon alpha (IFNa) response and IFNa was shown to abrogate PRRSV2 MLV replication and an inherent immune response. In this study, we evaluated the impacts of swIAV infection on the replication of a PRRSV1 MLV (MLV1), post-vaccine immune responses and post-challenge vaccine efficacy at both the systemic and pulmonary levels. Piglets were either swIAV inoculated and MLV1 vaccinated 6 h apart or singly vaccinated or mock inoculated and mock vaccinated. Four weeks after vaccination, the piglets were challenged with a PRRSV1 field strain. The results showed that swIAV infection delayed MLV1 viremia by six days and post-vaccine seroconversion by four days. After the PRRSV1 challenge, the swIAV enhanced the PRRSV1-specific cell-mediated immunity (CMI) but the PRRSV1 field strain viremia was not better controlled. High IFNa levels that were detected early after swIAV infection could have been responsible for both the inhibition of MLV1 replication and CMI enhancement. Thus, whereas swIAV infection had a negative impact on humoral responses post-vaccination, it did not interfere with the protective effectiveness of the PRRSV MLV1 in our experimental conditions.

Successive Inoculations of Pigs with Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) and Swine H1N2 Influenza Virus Suggest a Mutual Interference between the Two Viral Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A virus (swIAV) are major pathogens of the porcine respiratory disease complex, but little is known on their interaction in super-infected pigs. In this study, we investigated clinical, virological and immunological outcomes of successive infections with PRRSV-1 and H1N2 swIAV. Twenty-four specific pathogen-free piglets were distributed into four groups and inoculated either with PRRSV at study day (SD) 0, or with swIAV at SD8, or with PRRSV and swIAV one week apart at SD0 and SD8, respectively, or mock-inoculated. In PRRSV/swIAV group, the clinical signs usually observed after swIAV infection were attenuated while higher levels of anti-swIAV antibodies were measured in lungs. Concurrently, PRRSV multiplication in lungs was significantly affected by swIAV infection, whereas the cell-mediated immune response specific to PRRSV was detected earlier in blood, as compared to PRRSV group. Moreover, levels of interferon (IFN)-α measured from SD9 in the blood of super-infected pigs were lower than those measured in the swIAV group, but higher than in the PRRSV group at the same time. Correlation analyses suggested an important role of IFN-α in the two-way interference highlighted between both viral infections.

Coding-Complete Genome Sequence of an African Swine Fever Virus Strain Liv13/33 Isolate from Experimental Transmission between Pigs and Ornithodoros moubata Ticks.

Here, we report the coding-complete genome sequence of African swine fever (ASF) virus strain Liv13/33, isolated from experimentally infected pigs and Ornithodoros moubata ticks. The 11 sequences that we obtained harbored no notable differences to each other, and all of them were closely related to the genome sequence of the Mkuzi 1979 strain of genotype I.

Complete Genome Sequence of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Strain from Two Genotype 1 Modified Live Virus Vaccine Strains.

This paper provides information on the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) strain isolated on a French pig farm which was identified as a recombinant strain from two commercial modified live virus vaccine strains of genotype 1 (VP-046BIS and DV strains).

Immune responses after local administration of IgY loaded-PLGA microspheres in gut-associated lymphoid tissue in pigs.

Oral vaccination of large animals using PLGA MS (poly(D,L-lactide-co-glycolide)microspheres) appeared to be more challenging than immunization of mice. The purpose of this study was to deliver to GALT an immunogenic model protein (IgY), free or encapsulated by spray-drying in PLGA MS, and to evaluate systemic immune response in SPF Large White pigs. Pigs were surgically processed for local administration of IgY in three sets of experiments. In two sets of experiments, administration was locally performed in temporary ligatured intestinal segments, in jejunal Peyer's patches and in mesenteric lymph nodes. In the third experiment, pigs received IgY via an intestinal cannula. Total IgY-specific antibodies were detected in the sera of pigs after a single local immunization, but not in the sera of cannulated pigs. The study of IgG1 and IgG2 isotypes indicated that PLGA MS are able to elicit a combined serum IgG2/G1 response with a predominance of IgG1 response when locally administered. PLGA MS can be a potential oral delivery system for antigen but our results underlined the difficulty to immunize large animals like pigs. Transposition of data between small and large animals appears to be complex and suggests that physiological features need to be considered to increase intestinal availability of oral encapsulated vaccines.

Use of a polymerase chain reaction assay and an ELISA to monitor porcine circovirus type 2 infection in pigs from farms with and without postweaning multisystemic wasting syndrome.

OBJECTIVE: To determine whether correlations exist between viremia with porcine circovirus type 2 (PCV2) and serum antibody profiles and between detection of PCV2 in nasal cavities and viremia of pigs from farms with and without postweaning multisystemic wasting syndrome (PMWS). ANIMALS: 495 pigs, ranging from the late nursery stage to the early grower-finisher stage of production. PROCEDURE: Serum antibodies to PCV2 were studied with an ELISA that detects the ORF2 viral protein. Nasal swab specimens and serum samples were tested with a PCV2-specific PCR assay. RESULTS: PCV2 DNA and serum antibodies to PCV2 were detected in pigs from all farms, although in different proportions. Overall, PCV2 DNA was detected in greater percentages in serum samples and nasal swab specimens of pigs from farms with PMWS. Although viral DNA was detected in both serum samples and nasal swab specimens, PCV2 detection in nasal swab specimens was higher than in serum samples of pigs from all farms. Serum antibodies to PCV2 were detected in a greater percentage of pigs from farms with PMWS, compared with farms without PMWS. CONCLUSIONS AND CLINICAL RELEVANCE: A high prevalence of PCV2 infection was found in pigs from farms with and without PMWS. Besides the presence of PCV2, unknown additional factors may be necessary to induce the full expression of PMWS.

Validation of a commercial real-time PCR kit for specific and sensitive detection of Pseudorabies.

Pseudorabies virus is the causative agent of Aujeszky's disease, one of the OIE listed diseases that mainly affects swine, but also can affect other animal species, and which can lead to heavy economic losses in pig industry. This study was designed to evaluate the performance of the ADIAVET(®) PRV REALTIME kit, a new commercial real time PCR kit for Pseudorabies virus genome detection developed by the French manufacturer Adiagène. It can be used on pig biological samples such as nasal swab supernatant, tonsil, brain or lung samples, or on samples from other susceptible animals, such as domestic carnivores. This ready-to-use duplex PCR assay contains an external positive control, appropriate for assessing DNA extraction efficiency and the presence of PCR inhibitors. The analytical specificity and sensitivity, intra- and inter-assay repeatability and diagnostic characteristics of the kit were determined and compared with virus isolation, which is the gold standard. Based on these results, the ADIAVET(®) PRV REALTIME kit received full validation for diagnostic purposes.

Molecular tracing of classical swine fever viruses isolated from wild boars and pigs in France from 2002 to 2011.

There were three outbreaks of classical swine fever (CSF) in north-eastern France between 2002 and 2011. The first two occurred in April 2002 in the Moselle department, in a wild boar and pig herd, respectively, while the third occurred in April 2003, in the Bas-Rhin department, in a wild boar. A survey was subsequently implemented in wild boar and domestic pig populations, during which 43 CSF viruses (CSFVs) were genetically characterized to provide information on virus sources, trace virus evolution and help in the monitoring of effective control measures. Phylogenetic analyses, based on fragments of the 5'NTR, E2 and NS5B genes, showed that all French CSFVs could be assigned to genotype 2, subgenotype 2.3. CSFVs isolated in Moselle were classified in the "Rostock" lineage, a strain first described in 2001 in wild boar populations in the Eifel region of north-western Rhineland-Palatinate in Germany, and in Luxemburg. In contrast, the CSFVs isolated in Bas-Rhin were homologous to strains from the "Uelzen" lineage, a strain previously isolated from wild boars in south-eastern Rhineland-Palatinate, Germany, as well as in Vosges du Nord, France, during a previous outbreak that had occurred in wild boars between 1992 and 2001. The outbreak in Moselle domestic pigs was quickly resolved as it concerned only one herd. The infection in wild boars from Moselle was extinguished after a few months whereas wild boars from Bas-Rhin remained infected until 2007. Molecular tracing showed that the Bas-Rhin index virus strain evolved slightly during the period but that no strain from a novel lineage was introduced until this outbreak ended after application of a vaccination scheme for six years.

Acute induction of cell death-related IFN stimulated genes (ISG) differentiates highly from moderately virulent CSFV strains.

Classical swine fever (CSF) severity is dependent on the virulence of the CSF virus (CSFV) strain. The earliest event detected following CSFV infection is a decrease in lymphocytes number. With some CSFV strains this leads to lymphopenia, the severity varying according to strain virulence. This lymphocyte depletion is attributed to an induction of apoptosis in non-infected bystander cells. We collected peripheral blood mononuclear cells (PBMC) before and during 3 days post-infection with either a highly or moderately virulent CSFV strain and subjected them to comparative microarray analysis to decipher the transcriptomic modulations induced in these cells in relation to strain virulence. The results revealed that the main difference between strains resided in the kinetics of host response to the infection: strong and immediate with the highly virulent strain, progressive and delayed with the moderately virulent one. Also although cell death/apoptosis-related IFN stimulated genes (ISG) were strongly up-regulated by both strains, significant differences in their regulation were apparent from the observed differences in onset and extent of lymphopenia induced by the two strains. Furthermore, the death receptors apoptotic pathways (TRAILDR4, FASL-FAS and TNFa-TNFR1) were also differently regulated. Our results suggest that CSFV strains might exacerbate the interferon alpha response, leading to bystander killing of lymphocytes and lymphopenia, the severity of which might be due to the host's loss of control of IFN production and downstream effectors regulation.

Differentiation of C-strain "Riems" or CP7_E2alf vaccinated animals from animals infected by classical swine fever virus field strains using real-time RT-PCR.

Classical swine fever (CSF) is one of the most important diseases of pigs. Although prophylactic vaccination is banned within the European Union, emergency vaccination, allowing differentiation of vaccinated from infected animals, is an option for disease control. Up to now, these strategies are based on antibody detection. In this context, conventional modified live vaccines are not suitable. A promising perspective could be genetic differentiation of vaccinated from infected animals where field virus strains are differentiated from vaccine viruses by sequence differences. This concept could also be used with marker vaccines. To this end, a set of real-time reverse transcription-polymerase chain reaction (RT-PCR) assays was developed and validated. Specific primers and probes were designed for detection of the C-strain "Riems" vaccine virus or the chimeric marker vaccine candidate CP7_E2alf. A heterologous internal positive control was also included. The assays were then multiplexed to detect simultaneously either CSF field virus, C-strain "Riems", and the internal control or CSF field virus, CP7_E2alf, and the internal control. To validate both systems, samples from vaccination/challenge trials were tested. Only samples from vaccinated animals were found to be positive, while all samples from wild type virus-infected animals and a broad test panel of different pestiviruses were negative. Field application of the "C-strain Riems" specific assay was proven with wild boar samples from surveillance programs in Germany and France. In conclusion, ready-to-use RT-PCR sets are presented as reliable tools for genetic differentiation of vaccinated from infected animals for CSFV eradication strategies.