Elucidating the Onset of Cross-Protective Immunity after Intranasal Vaccination with the Attenuated African Swine Fever Vaccine Candidate BA71ΔCD2.

African swine fever (ASF) is a deadly disease of swine currently causing a worldwide pandemic, leading to severe economic consequences for the porcine industry. The control of disease spread is hampered by the limitation of available effective vaccines. Live attenuated vaccines (LAVs) are currently the most advanced vaccine prototypes, providing strong protection against ASF. However, the significant advances achieved using LAVs must be complemented with further studies to analyze vaccine-induced immunity. Here, we characterized the onset of cross-protective immunity triggered by the LAV candidate BA71ΔCD2. Intranasally vaccinated pigs were challenged with the virulent Georgia 2007/1 strain at days 3, 7 and 12 postvaccination. Only the animals vaccinated 12 days before the challenge had effectively controlled infection progression, showing low virus loads, minor clinical signs and a lack of the unbalanced inflammatory response characteristic of severe disease. Contrarily, the animals vaccinated 3 or 7 days before the challenge just showed a minor delay in disease progression. An analysis of the humoral response and whole blood transcriptome signatures demonstrated that the control of infection was associated with the presence of virus-specific IgG and a cytotoxic response before the challenge. These results contribute to our understanding of protective immunity induced by LAV-based vaccines, encouraging their use in emergency responses in ASF-affected areas.

Identification of Single Amino Acid Changes in the Rift Valley Fever Virus Polymerase Core Domain Contributing to Virus Attenuation In Vivo.

Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. RVF affects sheep, goats, cattle and camels, causing a high rate of abortions and death of newborn lambs. Also, humans can be infected, developing a usually self-limiting disease that can turn into a more severe illness in a low percentage of cases. Although different veterinary vaccines are available in endemic areas in Africa, to date no human vaccine has been licensed. In previous works, we described the selection and characterization of a favipiravir-mutagenized RVFV variant, termed 40Fp8, with potential as a RVF vaccine candidate due to the strong attenuation shown in immunocompromised animal models. Compared to the parental South African 56/74 viral strain, 40Fp8 displayed 7 amino acid substitutions in the L-protein, three of them located in the central region corresponding to the catalytic core of the RNA-dependent RNA polymerase (RdRp). In this work, by means of a reverse genetics system, we have analyzed the effect on virulence of these amino acid changes, alone or combined, both in vitro and in vivo. We found that the simultaneous introduction of two changes (G924S and A1303T) in the heterologous ZH548-RVFV Egyptian strain conferred attenuated phenotypes to the rescued viruses as shown in infected mice without affecting virus immunogenicity. Our results suggest that both changes induce resistance to favipiravir likely associated to some fitness cost that could be the basis for the observed attenuation in vivo. Conversely, the third change, I1050V, appears to be a compensatory mutation increasing viral fitness. Altogether, these results provide relevant information for the safety improvement of novel live attenuated RVFV vaccines.

Genotyping of Porcine Circovirus 2 (PCV-2) in Vaccinated Pigs Suffering from PCV-2-Systemic Disease between 2009 and 2020 in Spain.

Vaccination against porcine circovirus 2 (PCV-2) is a common practice all over the world. Vaccines can prevent PCV-2-systemic disease (PCV-2-SD) outbreaks but not PCV-2 infection, which can be detectable in a percentage of vaccinated animals. Occasionally, PCV-2-SD is diagnosed in vaccinated farms. The objective of this study was to genotype the PCV-2 strains detected in vaccinated animals diagnosed with PCV-2-SD. Additionally, the evolution of the frequency of PCV-2 genotype detection at Spanish, European, and world levels was assessed. Fifty cases diagnosed as PCV-2-SD between 2009 and 2020 were included in this study. PCV-2 genotype was determined by sequencing the Cap gene region. Among them, only PCV-2b (23/50, 46%) and PCV-2d (27/50, 54%) genotypes were detected. Although the frequency of detection of these two genotypes was similar, their temporal distribution was different. Whereas most PCV-2b sequences (17/23, 74%) were detected between 2009 and 2012, PCV-2d sequences were obtained from 2013 to 2020. Indeed, a predominance of the PCV-2d genotype was observed from 2013 onwards, a trend also noticed at European and world levels. The results suggest that detection of particular genotypes in vaccinated animals probably reflects the general prevalence of the genotypes over time rather than genotype-specific vaccine-immunity escaping.

Exploratory field study on the effects of porcine circovirus 2 (PCV-2) sow vaccination at different physiological stages mimicking blanket vaccination.

BACKGROUND: The objective of the present study was to explore the benefits of Porcine circovirus 2 (PCV-2) blanket vaccination in a sow herd on productive parameters, PCV-2 infection and immune status in sows and their progeny. For this purpose, 288 sows were distributed among four balanced experimental groups. One group remained as negative control group and the other three received 1 mL of PCV-2 Ingelvac Circoflex® intramuscularly at different productive cycle moments: before mating, mid gestation (42-49 days post-insemination) or late gestation (86-93 days post-insemination); phosphate buffered saline (PBS) was used as negative control item. Reproductive parameters from sows during gestation and body weight of their progeny from birth to weaning were recorded. Additionally, blood was collected from sows at each vaccination time and piglets at 3 weeks of age. Moreover, up to 4 placental umbilical cords (PUC) per sow were taken at peri-partum. Sera from sows and piglets were analysed for PCV-2 antibody detection using an enzyme-linked immunosorbent assay (ELISA). Sera from sows and PUC were tested to quantify viraemia using a real time quantitative polymerase chain reaction (qPCR) assay. RESULTS: Globally, results indicated that vaccinated sows showed heavier piglets at birth and at weaning, less cross-fostered piglets, lower viral load at farrowing as well as in PUC, and higher antibody levels at farrowing, compared to non-vaccinated ones. When all groups were compared among them, sows vaccinated at mid or late gestation had heavier piglets at birth than non-vaccinated sows, and lower proportion of PCV-2 positive PUC. Also, cross-fostering was less frequently practiced in sows vaccinated at pre-mating or mid gestation compared to non-vaccinated ones. CONCLUSIONS: In conclusion, the present study points out that PCV-2 sow vaccination at different time points of their physiological status (mimicking blanket vaccination) offers benefits at production and serological and virological levels.

Correlación de la distancia recorrida en la caminata de 6 minutos y puntajes de comorbilidad Charlson y Elixhauser / Correlation of distance traveled in the 6 minute walk and Charlson and Elihauser comorbidity scores

Introduction: The walking test of 6 minutes (6MW) is a test that merges the answer of different systems (respiratory, cardiovascular, metabolic, skeletal muscle and neurosensorial) and offers an useful objective result to lead therapeutic measurements and stablish a prognosis, it's possible that the comorbid patient lowers their functional reserve and alters the result of the test not only because of the presence of pathologies cardiorespiratory, nevertheless, information about the correlation between the scores of comorbidity and the traveled distance in the 6MW is limited. Objective: Determine the correlation between the traveled distance in the 6MW and the scores of comorbidities of Charlson and Elixhauser. Methods: A cross-sectional study was made, in patients taken to the 6MW made between 2006 until March 2020, in a hospital of high complexity; there were included patients older than 18 years old, whose clinic history record and walk of 6 minutes were available. The index of Charlson and Elixhauser were calculated in the 6MW, a bivariate analysis was made between the antecedents of pathologies and the traveled distance, independently and adjusted, the spearman correlation coefficient was calculated for the different scores and the distance in meters of the 6MW, was considerate a significative p: <0,05. Results: to the final analysis 491 subjects entered, the average age was of 69 years old (sd: 14,9), 54% male, the 15,3% had an abnormal walk less than the 80% of the expected, the diseases that were considered had a statistically significant relation with the decrease of the distance in the 6MW were arterial hypertension (p: <0,001), chronic heart failure (p=0,037), heart arrhythmia (p=0,003), smoking (p=0,022), chronic pulmonary obstruction disease (p: <0,001), dementia (p=0,03diabetes mellitus with target organ damage (p=0,01), moderate to severe chronic kidney disease (p=0,012), obesity (p=0,036) y lymphoma (p=0,038 the spearman correlation coefficient between the traveled distances and Charlson was of -0,343 (IC95%:-0,420 -0,264)(p: < 0,001) and -0,213(IC95%:-0,285 -0,116)(p: <0,001) with the Elixhauser index. Conclusion: The distances walked in meters in the 6MW has a reverse low correlation with the comorbidity index, the diseases that were not cardiopulmonary and that related independently with changes in the traveled dist ance are smoking, dementia, diabetes mellitus, chronic kidney disease, obesity, and lymphoma. Key words: Comorbidities, Walk, Test, Cardiopulmonary, Charlson, Elixhauser

Infection dynamics of porcine circovirus type 3 in longitudinally sampled pigs from four Spanish farms.

Porcine circovirus type 3 (PCV-3) is a recently discovered virus in domestic pigs and wild boar. The virus has been described in pigs with different clinical/pathological presentations and healthy animals, but the dynamics of infection is currently unknown. The aim of this study was to longitudinally monitor PCV-3 infection in 152 pigs from four different healthy farms (A, B, C and D) by means of PCR in serum. The selected animals were sampled five (farm A) or six (farms B-D) times from weaning until the end of the fattening period. PCV-3 genome was found in pigs from all tested ages and farms; few animals had an apparent long-term infection (4-23 weeks). PCV-3 frequency of detection remained fairly uniform along tested ages within farms A and C, but was more variable among sampling times in farms B and D. Eight partial genome sequences were obtained from six different animals. Phylogenetic tree and pairwise distance analysis showed high similarity among sequences and with available genomes from different countries. This is the first study on PCV-3 infection dynamics in longitudinally sampled pigs. Most pigs got infection during their life, although PCV-3 did not appear to be linked with any specific age.

Molecular epidemiology study of swine influenza virus revealing a reassorted virus H1N1 in swine farms in Cuba.

In this report, we describe the emergence of reassorted H1N1 swine influenza virus, originated from a reassortment event between the H1N1 pandemic influenza virus (H1N1p/2009) and endemic swine influenza virus in Cuban swine population. In November 2010, a clinical respiratory outbreak was reported on a pig fattening farm in Cuba. Phylogenetic analysis showed that all the genes of one of the isolate obtained, with the exception of neuraminidase, belonged to the H1N1p/2009 cluster. This finding suggests that H1N1pdm has been established in swine and has become a reservoir of reassortment that may produce new viruses with both animal and public health risks.

miRNA expression profile analysis in kidney of different porcine breeds.

microRNAs (miRNAs) are important post-transcriptional regulators in eukaryotes that target mRNAs repressing their expression. The uncertain process of pig domestication, with different origin focuses, and the selection process that commercial breeds suffered, have generated a wide spectrum of breeds with clear genetic and phenotypic variability. The aim of this work was to define the miRNAs expression profile in kidney of several porcine breeds. Small RNA libraries from kidney were elaborated and high-throughput sequenced with the 454 Genome Sequencer FLX (Roche). Pigs used were classified into three groups: the European origin group (Iberian breed and European Wild Boar ancestor), European commercial breeds (Landrace, Large White and Piétrain breeds) and breeds with Asian origin (Meishan and Vietnamese breeds). A total of 229 miRNAs were described in the pig kidney miRNA profile, including 110 miRNAs out of the 257 previously described pig miRNAs and 119 orthologous miRNAs. The most expressed miRNAs in pig kidney microRNAome were Hsa-miR-200b-3p, Ssc-miR-125b and Ssc-miR-23b. Moreover, 5 novel porcine miRNAs and 3 orthologous miRNAs could be validated through RT-qPCR. miRNA sequence variation was determined in 116 miRNAs, evidencing the presence of isomiRs. 125 miRNAs were differentially expressed between breed groups. The identification of breed-specific miRNAs, which could be potentially associated to certain phenotypes, is becoming a new tool for the study of the genetic variability underlying complex traits and furthermore, it adds a new layer of complexity to the interesting process of pig evolution.

Isolation and complete genomic characterization of pandemic H1N1/2009 influenza viruses from Cuban swine herds.

The emergence of the pandemic H1N1/2009 influenza virus poses a potential global threat for human and animal health. In this study, we carried out pandemic H1N1/2009 influenza virus surveillance in swine herds in Cuba intending to determine whether the virus was circulating among pig populations. As a result we describe, for the first time, the detection of pandemic H1N1/2009 influenza virus in swine herds in Cuba. In addition, phylogenetic analysis and molecular characterization of three viral isolates were performed. Phylogenetic relationships confirmed that all of the eight genes of the three isolates were derived from the pandemic H1N1/2009 virus. The Cuban isolates, formed an independent cluster within the pandemic H1N1/2009 influenza strains. Different molecular markers, previously described in pandemic H1N1/2009 influenza viruses, related with adaptive evolution, viral evasion from the host-immune response, virulence and dissemination were also present in Cuban pandemic H1N1/2009 isolates.

Development and validation of a novel SYBR Green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCR platforms.

Classical swine fever is a highly contagious viral disease that causes significant economic losses in pig production on a global scale. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate classical swine fever virus (CSFV) detection methods, which can assist in disease control. The development and evaluation of a novel quantitative real-time RT-PCR assay for CSFV detection, based on SYBR Green coupled to melting curve analysis, is described. The analytical and diagnostic performances of the method using two real-time PCR instruments were compared. The assay was specific and detected the major genotypes of CSFV. The limit of detection in cell culture medium and serum was 0.1 TCID50/reaction, while in tissue homogenate for both platforms, it was 1 TCID50/reaction. The limit of detection was 1, 10 and 10² gene copies/µL when nuclease-free water, serum and tissue homogenate, respectively, were used as sample matrices for both instruments. The analysis of 108 tissue homogenate and serum samples from animals infected with CSFV naturally and experimentally and non-infected animals showed that the assay provided a highly sensitive and specific method for classical swine fever.

Applying phylogenetic analysis to viral livestock diseases: moving beyond molecular typing.

Changes in livestock production systems in recent years have altered the presentation of many diseases resulting in the need for more sophisticated control measures. At the same time, new molecular assays have been developed to support the diagnosis of animal viral disease. Nucleotide sequences generated by these diagnostic techniques can be used in phylogenetic analysis to infer phenotypes by sequence homology and to perform molecular epidemiology studies. In this review, some key elements of phylogenetic analysis are highlighted, such as the selection of the appropriate neutral phylogenetic marker, the proper phylogenetic method and different techniques to test the reliability of the resulting tree. Examples are given of current and future applications of phylogenetic reconstructions in viral livestock diseases.

A DNA vaccine expressing the E2 protein of classical swine fever virus elicits T cell responses that can prime for rapid antibody production and confer total protection upon viral challenge.

Immunization of domestic pigs with a DNA vaccine expressing the complete E2 protein of classical swine fever virus (CSFV) conferred total protection against a severe viral challenge. Immunization with three doses of plasmid pcDNA3.1/E2 elicited a consistent and specific, MHC class II restricted T cell response in the three domestic pigs analyzed, in the absence of detectable anti-CSFV antibodies in serum. Upon challenge specific T cell responses were boosted in the three vaccinated pigs, and a rapid rise in the titers of CSFV neutralizing antibodies was noticed in two of them, which correlated with a total protection. In these two pigs, neither disease symptoms were observed nor was virus detected at any time after CSFV infection. Neutralizing antibody titers were lower in the third vaccine, which developed a mild and transient peak of pyrexia. As expected, similar analyses in three control pigs (injected with the empty vector or PBS) did not reveal the induction of specific T cells or viral antibodies and, upon challenge, animals developed severe symptoms of the disease, including high titers of viremia, hyperthermia and virus spread to different organs. Control pigs developed, also, a marked leucopenia, resulting in SWC3+ (myelomonocytic cells) being the major PBMC population, and a drastic decrease CD3+ T cells. This T cell depletion was prevented in animals immunized with pcDNA3.1/E2. The total protection achieved, in the absence of CSFV antibodies before challenge, supports the relevance in the antiviral response observed of specific T cell responses primed by pcDNA3.1/E2 vaccine, which, upon challenge, led to a rapid induction of neutralizing antibodies. The observation that CSFV antibodies could only be detected in protected animals after viral challenge opens the possibility of exploring the potential of the DNA vaccine approach used to develop marker vaccines against CSF.

Origin and evolution of viruses causing classical swine fever in Cuba.

We have analyzed the origin and evolution of viruses from the classical swine fever (CSF) epidemic that affects Cuba since 2001 by nucleotide sequencing of regions within the E2 glycoprotein and the NS5B (polymerase) genes. The sequence of 190 nucleotides from E2 gene was determined for 10 CSF viruses isolated at different locations of the island, and used for phylogenetic analyses, including sequences from viruses of the 1993--1997 epizootic, previously determined, as well as those from representatives of the different CSFV genotypes. The phylogenetic tree obtained indicates that viruses circulating at present belong to the subgroup 1.2 and are closely related to those isolated during the 1993--1997 epizootic, including the strain Margarita used for vaccine potency tests in Cuba. However, the pattern of evolution revealed by these analyses was different than that observed previously, in which western isolates were almost identical to Margarita strain, while eastern isolates showed a higher level of genetic diversification. In this case, all the viruses analyzed grouped in an independent, define cluster that is closely related, albeit distinguishable, from that of Margarita-related viruses that previously circulated in the western part of Cuba. In addition, the 2001--2003 viruses showed a branched pattern with a level of sequence diversification similar to that observed in the eastern 1993--1997 viruses. Interestingly, a significant fraction (about 54%) of the mutations found in the E2 sequence led to amino acid replacements. This high rate of non-synonymous mutations was not found in the previous Cuban epizootic and has not been reported for other CSF outbreaks. In spite of these amino acid replacements, no antigenic changes were observed in the reactivity of different isolates with CSFV-specific MAbs and polyclonal sera. The phylogenetic tree derived from 409 nucleotides of NS5B gene of seven isolates and Margarita strain, was consistent with that obtained from E2 sequences. In this region, encoding a non-structural protein, a low level of fixation of non-synonymous mutations was observed. The results obtained suggests that epidemiological factors affecting CSFV spread during the current epizootic in Cuba can favour the fixation of non-synonymous mutation in the E2 gene, which could be associated with a lower severity in the clinical signs developed by most of the affected animals.

Analysis of the immune response against mixotope peptide libraries from a main antigenic site of foot-and-mouth disease virus.

The design of vaccines for RNA viral diseases is complicated by the high genetic variability of the viruses, which favors the selection of escape mutants. A case in point is foot-and-mouth disease virus (FMDV), for which only limited protection has been observed in vaccination with single peptides. We have explored the potential of immunogens of higher sequence diversity, covering a broad range of field or culture-induced mutations at the immunodominant site A of FMDV, serotype C. Four mixotope-type peptide libraries, containing ca. 3 x 10(3) or ca. 3 x 10(5) peptides each, in either linear or cyclic form, and combining most significant mutations found or induced at site A have been synthesized and used to immunize guinea-pigs. Substantial levels of serum conversion have been observed for all four mixotope libraries, as well as for single peptides, linear or cyclic, corresponding to the consensus site A sequence. The specificity and neutralizing ability of the anti-mixotope and -peptide antibodies have been evaluated by direct ELISA and by plaque reduction and micro-neutralization assays, respectively. Challenge experiments with an infectious, guinea-pig-adapted FMDV strain, have shown higher protection rates in animals immunized with the cyclic versions, either in single sequence or in combinatorial mixotope form.

Towards a multi-site synthetic vaccine to foot-and-mouth disease: addition of discontinuous site peptide mimic increases the neutralization response in immunized animals.

Synthetic replicas of both antigenic sites A and D of foot-and-mouth disease virus have been tested as a first step towards a multicomponent peptide vaccine candidate. A first evaluation has been performed by neutralization assays on cells with serum mixtures from guinea pigs immunized independently with site A (A24) and site D (D8) peptides. The addition of site D antibodies to site A antibodies has a synergistic effect on neutralization. In a second group of experiments, guinea pigs have been immunized with a dendrimeric tetravalent (MAP) presentation of site A peptide, alone or in combination with D8, using the same total peptide dose. While the first inoculation gives a preferential response to site A-only antigen, specific response to site D and global neutralization levels significantly increase after reimmunization, reflecting a synergistic effect of site D.

Recovery of infectious foot-and-mouth disease virus from suckling mice after direct inoculation with in vitro-transcribed RNA.

We assayed the infectivity of naked foot-and-mouth disease virus (FMDV) RNA by direct inoculation of suckling mice. Our results demonstrate that transcripts generated from full-length cDNA clones were infectious, as was virion-extracted RNA. Interestingly, infectious virus could be recovered from a mutant transcript encoding amino acid substitution L-147-->P in capsid protein VP1, known to be noninfectious for BHK-21 cells. The model described here provides a useful tool for virulence studies in vivo, bypassing possible selection of variants during viral replication in cell culture.