Gerard E. Martín-Valls

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus.

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.

Torque teno sus virus 1 and 2 viral loads in postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) affected pigs

Torque teno viruses (TTV) are small, non-enveloped viruses with a circular single-stranded DNA genome, which are considered non-pathogenic. However, TTVs have been eventually linked to human diseases. TTVs infecting pigs, Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2), have been recently associated to porcine circovirus diseases (PCVD). To get more insights into such potential disease association, the aim of this study was to quantify TTSuV1 and TTSuV2 viral loads in serum of pigs affected by two PCVDs, postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). Such study was carried out by means of a newly developed real-time quantitative PCR (qPCR) method. Both TTSuVs were highly prevalent among studied pigs. TTSuV2 viral loads were significantly higher in PMWS affected animals, further supporting the previously suggested association between TTSuV2 and PMWS. On the contrary, TTSuV1 prevalence and loads were not related with the studied PCVDs.

Seroprevalence and risk factors of swine influenza in Spain.

Swine influenza is caused by type A influenza virus. Pigs can be infected by both avian and human influenza viruses; therefore, the influenza virus infection in pigs is considered an important public health concern. The aims of present study were to asses the seroprevalence of swine influenza subtypes in Spain and explore the risk factors associated with the spread of those infections. Serum samples from 2151 pigs of 98 randomly selected farms were analyzed by an indirect ELISA for detection of antibodies against nucleoprotein A of influenza viruses and by the hemagglutination inhibition (HI) using H1N1, H1N2 and H3N2 swine influenza viruses (SIV) as antigens. Data gathered in questionnaires filled for each farm were used to explore risk factors associated with swine influenza. For that purpose, data were analyzed using the generalized estimating equations method and, in parallel by means of a logistic regression. By ELISA, 92 farms (93.9%; CI(95%): 89.1-98.7%) had at least one positive animal and, in total, 1340/2151 animals (62.3%; CI(95%): 60.2-64.3%) were seropositive. A total of 1622 animals (75.4%; CI(95%): 73.6-77.2%) were positive in at least one of the HI tests. Of the 98 farms, 91 (92.9%; CI(95%): 87.7-98.1%) had H1N1 seropositive animals; 63 (64.3%; CI(95%): 54.6-73.9%) had H1N2 seropositive pigs and 91 (92.9%; CI(95%): 87.7-98.1%) were positive to H3N2. Mixed infections were detected in 88 farms (89.8; CI(95%): 83.7-95.9%). Three risk factors were associated with seroprevalences of SIV: increased replacement rates in pregnancy units and, for fatteners, existence of open partitions between pens and uncontrolled entrance to the farm.

Swine influenza virus infection dynamics in two pig farms; results of a longitudinal assessment

In order to assess the dynamics of influenza virus infection in pigs, serological and virological follow-ups were conducted in two whole batches of pigs from two different farms (F1 and F2), from 3 weeks of age until market age. Anti-swine influenza virus (SIV) antibodies (measured by ELISA and hemagglutination inhibition) and nasal virus shedding (measured by RRT-PCR and isolation in embryonated chicken eggs and MDCK cells) were carried out periodically. SIV isolates were subtyped and hemagglutinin and neuraminidase genes were partially sequenced and analyzed phylogenetically. In F1, four waves of viral circulation were detected, and globally, 62/121 pigs (51.2%) were positive by RRT-PCR at least once. All F1 isolates corresponded to H1N1 subtype although hemagglutination inhibition results also revealed the presence of antibodies against H3N2. The first viral wave took place in the presence of colostral-derived antibodies. Nine pigs were positive in two non-consecutive sampling weeks, with two of the animals being positive with the same isolate. Phylogenetic analyses showed that different H1N1 variants circulated in that farm. In F2, only one isolate, H1N2, was detected and all infections were concentrated in a very short period of time, as assumed for a classic influenza outbreak. These findings led us to propose that influenza virus infection in pigs might present different patterns, from an epidemic outbreak to an endemic form with different waves of infections with a lower incidence.

Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model.

Outbreaks involving either H5N1 or H1N1 influenza viruses (IV) have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM). This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1) from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1), two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

Transmission of Porcine reproductive and respiratory syndrome virus 1 to and from vaccinated pigs in a one-to-one model

The present study examined transmission by contact of Porcine reproductive and respiratory syndrome virus (PRRSV) 1 in a one-to-one model to vaccinated and unvaccinated pigs and from vaccinated infected pigs to other vaccinated pigs. The experiment started by randomly assigning weaned pigs to groups V (n=24) and U (n=26). V pigs were vaccinated with a commercial live attenuated PRRSV vaccine and the U animals were kept as unvaccinated controls. Twenty-eight days later, 6U pigs were separated and allocated in individual boxes. The remaining 20U pigs were intranasally inoculated with PRRSV isolate 3267 (from now on designated as seeder (S) pigs) and 48h later were distributed in boxes where they were commingled with either V or U pigs in 1:1 groups (first contact phase), resulting in 6S:U and 14S:V pairs. As soon as a V pig was detected to be viremic because of contact with a S, the infected V (from now on designated as Vinf) was transferred (<24h after detection) to a new pen where it was comingled with a new V pig (designated as V2) in a second contact phase. For the first contact phase, pigs were maintained 21days at maximum and for the second contact phase the maximum exposure period was 14days. Two V pigs tested positive for the vaccine virus (>99.5% similarity) when they were relocated with the corresponding V2 pigs and they were removed; thus, only 12Vinf were finally considered. All V pigs (12/12) exposed to S animals became infected although the first detection of viremia occurred at 13.6±3.6days, one week later than in U (p<0.05). Also, duration of viremia was shorter for Vinf compared to U, (5.5±4.3days versus 12.5±2.7days). The Vinf group showed remarkable individual variability: eight animals had a viremic period of 5 or less days (3.0±1.4) while the remaining four had a longer viremic period of more than one week (10.8±2.9). This situation was not observed in U. In the second contact phase, transmission from Vinf to V2 pigs occurred in 7/8 cases (87.5%). The mean duration of viremia for V2 was 4.8±3.4 and two different patterns were again observed: two animals had viremias of 9-10days and the rest averaged 3.0±1.4days (range: 2-5days). Vaccinated groups Vinf and V2 had a significantly lower PRRSV shedding in oral fluids for at least the first 9days after the onset of the viremia compared to U, and shedding for V2 was even significantly lower (p<0.05) than shedding for Vinf. Our experimental design reproduced the worst-case scenario for evaluating the effect of vaccination and, under such conditions; it was still efficacious in slowering PRRSV transmission and decreasing the global viral load and particularly oral shedding.

Phylogeny of Spanish swine influenza viruses isolated from respiratory disease outbreaks and evolution of swine influenza virus within an endemically infected farm

In the present study, outbreaks of respiratory disease were investigated for the presence of swine influenza virus (SIV). In 14 cases the circulating SIV strains were isolated, fully sequenced and compared with other known SIVs. The viruses causing the outbreaks belonged to the H1N1 (including human pandemic H1N1), H3N2 and H1N2 subtypes. In 11/14 cases the phylogenetic analyses indicated the occurrence of probable reassortment events. In the second part of the study, the genetic evolution of H1N1 SIV was assessed in a longitudinal study in closed groups of pigs over six months. Sequencing of the 22 isolates indicated co-circulation of two different variants for the same virus, as well as the emergence of SIV reassortants at certain time-points. These results indicate that reassortment events in SIV are common, and point towards the need for a better understanding of the epidemiology of SIV, particularly in endemic farms.

Comparison of two commercial enzyme-linked immunosorbent assays for the diagnosis of Porcine reproductive and respiratory syndrome virus infection

Early diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV) is critically important for control of the disease. Two new commercially available enzyme-linked immunosorbent assays (ELISAs) based on different methodologies have been developed. In the present report, the 2 ELISAs were compared using blood samples from experimentally and naturally infected pigs. One of the 2 ELISAs was shown to be more sensitive than the other. The higher sensitivity of one of the ELISAs could pose a problem in PRRS diagnosis in endemic farms, because it can detect maternally derived antibodies for a longer time, overlapping with the detection of antibodies developed after PRRSV infection. However, the ELISA with higher sensitivity could be suitable for early detection of PRRSV antibodies in individual pigs, especially in PRRS-free herds.

Testing of umbilical cords by real time PCR is suitable for assessing vertical transmission of porcine reproductive and respiratory syndrome virus under field conditions

The objective of this study was to test the suitability of umbilical cord (UC) sampling and ear vein swabbing (EVS) as alternatives to jugular vein bleeding (JVB) for the assessment of vertical transmission of porcine reproductive and respiratory syndrome virus (PRRSV). Twelve farms suspected to be PRRSV-positive unstable were selected and the three types of samples were obtained from 21 batches of newborn piglets (n=387). The proportions of positive results, viral loads and time spent to collect the samples were compared. UC yielded the highest detection rate, with 76 positives compared to 55 JVB- and 45 EV-positive results (P<0.05). Average Ct values were 26.6±8.5 for JVB, 30.8±6.4 for EV and 32.1±4.85 for UC (P<0.01). UC was the fastest collection method (mean 24s vs. 55s for EV and 72s for JVB; P<0.05). In this study, UC testing was a faster and more sensitive alternative to JVB or EV for the detection of PRRSV in newborn piglets.

Estimation of the transmission parameters for swine influenza and porcine reproductive and respiratory syndrome viruses in pigs from weaning to slaughter under natural conditions

In the present study, the transmission parameters of swine influenza virus (SIV) and porcine reproductive and respiratory virus (PRRSV) have been calculated using the basic reproductive rate (R) parameter in two commercial pig farms (F1 and F2). In order to do this, a serological (PRRSV genotype 1 and SIV) and virological (SIV) follow-up of a batch of animals was carried out weekly from 3 weeks of age until the age of slaughter on each farm. Results of the analysis for SIV and PRRSV showed different transmission profiles depending on the farm, the pathogen, and time of transmission. In F1, transmission of both viruses was detected throughout the sampling. The Rt (R for a given period of time) value for SIV ranged from 1.5 [0.9-2.3] to 3.6 [2.3-4.9] from farrowing to the beginning of the fattening period, and the Rt value for PRRSV was 3.3 [2.9-4.3] to 3.5 [2.8-4.1] from farrowing until the slaughter age. These results indicated that both viruses were transmitted enzootically in that farm for these periods of time. A different transmission pattern with a higher incidence was also observed during the fattening period in F1 (after 15 weeks of age) for SIV, coinciding with the entrance of a new subtype. In this case, R value for SIV reached 3.3 [1.65-4.9]. On the other hand, in F2, SIV and PRRSV seemed to be restricted to the fattening period. R reached a value of 6.4 [4.1-8.8] for SIV and 7.1 [3.5-10.6] for PRRSV. These findings suggest a different origin of the virus, as well as a more epidemic circulation, especially for SIV, where most of the new cases were observed in a one week period. In conclusion, the present study offers a reliable estimation of the range of Rt values for SIV and genotype 1 PRRSV transmission under field conditions, suggesting that enzootic circulations of both viruses are similar in terms of transmission, probably higher for PRRSV, but also that transmission of SIV is more efficient (or epidemic) than transmission of a genotype 1 PRRSV isolate in naïve animals given the new cases observed in only in F2.