Constantinos S. Kyriakis

Field Evaluation of the Effect of a Probiotic-containing Bacillus licheniformis and Bacillus subtilis Spores on the Health Status, Performance, and Carcass Quality of Grower and Finisher Pigs

Summary The aim of the study was to assess the efficacy of BioPlus 2B, a probiotic containing Bacillus licheniformis and B. subtilis spores, on the health status and productivity of pigs, during weaning, growing and finishing stages of growth. On a commercial farrow‐to‐finish farm, five experimental groups were formed, each of 54 weaned piglets. The pigs of the first group (double controls) received normal feed with no probiotic and the pigs of the second group (untreated controls) received BioPlus 2B only during the weaning stage. The pigs of the third, the fourth and the fifth group received the same as the second group feed but, at the growing and at a part of the finishing stages, supplemented with three different doses of Bioplus 2B, a low, medium and high dose, respectively. The results have shown that, compared with the double controls, BioPlus 2B‐treated pigs had a lower morbidity and mortality during the whole trial period, compared with the double controls (range from 9.26 to 14.81% versus 25.93% and from 0.00 to 3.70% versus 11.1%, respectively), as a result of the lower incidence of post‐weaning diarrhoea due mainly to Escherichia coli. Weight gain, feed conversion ratio and carcass quality of the BioPlus 2B‐treated pigs were significantly improved compared with the double controls, whilst the beneficial effects of the probiotic were more pronounced when the medium and high doses were used.

Virological Surveillance and Preliminary Antigenic Characterization of Influenza Viruses in Pigs in Five European Countries from 2006 to 2008

This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes – H1N1, H3N2 and H1N2 – at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as ‘avian‐like’ H1N1, 36 as human‐like H3N2 and 47 as human‐like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human‐like HA and three H1N2 viruses an avian‐like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross‐hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human‐like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections.

The M Segment of the 2009 Pandemic Influenza Virus Confers Increased Neuraminidase Activity, Filamentous Morphology, and Efficient Contact Transmissibility to A/Puerto Rico/8/1934-Based Reassortant Viruses

ABSTRACT The 2009 H1N1 lineage represented the first detection of a novel, highly transmissible influenza A virus genotype: six gene segments originated from the North American triple-reassortant swine lineage, and two segments, NA and M, derived from the Eurasian avian-like swine lineage. As neither parental lineage transmits efficiently between humans, the adaptations and mechanisms underlying the pandemic spread of the swine-origin 2009 strain are not clear. To help identify determinants of transmission, we used reverse genetics to introduce gene segments of an early pandemic isolate, A/Netherlands/602/2009 [H1N1] (NL602), into the background of A/Puerto Rico/8/1934 [H1N1] (PR8) and evaluated the resultant viruses in a guinea pig transmission model. Whereas the NL602 virus spread efficiently, the PR8 virus did not transmit. Swapping of the HA, NA, and M segments of NL602 into the PR8 background yielded a virus with indistinguishable contact transmissibility to the wild-type pandemic strain. Consistent with earlier reports, the pandemic M segment alone accounted for much of the improvement in transmission. To aid in understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility. IMPORTANCE Our work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor-destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that coadapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.

Serologic Cross- Reactivity with Pandemic (H1N1) 2009 Virus in Pigs, Europe

We tested serum samples from pigs infected or vaccinated with European swine influenza viruses (SIVs) in hemagglutination-inhibition assays against pandemic (H1N1) 2009 virus and related North American SIVs. We found more serologic cross-reaction than expected. Data suggest pigs in Europe may have partial immunity to pandemic (H1N1) 2009 virus.

European Surveillance Network for Influenza in Pigs: Surveillance Programs, Diagnostic Tools and Swine Influenza Virus Subtypes Identified in 14 European Countries from 2010 to 2013

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010–2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.

Efficacy of commercial swine influenza vaccines against challenge with a recent European H1N1 field isolate.

This study examines the immunogenicity and efficacy of four commercial swine influenza (SI) vaccines against challenge with a recent European H1N1 virus, Sw/Gent/112/07. The vaccines contained different H1N1 strains showing between 77% and 95% genetic homology with the haemagglutinin (HA) of the challenge virus. Four groups of 10 pigs each received a double vaccination, with a 4-week interval, with one of the vaccines; a fifth group served as unvaccinated controls. All pigs were challenged 3 weeks after the second vaccination intratracheally with 10(5.0)EID(50) of Sw/Gent/112/07. Sera were examined in haemagglutination inhibition (HI) tests against the homologous vaccine H1N1 strains, the challenge virus and a panel of five recent H1N1 isolates. Pigs were euthanized at 24 or 72h post-challenge and virus titres were determined in right and left lung halves. Two vaccines, in which the H1N1 strains showed a genetic homology of 93% and 89% to Sw/Gent/112/07, significantly reduced virus replication. The vaccine containing an H1N1 strain with 95% homology to Sw/Gent/112/07, did not offer significant protection, neither did it induce the highest HI titres. In general, pigs with HI antibody titres >or=20 against Sw/Gent/112/07 were virologically protected against challenge. HI titres against other viruses, however, differed compared to the challenge virus and between viruses. Our data clearly show that the genetic homology with the challenge virus is not the ultimate predictor for SI vaccine performance. The true reason for the differences in vaccine potency remains obscure because other factors, such as the antigen dose and/or the adjuvant, also differed between the vaccines.

Safety, immunogenicity and efficacy of poxvirus-based vector vaccines expressing the haemagglutinin gene of a highly pathogenic H5N1 avian influenza virus in pigs

This study investigates the safety, immunogenicity and efficacy of different pox-vector vaccines expressing the haemagglutinin of a highly pathogenic (HP) H5N1 avian influenza virus (AIV) (A/chicken/Indonesia/7/03) in pigs. Pigs were vaccinated twice, with a 4-week interval, with a fowlpox (TROVAC), a canarypox (ALVAC), or a vaccinia (NYVAC) vector vaccine combined with an oil-in-water adjuvant, with the unadjuvanted NYVAC, or left unvaccinated. Six weeks after the second vaccination, all pigs were challenged intra-tracheally with low pathogenic (LP) H5N2 AIV A/chicken/Belgium/150/99. Sera were examined in haemagglutination inhibition (HI) tests against the H5N1 AIV from which the vaccine haemagglutinin derived, the challenge virus and the human A/Vietnam/1194/04 HPAIV. After challenge pigs were compared for H5N2 virus replication in the trachea and 4 lung lobes at 24 or 72h post-challenge. Vaccination was well tolerated by all animals. Antibody titres peaked 2 weeks after the second vaccination and were 2- to 4-fold higher against the vaccine virus than heterologous H5 viruses. The NYVAC and ALVAC adjuvanted vaccines consistently induced higher antibody titres than TROVAC or NYVAC without adjuvant. Following challenge, the H5N2 challenge virus was isolated from all unvaccinated pigs, while 19 out of 21 vaccinates showed complete virological protection. Pox-vector vaccines were safe, immunogenic and efficacious against challenge with a heterologous H5 AIV, offering an alternative to classical inactivated vaccines. It remains to be seen whether they would protect against a swine-adapted H5 virus, which may replicate 100-1000 times better than our challenge virus.

Influenza A virus infection dynamics in swine farms in Belgium, France, Italy and Spain, 2006-2008.

Avian-like H1N1 and reassortant H3N2 and H1N2 influenza A viruses with a human-like haemagglutinin have been co-circulating in swine in Europe for more than a decade. We aimed to examine the infection dynamics of the three swine influenza virus (SIV) lineages at the farm level, and to identify possible regional and seasonal variations in their circulation. Sera were collected from six successive generations of fattening pigs (2006-2008) in a total 80 farrow-to-finish herds in Belgium, Italy, France and Spain and examined for antibodies against the three SIVs in haemagglutination inhibition tests. Overall, in all regions and periods, 9.7% of all farms were negative for SIV, 49% were infected with one subtype, 38% with two subtypes and 3.9% with all three SIVs. We found serological evidence for the circulation of all three subtypes in Belgium, Italy and Spain, while only infections with H1N1 and H1N2 SIVs were detected in France. Despite temporary changes in the circulation of H1N2 in Belgium and in Spain, there was no true seasonal variation. The exact combination of subtypes on the same farm differed in each of the sampling periods. On the other hand, 21 farms were found to be consistently infected with the same SIV subtype throughout the study. This can either be explained by the persistence of the virus in a farm, or by the periodical re-introduction of SIVs of the same subtype.

Residue 41 of the Eurasian avian-like swine influenza a virus matrix protein modulates virion filament length and efficiency of contact transmission.

ABSTRACT Position 41 of the influenza A virus matrix protein encodes a highly conserved alanine in human and avian lineages. Nonetheless, strains of the Eurasian avian-like swine (Easw) lineage contain a change at this position: position 41 of A/swine/Spain/53207/04 (H1N1) (SPN04) encodes a proline. To assess the impact of this naturally occurring polymorphism on viral fitness, we utilized reverse genetics to produce recombinant viruses encoding wild-type M1 41P (rSPN04-P) and consensus 41A (rSPN04-A) residues. Relative to rSPN04-A, rSPN04-P virus displayed reduced growth in vitro. In the guinea pig model, rSPN04-P was transmitted to fewer contact animals than rSPN04-A and failed to infect guinea pigs that received a low-dose inoculum. Moreover, the P41A change altered virion morphology, reducing the number and length of filamentous virions, as well as reducing the neuraminidase activity of virions. The lab-adapted human isolate, A/PR/8/34 (H1N1) (PR8), is nontransmissible in the guinea pig model, making it a useful background in which to identify certain viral factors that enhance transmissibility. We assessed transmission in the context of single-, double-, and triple-reassortant viruses between PR8 and SPN04; PR8/SPN04 M, PR8/SPN04 M+NA, and PR8/SPN04 M+NA+HA, encoding either matrix 41 A or P, were generated. In each case, the virus possessing 41P transmitted less well than the corresponding 41A-encoding virus. In summary, we have identified a naturally occurring mutation in the influenza A virus matrix protein that impacts transmission efficiency and can alter virion morphology and neuraminidase activity. IMPORTANCE We have developed a practical model for examining the genetics underlying transmissibility of the Eurasian avian-like swine lineage viruses, which contributed M and NA segments to the 2009 pandemic strain. Here, we use our system to investigate the impact on viral fitness of a naturally occurring polymorphism at matrix (M1) position 41 in an Easw isolate. Position 41 has been implicated previously in adaptation to laboratory substrates and to mice. Here we show that the polymorphism at M1 41 has a limited effect on growth in vitro but changes the morphology of the virus and impacts growth and transmission in the guinea pig model.

MicroRNA-555 has potent antiviral properties against poliovirus.

Vaccination with live-attenuated polio vaccine has been the primary reason for the drastic reduction of poliomyelitis worldwide. However, reversion of this attenuated poliovirus vaccine occasionally results in the emergence of vaccine-derived polioviruses that may cause poliomyelitis. Thus, the development of anti-poliovirus agents remains a priority for control and eradication of the disease. MicroRNAs (miRNAs) have been shown to regulate viral infection through targeting the viral genome or reducing host factors required for virus replication. However, the roles of miRNAs in poliovirus (PV) replication have not been fully elucidated. In this study, a library of 1200 miRNA mimics was used to identify miRNAs that govern PV replication. High-throughput screening revealed 29 miRNAs with antiviral properties against Sabin-2, which is one of the oral polio vaccine strains. In particular, miR-555 was found to have the most potent antiviral activity against three different oral polio attenuated vaccine strains tested. The results show that miR-555 reduced the level of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) required for PV replication in the infected cells, which in turn resulted in reduction of PV positive-strand RNA synthesis and production of infectious progeny. These findings provide the first evidence for the role of miR-555 in PV replication and reveal that miR-555 could contribute to the development of antiviral therapeutic strategies against PV.