Gaëlle Simon

Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross-sectional study

A study was carried out in 125 farrow-to-finish pig herds to assess the relationships between pathogens involved in respiratory disorders and to relate these findings to clinical signs of respiratory diseases and pneumonia and pleuritis at slaughter. Clinical examination and sampling were carried out on four different batches in each herd (pigs aged 4, 10, 16 and 22 weeks). Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine influenza viruses (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were detected by serological or PCR tests. Pneumonia-like gross lesions and pleuritis were scored at the slaughterhouse. The results indicate that the percentage of pigs PCR-positive for PCV2 at 4, 10 and 16 weeks old was associated with the percentage of pigs PCR-positive for M. hyopneumoniae at these ages. On the other hand, the percentage of pigs with antibodies against PRRSV at 10, 16 and 22 weeks was positively correlated with the percentage of pigs seropositive for M. hyopneumoniae at 22 weeks, with the percentage of pigs with antibodies against SIV H1N1 and SIV H1N2 and the percentage of pigs sero-positive for A. pleuropneumoniae serotype 2. The findings also indicate that, within the five studied pathogens, M. hyopneumoniae, PRRSV and SIV H1N1 are the major pathogens involved in pneumonia-like gross lesions even though PCV2 may play a role. A. pleuropneumoniae serotype 2, in association with PRRSV, is significantly associated with extensive pleuritis. Respiratory diseases could be significantly reduced by implementing measures including appropriate management practices to control these pathogens.

Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013

ABSTRACT The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like (“avian-like”) genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level. IMPORTANCE The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like (“avian-like”) lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

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.

The global antigenic diversity of swine influenza A viruses

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans. DOI: http://dx.doi.org/10.7554/eLife.12217.001

Valproic acid reduces the intracellular level of glutathione and stimulates human immunodeficiency virus

Modifications of the glutathione (GSH) intracellular level have been implicated in the regulation of human immunodeficiency virus (HIV) transcription and expression. In regard to this hypothesis, we have investigated the effects of valproic acid (VPA) on HIV replication. Indeed, it has been recently reported that VPA inhibits the human red blood cell glutathione reductase. In the supernatant of a CEM-SS T-lymphocytic cell line infected with the LAI strain of HIV-1, we observed an increase, in a dose-dependent fashion, of the reverse transcriptase activity after treatment of cells with VPA. VPA also induced HIV expression in the chronically infected monocytic U1 cell line which constitutively expresses low levels of virus, enhanced the HIV-long terminal repeat (LTR)-directed expression of beta-galactosidase in transiently transfected Jurkat T-cells, and potentiated the PMA effect on the LTR transactivation. GSH assays showed that VPA treatment led to a decrease in the intracellular level of this thiol compound in U937 (U1 parent-cell line) and in Jurkat T-cells. Work to understand the molecular mechanism of VPA-induced HIV transcription and expression are now in progress. VPA seems to be an adequate molecule to study the implications of a GSH decrease in the stimulation of HIV replication. However, a modification of the intracellular balance between reduced and oxidized glutathione, rather than a simple reduction of the intracellular glutathione level, could be of importance in the regulation of HIV replication and we are now testing this hypothesis. Finally, these findings already suggest that VPA, which is an anticonvulsive drug frequently prescribed for the management of various seizure disorders, should not be recommended for treatment of epilepsy or other related illnesses in HIV-positive individuals.

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.

Effects of glutathione precursors on human immunodeficiency virus replication

Asymptomatic human immunodeficiency virus (HIV)-seropositive individuals have reduced glutathione (GSH) levels. This has led to the suggestion that elevated intracellular thiols levels may inhibit HIV replication and progression of the disease. We confirmed that N-acetyl-L-cysteine (NAC), a cysteine prodrug which maintains intracellular GSH levels during oxidative stress, inhibits in the chronically infected U1 cells, the stimulation of HIV replication induced by phorbol 12-myristate 13-acetate (PMA), interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF). However, we found no significant inhibition of PMA-mediated long terminal repeat (LTR)-directed beta-galactosidase expression in transiently transfected Jurkat T-cells. We have compared NAC effects with the effects of other GSH precursors on HIV expression. Treatment of the U1 cell line by L-2-oxo-4-thiazolidine carboxylic acid (OTC), which is converted to cysteine by 5-oxoprolinase, or by homocysteine (HC), a natural cysteine precursor, reduced the PMA-induced HIV expression, but surprisingly, markedly stimulated the expression mediated by IL-6 and GM-CSF. Several experiments to investigate the effect of OTC on LTR transactivation were carried out, but beta-galactosidase activity was never modified in a significant fashion in PMA-induced Jurkat T-cells after OTC treatment. Furthermore, HC stimulated the PMA-mediated HIV-LTR transactivation in Jurkat T-cells. GSH assays showed that treatment of U937 and Jurkat T-cells with NAC and OTC moderately increased the GSH level, while HC led to a significantly higher increase of the thiol level. In conclusion, it appeared that an increase of the GSH intracellular level did not lead solely to an inhibition of HIV replication but could also lead to an activation of viral expression. This seemed the case when HIV replication was stimulated by compounds which act mainly at a post-transcriptional level.

In vitro and ex vivo analyses of co-infections with swine influenza and porcine reproductive and respiratory syndrome viruses.

Abstract Viral respiratory diseases remain problematic in swine. Among viruses, porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV), alone or in combination, are the two main known contributors to lung infectious diseases. Previous studies demonstrated that experimental dual infections of pigs with PRRSV followed by SIV can cause more severe disease than the single viral infections. However, our understanding of the impact of one virus on the other at the molecular level is still extremely limited. Thus, the aim of the current study was to determine the influence of dual infections, compared to single infections, in porcine alveolar macrophages (PAMs) and precision cut lung slices (PCLS). PAMs were isolated and PCLS were acquired from the lungs of healthy 8-week-old pigs. Then, PRRSV (ATCC VR-2385) and a local SIV strain of H1N1 subtype (A/Sw/Saskatchewan/18789/02) were applied simultaneously or with 3h apart on PAMs and PCLS for a total of 18h. Immuno-staining for both viruses and beta-tubulin, real-time quantitative PCR and ELISA assays targeting various genes (pathogen recognition receptors, interferons (IFN) type I, cytokines, and IFN-inducible genes) and proteins were performed to analyze the cell and the tissue responses. Interference caused by the first virus on replication of the second virus was observed, though limited. On the host side, a synergistic effect between PRRSV and SIV co-infections was observed for some transcripts such as TLR3, RIG-I, and IFNβ in PCLS. The PRRSV infection 3h prior to SIV infection reduced the response to SIV while the SIV infection prior to PRRSV infection had limited impact on the second infection. This study is the first to show an impact of PRRSV/SIV co-infection and superinfections in the cellular and tissue immune response at the molecular level. It opens the door to further research in this exciting and intriguing field.

Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events

Concomitant infections by different influenza A virus subtypes within pig farms increase the risk of new reassortant virus emergence. The aims of this study were to characterize the epidemiology of recurrent swine influenza virus infections and identify their main determinants. A follow-up study was carried out in 3 selected farms known to be affected by repeated influenza infections. Three batches of pigs were followed within each farm from birth to slaughter through a representative sample of 40 piglets per batch. Piglets were monitored individually on a monthly basis for serology and clinical parameters. When a flu outbreak occurred, daily virological and clinical investigations were carried out for two weeks. Influenza outbreaks, confirmed by influenza A virus detection, were reported at least once in each batch. These outbreaks occurred at a constant age within farms and were correlated with an increased frequency of sneezing and coughing fits. H1N1 and H1N2 viruses from European enzootic subtypes and reassortants between viruses from these lineages were consecutively and sometimes simultaneously identified depending on the batch, suggesting virus co-circulations at the farm, batch and sometimes individual levels. The estimated reproduction ratio R of influenza outbreaks ranged between 2.5 [1.9-2.9] and 6.9 [4.1-10.5] according to the age at infection-time and serological status of infected piglets. Duration of shedding was influenced by the age at infection time, the serological status of the dam and mingling practices. An impaired humoral response was identified in piglets infected at a time when they still presented maternally-derived antibodies.

The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model.

Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCɛRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.