Aurélie Oger

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication.

Individual risk factors for Post-weaning Multisystemic Wasting Syndrome (PMWS) in pigs: A hierarchical Bayesian survival analysis

Risk factors for Post-weaning Multisystemic Wasting Syndrome (PMWS) at the pig level were identified using data from a longitudinal study in seven PMWS-affected farms in France. In each farm, a representative sample of 120 pigs (180 in one farm) was randomly selected after farrowing and followed from birth to slaughter. Individual information included serological status for Porcine Circovirus type 2 (PCV-2), Porcine Reproductive and Respiratory Syndrome (PRRS) virus, and Porcine Parvovirus (PPV), individual weight, rearing conditions, and clinical observations recorded at 7, 13, 16 and 21 weeks of age and at slaughter. Two different Bayesian frailty models were used to identify variables related to time-to-PMWS: (i) a logistic-survival model and (ii) an accelerated failure time model (different survival time distributions) both with two levels of clustering (litter and farm). Similar results in terms of variables related to time-to-PMWS were obtained with both models. However, information provided by the different approaches were complementary. Piglets were more likely to exhibit PMWS after early infection by PCV-2 (i.e. before 7 weeks old) and if they were weaned early (before 21 days). Piglets born to PCV-2 seronegative sows and/or to sows with neck injuries due to poorly performed injections were also more at risk. With the accelerated failure time model, time ratios were obtained giving an estimation of the expected survival time (increased or decreased) after exposure to the factor. The logistic-survival model showed that the majority of the risk factors were mostly related to the odds of PMWS whereas the PCV-2 passive immunity derived from the dam also tended to postpone PMWS appearance later.

Effect of granulocyte-macrophage colony-stimulating factor on post-weaning multisystemic wasting syndrome in porcine circovirus type-2-transfected piglets.

Post‐weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type‐2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2‐affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem‐cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co‐factor, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). A single intramuscular injection of tandem‐cloned PCV2 DNA into 5‐week‐old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post‐injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper‐right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs – the tonsil and the lung – and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co‐injected with plasmid containing GM‐CSF DNA or had received plasmid containing PCV2 DNA alone. GM‐CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.

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

Pseudorabies virus is the causative agent of Aujeszkys 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.

Differential cellular gene expression in duck trachea infected with a highly or low pathogenic H5N1 avian influenza virus.

BackgroundAvian influenza A (AI) viruses of subtypes H5 can cause serious disease outbreaks in poultry including panzootic due to H5N1 highly pathogenic (HP) viruses. These viruses are a threat not only for animal health but also public health due to their zoonotic potential. The domestic duck plays a major role in the epidemiological cycle of influenza virus subtypes H5 but little is known concerning host/pathogen interactions during influenza infection in duck species. In this study, a subtracted library from duck trachea (a primary site of influenza virus infection) was constructed to analyse and compare the host response after a highly or low pathogenic (LP) H5N1-infection.ResultsHere, we show that more than 200 different genes were differentially expressed in infected duck trachea to a significant degree. In addition, significant differentially expressed genes between LPAI- and HPAI-infected tracheas were observed. Gene ontology annotation was used and specific signalling pathways were identified. These pathways were different for LPAI and HPAI-infected tracheas, except for the CXCR4 signalling pathway which is implicated in immune response. A different modulation of genes in the CXCR4 signalling pathway and TRIM33 was induced in duck tracheas infected with a HPAI- or a LPAI-H5N1.ConclusionFirst, this study indicates that Suppressive Subtractive Hybridization (SSH) is an alternative approach to gain insights into the pathogenesis of influenza infection in ducks. Secondly, the results indicate that cellular gene expression in the duck trachea was differently modulated after infection with a LPAI-H5N1 or after infection with a HPAI-H5N1 virus. Such difference found in infected trachea, a primary infection site, could precede continuation of infection and could explain appearance of respiratory symptoms or not.

Evaluation of the transmission of porcine circovirus type 2 (PCV-2) genogroups a and b with semen from infected specific-pathogen-free boars.

The porcine circovirus type 2 (PCV-2) is associated with several diseases including reproductive failure. This syndrome has been experimentally reproduced twice with two PCV-2 isolates representative of each major PCV-2 genogroup, i.e. PCV-2a and PCV-2b (Cariolet et al., 2002; Rose et al., 2007). In these two previous studies, the sows were infected by intra-uterine inoculation at insemination with 10(4.3) and 10(3.18) TCID(50) of PCV-2a and PCV-2b, respectively, corresponding to 1.2 × 10(11) and 3 × 10(10) genome copies, respectively. The aim of this present study was to quantify viral shedding in semen from specific-pathogen-free (SPF) boars infected with isolates from the two major PCV-2 genogroups a and b. We studied the transmission of the PCV-2 virus through contaminated semen to SPF sows and their offspring. The four inoculated boars developed sub-clinical PCV-2 infections and PCV-2 genomes were occasionally detected in semen after nasal infection of boars, with up to 1.2 × 10(6)copies/mL in the sperm-rich fraction. When PCV-2-contaminated semen was inoculated in SPF sows at artificial insemination, the sows and their offspring did not show any signs of PCV-2 infection or PCV-2 antibodies or genomes. In the present study, sows were inoculated with a maximal dose of 1.7 × 10(7) viral genome copies, which is lower than the genomic loads (i.e. 1.2 × 10(11) and 3 × 10(10) genome copies) that have been shown to induce reproductive troubles in intra-uterine inoculated sows. Our results together with the previous experiment findings suggest that PCV-2-induced reproductive disorders depend on the infectious dose inoculated to sows by the intra-uterine route.

Naked PCV-2 cloned genomic DNA is infectious by mucosal (intratracheal or oro-nasal) inoculation

Porcine circovirus type 2 (PCV-2) is involved in several diseases named porcine circovirus-associated diseases and is transmitted by oro-faecal route. In this study we inoculated porcine-circovirus free piglets by mucosal routes (intratracheal or oro-nasal routes) with a plasmid carrying two copies of PCV-2 genomic DNA and compared the results to the intramuscular route. We observed that this PCV-2 naked DNA serves as template for viral replication and infectious PCV-2 particles are detected in the whole body after parenteral (intramuscular) or mucosal (intratracheal or oro-nasal) delivery. These results suggest that PCV-2 genome could play a role in in vivo transmission.