In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3·9 kb (M39). Expression of the reporter gene {beta}-glucuronidase (GUS) (2–8 µg per 106 cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1–2 µg per 106 cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development

Apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines

International audienceApoptosis was studied in the lungs of pigs during an infection with a European strain of porcine reproductive and respiratory syndrome virus (PRRSV) and it was examined if cytokines were involved in the induction of apoptosis. Twenty-two 4- to 5-week-old gnotobiotic pigs were inoculated intranasally with 10$^{6.0}$ TCID$_{50}$ of the Lelystad virus and euthanised between 1 and 52 days post inoculation (PI). The lungs and broncho-alveolar lavage (BAL) cells were assessed both for virus replication and apoptosis; BAL fluids were examined for interleukin (IL)-1, tumour necrosis factor-alpha and IL-10. Double-labellings were conducted to determine the relation between virus replication and apoptosis and to identify the apoptotic cells. Apoptosis occurred in both infected and non-infected cells. The percentages of infected cells, which were apoptotic, ranged between 9 and 39% in the lungs and between 13 and 30% in the BAL cells. The majority of apoptotic cells were non-infected. Non-infected apoptotic cells in the lungs were predominantly monocytes/macrophages, whereas those in the broncho-alveolar spaces were predominantly lymphocytes. The peak of apoptosis in the lungs at 14 days PI was preceded by a peak of IL-1 and IL-10 production at 9 days PI, suggesting a possible role of these cytokines in the induction of apoptosis in non-infected interstitial monocytes/macrophages. However, the latter hypothesis was not confirmed in vitro, since blood monocytes or alveolar macrophages did not undergo apoptosis after treatment with recombinant porcine IL-1 or IL-10

Viable porcine arteriviruses with deletions proximal to the 3 ' end of the genome

In order to obtain attenuated live vaccine candidates of porcine reproductive and respiratory syndrome virus (PRRSV), a series of deletions was introduced at the 3′ end of the viral genome using an infectious cDNA clone of the Lelystad virus isolate. RNA transcripts from the full-length cDNA clones were transfected into BHK-21 cells. The culture supernatant of these cells was subsequently used to infect porcine alveolar macrophages to detect the production of progeny virus. It is shown that C-terminal truncation of the nucleocapsid (N) protein, encoded by ORF7, was tolerated for up to six amino acids without blocking the production of infectious virus. Mutants containing larger deletions produced neither virus nor virus-like particles containing viral RNA. Deletion analysis of the 3′ UTR immediately downstream of ORF7 showed that infectious virus was still produced after removal of seven nucleotides behind the stop codon of ORF7. Deletion of 32 nucleotides in this region abolished RNA replication and, consequently, no infectious virus was formed. Serial passage on porcine alveolar macrophages demonstrated that the viable deletion mutants were genetically stable at the site of mutation. In addition, the deletions did not affect the growth properties of the recombinant viruses in vitro, while their antigenic profiles were similar to that of wild-type virus. Immunoprecipitation experiments with the six-residue N protein-deletion mutant confirmed that the truncated protein was indeed smaller than the wild-type N protein. The deletion mutants produced in this study are interesting candidate vaccines to prevent PRRS disease in pigs

Genetic markers associated with field PRRSV-induced abortion rates

In gilts and sows, the more severe clinical manifestation of PRRSV occurs in late gestation and can result in up to 40% of abortion incidence. Despite the known genetic component in the resilience to PRRSV, there is scarce information regarding the abortive outcome of this disease. We have tested the relationship between eight molecular markers (six from published studies and two identified in the present study in the HDAC6 gene) and the probability of abortion during a PRRSV outbreak, using data of two commercial Landrace x Large White sow farms with an incidence of abortion of 35% and 17%. From the markers tested, the USP18_-1533G>A did not segregate in these populations and CD163_c.3534C>T and HDAC6_g.2360C>T did not affect abortion rate. In contrast, the minor allele of two markers in SSC4 (WUR1000125 in GBP1 and rs340943904 in GBP5), which lower viremia in growing pigs, and the major alleles of CD163_rs1107556229 and HDAC6_rs325981825 were associated with lower probability of abortion during PRRSV outbreaks. The more striking result was for the MX1 gene, where odds ratio of aborting vs not was 9 times lower in the sows homozygous for a 275bp insertion than in the other genotypes. Interactions between markers were not relevant. All together, we bring here the first evidence that mutations in the host genome can predispose or protect from complete reproductive failure in sows infected with PRRSV.This research and the APC were partially funded by FEDER projects COMRDI16-1-0035-03 and RTI2018-097700-B-I00 from the Spanish Ministry of Science, Innovation, and Universities

Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena)

Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163+Sialoadhesin (Sn)+, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163+Sn+, CD163+Sn- and to a lesser extent CD163-Sn- monocytic subtypes were involved in infection. CD163+Sn- cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163+Sn+ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163+Sn- cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena

Performance of assays for testing antibodies against porcine reproductive and respiratory syndrome virus in sera collected from swine farms in a region with an extreme virus heterogeneity

Porcine reproductive and respiratory syndrome is the most economically important viral disease in the swine industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) strains are classified into two distinct genotypes, the European genotype and the North American genotype. The European PRRSV genotype has been divided into three subtypes: a pan-European subtype 1 and East European subtypes 2 and 3. The aim of this study was to evaluate the performance of commercial and homemade serological assays to test field sera from a geographical region with an extreme PRRSV heterogeneity. Belarus became the country of choice for sample collection because heterologous PRRSV strains of all known European subtypes circulate in this country. Sera from Belarusian swine farms were tested in immunoperoxidase monolayer assays based on pan-European subtype 1, East European subtype 3 and North American strains as antigens and commercial enzyme-linked immunosorbent assays (IDEXX and INGEZIM). The obtained results suggest that none of the serological tools for PRRSV diagnosis can guarantee a flawless detection of antibodies at the individual animal level. Considering heterogeneity of recently isolated European PRRSV strains the problem can be relevant in many countries

Resilience effects of SGK1 and TAP1 DNA markers during PRRSV outbreaks in reproductive sows

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious stressor that causes serious health problems and productivity drops. Based on previous genome-wide analyses, we selected SGK1 and TAP1 as candidate genes for resilience, and genotyped three mutations, including a 3′UTR variant SGK1_rs338508371 and two synonymous variants TAP1_rs1109026889 and TAP1_rs80928141 in 305 Landrace × Large White sows. All polymorphisms affected the reproductive performance in the outbreak, but not during the endemic phase, thereby indicating a potential use of these markers for resilience. Moreover, some genotypes were associated with a stable performance across PRRSV phases. Thus, in the outbreak, the SGK1_rs338508371 AA sows had less piglets born alive (p < 0.0001) and more stillborns (p < 0.05) while other sows were able to keep their productivity. During the outbreak, TAP1_rs80928141 GG sows had less piglets born alive (p < 0.05) and both TAP1 polymorphisms influenced the number of mummies in an additive manner (p < 0.05). Remarkably, TAP1_rs80928141 AA sows had around one mummy more than GG sows (p < 0.01). Resilience to PRRSV could be improved by including the SGK1 and TAP1 markers in crossbreeding and/or selection schemes, as they contribute to maintaining a stable number of piglets born alive and lost, particularly mummies, despite the outbreak.This research and the APC were partially funded by FEDER projects COMRDI16-1-0035-03 and RTI2018-097700-B-I00 from the Spanish Ministry of Science, Innovation, and Universities. M.L. received a postdoctoral grant from UdL-Impuls programme

Health advantages of transition to batch management system in farrow-to-finish pig herds

Sow batch management systems have become more popular due to advantages in labour planning, piglet batch sizes, all-in all-out practices and health management. The present study investigated the potential health advantages of 10 selected farrow-to-finish pig herds before and after transition from a one week batch management system to a four or five week batch management system. Five different animal categories (gilts, sows, piglets, growers and finishers) were sampled at three time points (T0, T1 and T2) before and after transition to a four or five week batch management system. Different matrices of the animals were collected: blood, nasal swabs and faeces. Several economically important diseases were monitored through serology: Lawsonia intracellularis, Porcine Reproductive and Respiratory Syndrome virus (PRRSv), Mycoplasma hyopneumoniae, Actinobacillus pleuropneurnoniae; and PCR-testing: Pasteurella multocida dermonecrotic toxin (DNT) and Brachyspira species, especially the major pathogenic Brachyspira hyodysenteriae. Following serological analysis, the percentage of positive animals per category and sampling occasion were calculated. Health improvement based on serology was defined as the reduction in the percentage of positive animals for a specific disease in a specified animal category. All samples were negative for P. multocida DNT and B. hyodysenteriae. Little to no improvement could be observed for PRRSv. For L. intracellularis an improvement could be observed in piglets (71%) and growers (56%; P < 0.05). For both of the respiratory pathogens, M. hyopneumoniae and A. pleuropneumoniae, significant improvement was observed in finishers (34 and 24%, respectively). In growers, only M. hyopneumoniae showed a significant improvement (34%). In conclusion, the transition from a one week batch management system to a four or five week batch management system in the present herds resulted in a reduction of the percentage of seropositive animals for three of the monitored economically important diseases: L. intracellularis, M. hyopneumoniae and A. pleuropneumoniae