Reverse Genetic Assessment of the Roles Played by the Spike Protein and ORF3 in Porcine Epidemic Diarrhea Virus Pathogenicity.

Porcine epidemic diarrhea virus is a swine pathogen that has been responsible for significant animal and economic losses worldwide in recent years. In this manuscript, we report the generation of a reverse genetics system C(RGS) for the highly virulent US PEDV strain Minnesota (PEDV-MN; GenBank accession number KF468752), which was based on the assembly and cloning of synthetic DNA, using vaccinia virus as a cloning vector. Viral rescue was only possible following the substitution of 2 nucleotides within the 5'UTR and 2 additional nucleotides within the spike gene, based on the sequence of the cell culture-adapted strains. Besides displaying a highly pathogenic phenotype in newborn piglets, in comparison with the parental virus, the rescued recombinant PEDV-MN was used to confirm that the PEDV spike gene has an important role in PEDV virulence and that the impact of an intact PEDV ORF3 on viral pathogenicity is modest. Moreover, a chimeric virus with a TGEV spike gene in the PEDV backbone generated with RGS was able to replicate efficiently in vivo and could be readily transmitted between piglets. Although this chimeric virus did not cause severe disease upon the initial infection of piglets, there was evidence of increasing pathogenicity upon transmission to contact piglets. The RGS described in this study constitutes a powerful tool with which to study PEDV pathogenesis and can be used to generate vaccines against porcine enteric coronaviruses. IMPORTANCE PEDV is a swine pathogen that is responsible for significant animal and economic losses worldwide. Highly pathogenic variants can lead to a mortality rate of up to 100% in newborn piglets. The generation of a reverse genetics system for a highly virulent PEDV strain originating from the United States is an important step in phenotypically characterizing PEDV. The synthetic PEDV mirrored the authentic isolate and displayed a highly pathogenic phenotype in newborn piglets. With this system, it was possible to characterize potential viral virulence factors. Our data revealed that an accessory gene (ORF3) has a limited impact on pathogenicity. However, as it is also now known for many coronaviruses, the PEDV spike gene is one of the main determinants of pathogenicity. Finally, we show that the spike gene of another porcine coronavirus, namely, TGEV, can be accommodated in the PEDV genome background, suggesting that similar viruses can emerge in the field via recombination.

A TLR7 antagonist restricts interferon-dependent and -independent immunopathology in a mouse model of severe influenza.

Cytokine-mediated immune-cell recruitment and inflammation contribute to protection in respiratory virus infection. However, uncontrolled inflammation and the "cytokine storm" are hallmarks of immunopathology in severe infection. Cytokine storm is a broad term for a phenomenon with diverse characteristics and drivers, depending on host genetics, age, and other factors. Taking advantage of the differential use of virus-sensing systems by different cell types, we test the hypothesis that specifically blocking TLR7-dependent, immune cell-produced cytokines reduces influenza-related immunopathology. In a mouse model of severe influenza characterized by a type I interferon (IFN-I)-driven cytokine storm, TLR7 antagonist treatment leaves epithelial antiviral responses unaltered but acts through pDCs and monocytes to reduce IFN-I and other cytokines in the lung, thus ameliorating inflammation and severity. Moreover, even in the absence of IFN-I signaling, TLR7 antagonism reduces inflammation and mortality driven by monocyte-produced chemoattractants and neutrophil recruitment into the infected lung. Hence, TLR7 antagonism reduces diverse types of cytokine storm in severe influenza.

Critical requirement for BCR, BAFF, and BAFFR in memory B cell survival.

Memory B cells (MBCs) are long-lived cells that form a critical part of immunological memory, providing rapid antibody responses to recurring infections. However, very little is known about signals controlling MBC survival. Previous work has shown that antigen is not required for MBC survival, but a requirement for the B cell antigen receptor (BCR) has not been tested. Other studies have shown that, unlike naive B cells, MBCs do not express BAFFR and their survival is independent of BAFF, the ligand for BAFFR. Here, using inducible genetic ablation, we show that survival of MBCs is critically dependent on the BCR and on signaling through the associated CD79A protein. Unexpectedly, we found that MBCs express BAFFR and that their survival requires BAFF and BAFFR; hence, loss of BAFF or BAFFR impairs recall responses. Finally, we show that MBC survival requires IKK2, a kinase that transduces BAFFR signals. Thus, MBC survival is critically dependent on signaling from BCR and BAFFR.

A Triple Amino Acid Substitution at Position 88/94/95 in Glycoprotein GP2a of Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV1) Is Responsible for Adaptation to MARC-145 Cells.

The Meat Animal Research Center-145 (MARC-145) cell line has been proven to be valuable for viral attenuation regarding vaccine development and production. Cell-adaptation is necessary for the efficient replication of porcine reproductive and respiratory syndrome virus (PRRSV) in these cells. Multiple sequence analysis revealed consistent amino acid substitutions in GP2a (V88F, M94I, F95L) of MARC-145 cell-adapted strains. To investigate the putative effect of these substitutions, mutations at either position 88, 94, 95, and their combinations were introduced into two PRRSV1 (13V091 and IVI-1173) infectious clones followed by the recovery of viable recombinants. When comparing the replication kinetics in MARC-145 cells, a strongly positive effect on the growth characteristics of the 13V091 strain (+2.1 log10) and the IVI-1173 strain (+1.7 log10) compared to wild-type (WT) virus was only observed upon triple amino acid substitution at positions 88 (V88F), 94 (M94I), and 95 (F95L) of GP2a, suggesting that the triple mutation is a determining factor in PRRSV1 adaptation to MARC-145 cells.

Antiviral activity of K22 against members of the order Nidovirales.

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Heterogeneous antigenic properties of the porcine reproductive and respiratory syndrome virus nucleocapsid.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus responsible for a widespread contagious disease of domestic pigs with high economic impact. Switzerland is one of the rare PRRSV-free countries in Europe, although sporadic outbreaks have occurred in the past. The PRRSV isolate IVI-1173 from the short outbreak in Switzerland in 2012 was entirely sequenced, and a functional full-length cDNA clone was constructed. Genetic and antigenic characterization of IVI-1173 revealed the importance of amino acid 90 of the nucleocapsid protein N as part of a conformational epitope. IVI-1173 was not detected by SDOW17, a monoclonal antibody against N widely used to detect PRRSV-infected cells. Substitution of alanine at position 90 of N [N(A90)] with a threonine [N(T90)] restored reactivity of vIVI1173-N(T90) to SDOW17 completely. The relevance of this amino acid for the conformational SDOW17 epitope of PRRSV N was further confirmed by the opposite substitution in a functional cDNA clone of the genotype 2 isolate RVB-581. Finally, N proteins from ten genotype 1 strains differing from threonine at position 90 were analysed for reactivity with SDOW17. N(A90) totally disrupted or severely affected the epitope in 7 out of 8 strains tested. Based on these findings, 225 genotype 1 strains were screened for the prevalence of N(A90). N(A90) is rare in classical subtype 1 and in subtype 3 strains, but is frequent in Russian subtype 1 (70%) and in subtype 2 (45%) isolates. In conclusion, this study highlights the variable antigenic properties of N among genotype 1 PRRSV strains.

Sensing of Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages by Plasmacytoid Dendritic Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) represents a macrophage (MØ)-tropic virus which is unable to induce interferon (IFN) type I in its target cells. Nevertheless, infected pigs show a short but prominent systemic IFN alpha (IFN-α) response. A possible explanation for this discrepancy is the ability of plasmacytoid dendritic cells (pDC) to produce IFN-α in response to free PRRSV virions, independent of infection. Here, we show that the highly pathogenic PRRSV genotype 1 strain Lena is unique in not inducing IFN-α production in pDC, contrasting with systemic IFN-α responses found in infected pigs. We also demonstrate efficient pDC stimulation by PRRSV Lena-infected MØ, resulting in a higher IFN-α production than direct stimulation of pDC by PRRSV virions. This response was strain-independent, required integrin-mediated intercellular contact, intact actin filaments in the MØ and was partially inhibited by an inhibitor of neutral sphingomyelinase. Although infected MØ-derived exosomes stimulated pDC, an efficient delivery of the stimulatory component was dependent on a tight contact between pDC and the infected cells. In conclusion, with this mechanism the immune system can efficiently sense PRRSV, resulting in production of considerable quantities of IFN-α. This is adding complexity to the immunopathogenesis of PRRSV infections, as IFN-α should alert the immune system and initiate the induction of adaptive immune responses, a process known to be inefficient during infection of pigs.