Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.

The Cholera Toxin B Subunit (CTB) Fused to the Porcine Arterivirus Matrix M and GP5 Envelope Proteins Fails to Enhance the GP5-Specific Antibody Response in Pigs Immunized with Adenovectors.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of the Arteriviridae family. As the current commercial vaccines are incompletely protective effective against PRRSV infection, we developed a vaccine strategy using replicating but non-disseminating adenovectors (rAdVs) expressing the PRRSV M matrix protein in fusion with the neutralizing major epitope-carrying GP5 envelope protein (Roques et al. in Vet Res 44:17, 2013). Although production of GP5-specific antibodies (Abs) was observed, no PRRSV-specific neutralizing Abs (NAbs) were induced in pigs given the rAdVs expressing M-GP5 or M-GP5m (GP5m being a mutant form of GP5). Nevertheless, partial protection was observed in the M-GP5m-rAdV-inoculated pigs experimentally infected with PRRSV. Here, we determined the impact of the cholera toxin B subunit (CTB, known for its adjuvant effect) in fusion with the C-terminus of M-GP5m on the Ab response to PRRSV. Three-week-old pigs were immunized twice both intramuscularly and intranasally at 3-week intervals with rAdV-expressing the green fluorescent protein (rAdV-GFP), rAdV-M-GP5m, or rAdV-M-GP5m-CTB. Pigs immunized with rAdV-M-GP5m showed a high level of serum GP5-specific Abs (as determined by an indirect ELISA). In contrast, CTB in fusion with M-GP5m had an unexpected severe negative impact on GP5-specific Ab production. PRRSV-specific NAbs could not be detected in any pigs of all groups.

Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting.

Arteriviruses are enveloped, positive-strand RNA viruses for which the two major envelope proteins GP(5) and M occur as disulfide-linked heterodimers. These were assumed to serve the viral targeting functions, but recent ectodomain swapping studies with equine arteritis virus (EAV) indicate that the GP(5) protein does not determine arteriviral tropism. Here, we focused on the short, 13- to 18-residue ectodomain of the M protein. Using an infectious cDNA clone of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV), we substituted the genomic sequence encoding the M ectodomain by that of murine lactate dehydrogenase-elevating virus, EAV, and the US PRRSV-isolate, VR2332. Viable viruses with a chimeric M protein were obtained in all three cases, but for the latter two only after removal of the genomic overlap between the M and GP(5) genes. Characterization of the chimeric viruses revealed that they could be distinguished immunologically from wild-type virus, that they were genetically stable in vitro, but that they were impaired in their growth, reaching lower titers than the parental virus. The latter appeared to be due to an increased particle-to-infectivity ratio of the chimeric virus particles. Interestingly, the chimeric viruses had retained their ability to infect porcine cells and had not acquired tropism for cells susceptible to the viruses from which the foreign ectodomains were derived. We conclude that the surface structures composed by the arterivirus M and GP(5) ectodomains do not determine viral tropism.

Autoantibodies against golgi apparatus induced by arteriviruses.

Members of the genus Arterivirus within the monogeneric family Arteriviridae are lactate dehydrogenase-elvating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus (EAV) and simian hemorrhagic fever virus. In LDV-infected mice the appearance of autoantibodies against Golgi-antigen dominated the early immune response. Shared antigenicity between LDV and Golgi-antigen of normal cells could not be demonstrated. Monoclonal antibodies (MAbs) reacted either with LDV or with Golgi-antigen but not with both. Immunization of mice with the porcine arterivirus PRRSV, however, led to the establishment of MAbs that recognized the structural glycoprotein GP3 as well as Golgi-antigen of normal porcine cells indicating molecular mimicry of viral and cellular antigen. In addition to cross-reactive antibodies MAbs solely reactive with Golgi-antigen were observed. After immunization of mice with EAV, the equine arterivirus, clones were isolated producing Golgi-antigen recognizing autoantibodies. Morphogenesis of arteriviruses occurs in the Golgi region. The autoimmune responses following immunization with arteriviruses may offer an approach for determining the mechanism by which such responses develop and become of biologic importance.

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.

Detection of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus in swine sera by enzyme-linked immunosorbent assay.

We developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against PRRS virus in swine sera. The ELISA antigen was prepared from MARC-145 cells infected with PRRS virus. The results from serial serum samples from experimentally infected and random swine indicated that the ELISA was more sensitive than indirect fluorescent and immunoperoxidase monolayer assays. Since the ELISA enables many sera to be simultaneously and rapidly tested, it was useful for detection antibodies against PRRS virus in swine sera.

Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine respiratory and reproductive syndrome virus (PRRSV) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used were collected from pigs experimentally infected with either the American or European antigenic type of PRRSV, and also from piglets born to sows that had been experimentally infected with the European antigenic type of PRRSV. In addition, three sets of European field sera (n = 275, n = 68, n = 349) were tested and evaluated using the IPMA as the gold standard. Results showed that both the IPMA and the ELISA were able to detect antibodies against the two antigenic types of PRRSV. When sera of experimentally infected pigs were tested, the IPMA with homologous antigen detected antibodies 2 to 3 days earlier than the ELISA, and was more sensitive in detecting maternal antibodies. The ELISA was slightly more sensitive for detecting antibodies against the American type than for the European type. When sets of field sera were tested, the relative sensitivity of the ELISA ranged between 0.68 and 0.91, and the relative specificity ranged between 0.75 and 0.97. However, in two of these sets (n = 275, n = 349) we determined that a decrease of the threshold value of ELISA (from 0.4 to 0.3) increased sensitivity without loss of specificity. We concluded that the ELISA is an easy, quick and reliable test to diagnose PRRSV infection in swine herds.

Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus.

The kinetics of appearance of antibodies directed to the major structural proteins N, M and E of porcine reproductive and respiratory syndrome virus (PRRSV) was followed in pigs naturally- and experimentally-exposed to the virus. Specific IgM antibody titers were first detected by indirect immunofluorescence (IIF) at the end of the first week of PRRSV infection, peaked by day 14 to 21 post-inoculation (p.i.), then rapidly decreased to undetectable levels by day 35 to 42 p.i. On the other hand, specific IgG antibody titers peaked by day 21 to 28 p.i. and remained unchanged to the end of the 6- or 9-week observation period; in addition, a persistent viremia was observed. Virus neutralizing (VN) antibody titers > 8 were not detected until 3 to 4 weeks p.i. Taken together, the results obtained by Western blotting analyses using purified virus and E. coli-expressed ORFs 5 to 7 gene products, suggested that antibodies directed against the envelope E protein appear by day 7 p.i., whereas antibodies directed against the nucleocapsid N and membrane M proteins can only be detected by the end of the second week p.i. No correlation could be demonstrated between VN and IIF antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i.

Comparative serology of porcine reproductive and respiratory syndrome in eight European laboratories, using immunoperoxidase monolayer assay and enzyme-linked immunosorbent assay.

The members of a European Community Concerted Action participated in a study to compare in-house and commercial tests for the serodiagnosis of porcine reproductive and respiratory syndrome (PRRS). These tests included the immunoperoxidase monolayer assay (IPMA) and enzyme-linked immunosorbent assay (ELISA). The trials involved assays on experimentally-produced reference sera, a blind trial of sera of known status, and a comparative study of negative and 'problem' field sera. The results showed a high level of agreement among IPMA tests used in the participating laboratories at the herd level, with only minor inconsistencies in testing early post-infection sera. A blocking ELISA, used in experimental work by one laboratory, was almost as sensitive as IPMA in assays of reference and validation trial sera. Commercial ELISAs were generally less sensitive than IPMA when used to assay ten-day post-infection sera. Discrepant results among certain field sera are likely to be due to antigenic variation among PRRS viruses in Europe. This may lead to increasing instances of misdiagnosis using tests which rely solely on test antigens derived from single PRRS virus isolates.

Comparative study of a blocking enzyme-linked immunosorbent assay and the immunoperoxidase monolayer assay for the detection of antibodies to the porcine reproductive and respiratory syndrome virus in pigs.

A blocking enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to the porcine reproductive and respiratory syndrome virus (PRRSV) in pig sera was developed and compared with the immunoperoxidase monolayer assay (IPMA), the most widely used serological diagnostic test in Europe. The blocking ELISA was specific and more sensitive than the IPMA when applied to field sera and to sera which were collected early after an experimental infection with PRRSV. Problems with high background activity as observed in IPMA or indirect ELISA were not encountered.