Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Parainfluenza virus 5 upregulates CD55 expression to produce virions with enhanced resistance to complement-mediated neutralization.

Many enveloped RNA viruses recruit host cell proteins during assembly as a mechanism to limit antiviral effects of complement. Using viruses which incorporated CD46 alone, CD55 alone or both CD46 and CD55, we addressed the role of these two host cell regulators in limiting complement-mediated neutralization of Parainfluenza virus 5 (PIV5). PIV5 incorporated functional forms of both CD55 and CD46 into virions. PIV5 containing CD55 was highly resistant to complement-mediated neutralization, whereas CD46-containing PIV5 was as sensitive to neutralization as virus lacking both regulators. PIV5 infected cells had increased levels of cell surface CD55, which was further upregulated by exogenous treatment with tumor necrosis factor alpha. PIV5 derived from cells with higher CD55 levels was more resistant to complement-mediated neutralization in vitro than virus from control cells. We propose a role for virus induction of host cell complement inhibitors in defining virus growth and tissue tropism.

In vivo modulation of avidity in highly sensitive CD8(+) effector T cells following viral infection.

Numerous studies have demonstrated a critical role for T cell avidity in predicting in vivo efficacy. Even though the measurement of avidity is now a routine assessment for the analysis of effector and memory T cell populations, our understanding of how this property is controlled in vivo at both the population and individual cell levels is limited. Our previous studies have identified high avidity as a property of the initial effector population generated in mice following respiratory virus infection. As the response progresses, lower avidity cells appear in the effector pool. The studies described here investigate the mechanistic basis of this in vivo regulation of avidity. We present data supporting in vivo avidity modulation within the early high avidity responders that results in a population of lower avidity effector cells. Changes in avidity were correlated with decreased lck expression and increased sensitivity to lck inhibitors in effector cells present at late versus early times postinfection. The possibility of tuning within select individual effectors is a previously unappreciated mechanism for the control of avidity in vivo.

The paramyxoviruses simian virus 5 and mumps virus recruit host cell CD46 to evade complement-mediated neutralization.

The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization.

Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

Parainfluenza virus 5 (PIV5 or SV5) infects several mammalian species but is restricted from efficient replication in mice. In humans, PIV5 evades IFN signaling by targeting STAT1 for proteasomal degradation in a STAT2-dependent reaction. In contrast, cell culture experiments have demonstrated that the divergent murine STAT2 protein fails to support STAT1 targeting. Expression of human STAT2 in mouse cells can overcome the species restriction to enable PIV5-induced STAT1 degradation and subsequent IFN antagonism. Here, we describe a transgenic mouse that ubiquitously expresses human STAT2. PIV5 infection induces STAT1 degradation leading to enhanced virus replication and protein expression in the cells from the transgenic mouse but not from the non-transgenic littermates. Importantly, intranasal inoculation with PIV5 results in increased viral load in the lungs of the transgenic mice compared to wild-type littermates. These transgenic mice provide a small animal model to study the role of innate immune evasion in paramyxovirus pathogenesis.

Identification of potential B cell epitope determinants by computer techniques, in hemagglutinin-neuraminidase from the porcine rubulavirus La Piedad Michoacan.

Hemagglutinin-neuraminidase (HN) from porcine rubulavirus La Piedad Michoacan (RvpLPM) is one of the most antigenic proteins known, and is responsible for virus-host cell interaction. We analyzed the amino acid sequence of HN, using computer-assisted techniques to identify B cell epitopes. From a pool of 18 possible antigenic peptides, we evaluated the antigenicity of the 2 peptides with the highest scores and the 1 with lowest score. Antibodies from RvpLPM-infected pigs recognized the synthesized HN-A, HN-B, and HN-R peptides (optical density [OD]: 0.33 +/- 0.02 for HN-A, 0.20 +/- 0.02 for HN-B, and 0.07 +/- 0.01 for HN-R); bovine serum albumin-coupled HN-A and HN-B induced rabbit anti-RvpLPM antibodies (OD: 0.39 +/- 0.01 for HN-A and 0.35 +/- 0.02 for HN-B). Loop 5 from the outer membrane protein, OmpC, from Salmonella typhi was replaced with HN-B; this protein was then expressed in Escherichia coli UH302. BALB/c mice were challenged intraperitoneally or orogastrically with the fusion protein expressed in E. coli and murine antibodies obtained from both types of administration inhibited virus-hemagglutinating activity, as did the antibodies from RvpLPM-infected swine. These results suggest that HN-A and HN-B are peptides involved in RvpLPM cell carbohydrate recognition, and could therefore be considered potential targets for vaccine and diagnostic procedures development.

Distinct pathways for signaling maturation in macrophages and dendritic cells after infection with paramyxovirus simian virus 5.

Professional antigen-presenting cells are critical components of both the innate and adaptive immune responses. Although dendritic cells (DCs) are generally thought to be the primary activators of naive T cells, macrophages have also been shown to fulfill this role. As with DCs, the capacity to induce optimal activation of T cells requires that macrophages undergo a process that results in the increased expression of costimulatory molecules, such as CD40, CD80, and CD86, and the production of cytokines. In this study we analyzed the effect of infection of macrophages generated from BALB/c mice with the paramyxovirus simian virus 5 (SV5). Here we have shown that bone marrow-derived macrophages (BMMs) are not productively infected at any multiplicity of infection tested. Analysis of activation markers revealed that SV5-infected BMMs robustly upregulated CD40 and modestly upregulated CD86, but did not upregulate the expression of CD80. Further, SV5-infected BMMs secreted low levels of interferon-beta and interleukin (IL)-12p40, but high levels of tumor necrosis factor-alpha and IL-6. Intriguingly, upregulation of these molecules on BMMs, unlike our previous results using bone marrow-derived dendritic cells, was not dependent on live virus. These findings provide evidence that different professional antigen-presenting cells can detect and respond to virus via distinct mechanisms.

Virological and immunological characteristics of fatal virus-associated haemophagocytic syndrome (VAHS).

We report three infants and one teenager with fatal virus-associated haemophagocytic syndrome (VAHS). Two infants were admitted to our hospital because of cardio-pulmonary arrest (CPA). The third infant was admitted to our department because of fever and liver dysfunction, and he was diagnosed as combined immunodeficiency with predominant T cell defect. The teenager was diagnosed as systemic lupus erythema (SLE) when she was 10 years old and admitted to our department because of fever and thrombocytopenia . The histological findings for the four patients' organs revealed many haemophagocytic cells . The patients were infected by Parainfluenza virus type 2 (PIV2), Enterovirus (EV), Cytomegalovirus (CMV), and Epstein-Barr virus (EBV), respectively. Their laboratory data revealed elevated levels of ferritin and IL-6, which also suggested virus-associated haemophagocytic syndrome (VAHS). Although aggressive therapies were performed in all cases, the outcome was fatal. Further investigation would be needed to clarify the mechanism of VAHS and an effective therapeutic regimen is needed.

Abortive versus productive viral infection of dendritic cells with a paramyxovirus results in differential upregulation of select costimulatory molecules.

Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.

Exaggerated IL-8 and IL-6 responses to TNF-alpha by parainfluenza virus type 4-infected NCI-H292 cells.

Respiratory viruses induce and potentiate airway inflammation, which is related to the induction of proinflammatory mediators such as interleukin (IL)-8 and IL-6. Here we report on mechanisms implicated in IL-8 and IL-6 production by airway epithelium-like NCI-H292 cells exposed to parainfluenza virus type 4a (PIV-4). PIV-4 readily infected NCI-H292 cells as reflected by intracellular PIV-4 antigen expression. PIV-4 infection triggered a biphasic IL-8 and IL-6 mRNA response. Transient transfection with truncated and mutated promoter constructs identified NF-kappaB and activator protein (AP)-1, and CCAAT-enhancer binding protein (C/EBP) as the relevant transcription factors for PIV-4-induced IL-8 and IL-6 gene transcription, respectively. An increase of DNA-binding activities for NF-kappaB and C/EBP paralleled the induction of the first and second IL-8 and IL-6 mRNA peaks, whereas the onset of AP-1 paralleled the first IL-8 mRNA peak only. The second mRNA peak, apparently dependent on viral replication, coincided also with a marked reduction of IL-8 and IL-6 mRNA degradation. Importantly, cells at the time of the reduced mRNA degradation displayed an exaggerated IL-8 and IL-6 protein production to a secondary stimulus, as exemplified by steeper dose-response curves to TNF-alpha. Thus PIV-4 infection enhances epithelial IL-8 and IL-6 production by transcriptional and posttranscriptional mechanisms. The previously unrecognized phase of reduced IL-8 and IL-6 mRNA degradation and the concurrent amplified epithelial IL-8 and IL-6 responses may play an important role in virus-induced potentiation of airway inflammation.

Lesions in the reproductive tract of boars experimentally infected with porcine rubulavirus.

"Blue eye" disease of pigs in Mexico is caused by porcine rubulavirus and characterized by infertility in sows and boars, nervous signs in young pigs, and corneal opacity in pigs of all ages. The pathogenesis of reproductive tract lesions in rubulavirus-infected boars has not previously been investigated. In a first experiment, four 9-month-old boars were inoculated with porcine rubulavirus and killed 5, 15, 30 or 45 days post-inoculation (pi). In a second experiment, four similar boars were inoculated with the same virus and two animals were killed on each of days 70 and 80 pi. Swelling of the head of the epididymis developed in all inoculated boars at approximately day 15 pi. Reduced spermatozoan motility and concentration were detected in semen samples collected from one boar from day 21 pi. At post-mortem examination, nodules were seen in the head of the epididymis of the boars killed 15, 30 or 45 days pi and the right testis of the pig killed 30 days pi was atrophic. Corresponding histopathological epididymal alterations included formation of spermatic granulomas and vacuolar degeneration of ductular epithelium. These lesions were associated with mononuclear cell infiltration and interstitial fibroplasia. Degeneration of seminiferous tubules and interstitial mononuclear cell infiltration were seen in the atrophic testis of the pig killed 30 days pi. There was fibrosis of the head of the epididymis in all boars killed 70 or 80 days pi and one of these animals also had right testicular atrophy associated with degeneration of seminiferous tubules, lymphocytic infiltration and giant cell formation. Porcine rubulavirus antigen was detected by immunofluorescence labelling in the head of the epididymis of the pigs killed 15, 30 or 45 days pi and in one animal killed on day 70 pi. These results indicate that porcine rubulavirus can cause severe epididymo-orchitis and reduced semen quality in sexually mature boars.