The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure

AbstractWe have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five α helices and a β-hairpin. The dimer interface consists of a continuous four-stranded β-sheet superposed by two long α helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the β-sheet and the α helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology

Direct observation of membrane insertion by enveloped virus matrix proteins by phosphate displacement

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes

Kinetics of UV254 inactivation of selected viral pathogens in a static system

Aims:  The objective of this study was to estimate UV254 inactivation constants for four viral pathogens: influenza virus type A, porcine respiratory and reproductive syndrome virus (PRRSV), bovine viral diarrhoea virus (BVDV) and reovirus. Methods and Results:  Viruses in culture medium were exposed to one of nine doses of UV254 and then titrated for infectious virus. Analysis showed that viral inactivation by UV254 was more accurately described by a two-stage inactivation model vs a standard one-stage inactivation model. Conclusions:  The results provided evidence for the existence of two heterogeneous viral subpopulations among the viruses tested, one highly susceptible to UV254 inactivation and the other more resistant. Importantly, inactivation constants based on the one-stage inactivation model would have underestimated the UV254 dose required for the inactivation of these viruses under the conditions of the experiment. Significance and Impact of the Study:  To improve the accuracy of estimates, it is recommended that research involving the inactivation of micro-organisms evaluates inactivation kinetics using both one-stage and two-stage models. These results will be of interest to persons responsible for microbial agents under laboratory or field conditions

Systems Immunology Analyses Following Porcine Respiratory and Reproductive Syndrome Virus Infection and Vaccination.

This study was initiated to better understand the nature of innate immune responses and the relatively weak and delayed immune response against porcine reproductive and respiratory syndrome virus (PRRSV). Following modified live virus (MLV) vaccination or infection with two PRRSV-2 strains, we analyzed the transcriptome of peripheral blood mononuclear cells collected before and at three and seven days after vaccination or infection. We used blood transcriptional modules (BTMs)-based gene set enrichment analyses. BTMs related to innate immune processes were upregulated by PRRSV-2 strains but downregulated by MLV. In contrast, BTMs related to adaptive immune responses, in particular T cells and cell cycle, were downregulated by PRRSV-2 but upregulated by MLV. In addition, we found differences between the PRRSV strains. Only the more virulent strain induced a strong platelet activation, dendritic cell activation, interferon type I and plasma cell responses. We also calculated the correlations of BTM with the neutralizing antibody and the T-cell responses. Early downregulation (day 0-3) of dendritic cell and B-cell BTM correlated to both CD4 and CD8 T-cell responses. Furthermore, a late (day 3-7) upregulation of interferon type I modules strongly correlated to helper and regulatory T-cell responses, while inflammatory BTM upregulation correlated more to CD8 T-cell responses. BTM related to T cells had positive correlations at three days but negative associations at seven days post-infection. Taken together, this work contributes to resolve the complexity of the innate and adaptive immune responses against PRRSV and indicates a fundamentally different immune response to the less immunogenic MLV compared to field strains which induced robust adaptive immune responses. The identified correlates of T-cell responses will facilitate a rational approach to improve the immunogenicity of MLV

Evaluation of porcine respiratory and reproductive syndrome virus (PRRSV) ante-mortem diagnostic techniques

Objective. The specific objective of this thesis was to address the U.S. pork industry\u27s need for evaluation of alternative ante-mortem diagnostic samples for PRRSV. To accomplish this objective, three trials were untaken with the following goals: (1) Develop a sampling protocol for the blood swab method. (2) Analyze the diagnostic accuracies of the blood swab, the capillary tube and the jugular sampling methods on two standard PRRS diagnostic tests: quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) and ELISA in known positive and negative animals. (3) Analyze the diagnostic accuracy of the blood swab method for ELISA testing in commercial finishing swine.;Trial one. Trial 1 showed that under laboratory conditions, the Fisherbrand swabs absorbed a significantly higher volume of blood than the Puritan swabs. Trial 2 showed that when the 20g x 1/2&inches; needle was used, significantly more blood was absorbed than when the 25g x 5/8&inches; needle was used regardless of the swab type. Trial 3 confirmed that when the 20g x 1/2&inches; needle was used, the swab absorbed significantly more blood. Trial two. For qRT-PCR testing, the sensitivity and specificity for all sampling methods ranged from 93%--100% for weeks 1--3 PI. Results of ELISA testing depended on cut-off selection. Optimization of ELISA S/P cut-off points for swab sample data was substantially lower (S/P ratio of 0.08 +/- 0.05) than the industry standard (S/P ratio of 0.4). Trial three. The sensitivity and specificity of the swab sampling method was 22.3% (95% probability intervals = 16.0%, 29.2%) and 94.3% (80.1%, 99.8%) respectively when an S/P ratio cut-off of 0.4 was used. The sensitivity and specificity were 94% (89.8%, 97.2%) and 93.5% (77.8%, 99.8%) respectively when an S/P ratio cut-off of 0.08 was used.;Implications. (1) Fisherbrand swabs absorb a significantly greater blood volume (167mul) then Puritan swabs (142mul) under ideal sampling conditions. (2) Under field conditions, the Fisherbrand swabs absorbed numerically less blood (118mul) then under laboratory conditions (167mul). (3) A Fisherbrand swab and a 20g x 1/2&feet; needle combination would be the best diagnostic sample for sows and finisher pigs when collection time is less than 15 seconds. (4) The capillary tube method suffers from neither inadequate volume nor differences in sample type collected (as compared to the blood swab method). This study indicated that the capillary sampling method can be used with ELISA and real-time qRT-PCR testing with diagnostic accuracy equal to the jugular sampling method. The total expense for the capillary sampling method was {dollar}1.44/sample ({dollar}0.94/tube in product and {dollar}0.50/sample in labor cost (calculated for 3 minutes at {dollar}10/hr)). In comparison, the total cost of the jugular sample was {dollar}0.91/sample. (5) Early diagnosis (weeks 1--3 PI) of PRRSV infected nursery pigs using real-time qRT-PCR under study conditions can be equivalently accomplished using the capillary, swab, or jugular sampling methods. (6) No change in cut-off values for qRT-PCR data dichotomization is necessary for data obtained via any of the sampling methods. (7) The diagnostic accuracy of PRRSV ELISA was poor for the swab sampling method when an S/P ration cut-off of 0.4 was utilized (sensitivity ranged from 20%--55.6% over weeks 2--7 PI). (8) When optimal cut-off values are employed, as determined by AUC analysis, all sampling methods are capable of achieving very high diagnostic accuracy on PRRSV ELISA testing. These cut-off values may not be clinically useful. (9) Under commercial (field) conditions, the sensitivity of the swab sampling method was low (22.3%) for ELISA results dichotomized at an S/P ratio of 0.4. (10) The sensitivity of the swab method improved when a lower S/P cut-off was used (94%) indicating this method may have application in routine ELISA diagnostic monitoring programs. (11) In comparison to the jugular sampling method, the sensitivity and specificity of the swab method is lower when used in commercial settings; this will result in more false negative and false positive test results. (12) Under the assumptions of the partial budget, the jugular sampling method would cost a 1000-head sow operation {dollar}0.15/pig produced while the swab sampling method would cost {dollar}0.36/pig produced. (Abstract shortened by UMI.

Ability of ELISAs to detect antibodies against porcine respiratory and reproductive syndrome virus in serum of pigs after inactivated vaccination and subsequent challenge: Ability of ELISAs to detect antibodies against porcine respiratory andreproductive syndrome virus in serum of pigs after inactivated vaccination and subsequent challenge

Background: In this study, six enzyme-linked immunosorbent assays (ELISA), intended for routine porcine reproductive and respiratory syndrome virus (PRRSV) herd monitoring, are tested for their ability to detect PRRSV specific antibodies in the serum of pigs after vaccination with an inactivated PRRSV type 1 vaccine and subsequent infection with a highly pathogenic (HP) PRRSV field strain. For this reason, ten piglets (group V) from a PRRSV negative herd were vaccinated twice at the age of 2 and 4 weeks with an inactivated PRRSV vaccine. Ten additional piglets (group N) from the sameherd remained unvaccinated. Three weeks after second vaccination, each of the piglets received an intradermal application of an HP PRRSV field strain. Serum samples were taken before first vaccination as well as before and 3, 7, 10 and 14 days after HP PRRSV application. All serum samples were tested for PRRSV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as well as for PRRSV antibodies with all six study ELISAs. Results: At the beginning of the study (before vaccination), all of the piglets were PRRSV antibody negative with all study ELISAs. They also tested negative for PRRSV RNA measured by RT-qPCR. From day 3 after HP PRRSV application until the end of the study, a viremia was detected by RT-qPCR in all of the piglets. On day 0 (day of HP PRRSV application), nine out of ten piglets of the pre-vaccinated group tested PRRSV antibody positive with one of the tested ELISAs, although with lower S/P values than after infection. On day 10 after HP PRRSV application, all study ELISAs except one had significantly higher S/P or OD values, respectively more positive samples, in group V than in group N. Conclusions: Only one of the tested ELISAs was able to detect reliably PRRSV antibodies in pigs vaccinated with an inactivated PRRSV vaccine. With most of the tested ELISAs, higher S/P values respectively more positive samples after PRRSV infection were seen in the pre-vaccinated group than in the non-vaccinated

Comparison of different commercial ELISAs for detection of antibodies against porcine respiratory and reproductive syndrome virus in serum

Background: In recent years, several new ELISAs for the detection of antibodies against the porcine reproductive and respiratory disease virus (PRRSV) in pig serum have been developed. To interpret the results, specificity and sensitivity data as well as agreement to a reference ELISA must be available. In this study, three commercial ELISAs (INgezim PRRS 2.0 - ELISA II, Priocheck® PRRSV Ab porcine – ELISA III and CIVTEST suis PRRS E/S PLUS - ELISA IV, detecting PRRSV type 1 antibodies) were compared to a standard ELISA (IDEXX PRRS X3 Ab Test - ELISA I). The serum of three pigs vaccinated with an attenuated PRRSV live vaccine (genotype 2) was tested prior to and several times after the vaccination. Furthermore, serum samples of 245 pigs of PRRSV positive herds, 309 pigs of monitored PRRSV negative herds, 256 fatteners of assumed PRRSV negative herds with unknown herd history and 92 wild boars were tested with all four ELISAs. Results: ELISAs II and III were able to detect seroconversion of vaccinated pigs with a similar reliability. According to kappa coefficient, the results showed an almost perfect agreement between ELISA I as reference and ELISA II and III (kappa > 0.8), and substantial agreement between ELISA I and ELISA IV (kappa = 0.71). Sensitivity of ELISA II, III and IV was 96.0%, 100% and 91.5%, respectively. The specificity of the ELISAs determined in samples of monitored PRRSV negative herds was 99.0%, 95.1% and 96.4%, respectively. In assumed negative farms that were not continually monitored, more positive samples were found with ELISA II to IV. The reference ELISA I had a specificity of 100% in this study. Conclusions: All tested ELISAs were able to detect a PRRSV positive herd. The specificity and sensitivity of the tested commercial ELISAs, however, differed. ELISA II had the highest specificity an ELISA III had the highest sensitivity in comparison to the reference ELISA. ELISA IV had a lower sensitivity and specificity than the other ELISAs