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Zika, yellow fever and dengue viruses are three important flaviviruses that are epidemic and endemic in Central Africa, Middle and South America, Southeast Asia and Pacific Islands. Since these three viral diseases present with similar symptoms and there is a possibility of multiple flaviviruses co-infection, it is important to develop an effective and quick diagnostic tool. Here, we designed and validated a one-step triplex real time reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probes targeting NS5 region for detecting zika virus (ZIKV), dengue virus-4 (DENV-4) and yellow fever virus (YFV). The assay was highly specific for the target viral RNA corresponding to ZIKV, DENV-4 and YFV strains. The sensitivity of the assay was compared to one-step, RT-PCR, it showed a 100-fold higher sensitivity than RT-PCR with ZIKV and DENV and 10-fold higher sensitivity with YFV. In conclusion, the one-step, triplex, TaqMan, real time RT-PCR assay identified and distinguished ZIKV, DENV-4 and YFV by targeting single gene. Key words: Zika, yellow fever, dengue, multiplex real time reverse transcription-polymerase chain reaction (RT-PCR).

Background Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease. Methods Through artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs). Results Using the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection. Conclusions Our study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.

Background Recently we reported IgA anti-Chlamydia antibodies in patients with Crohn's disease (CD), in particular in four patients from a single family of six with CD. Methods We studied sera from four cohorts from the north of France. These were identified as: EPIMAD (80 pediatric onset CD and 20 pediatric onset ulcerative colitis), MINOTOR (148 adult onset sporadic CD and 50 adult onset ulcerative colitis), Grande Famillies (50) and matched controls for the Grande Famillies cohort (49). Sera were tested using commercial anti-Chlamydia trachomatis (LGV2:434) IgG and IgA human enzyme-linked immunosorbent assay (ELISA) kits. Cutoff for positivity was 11.0 standard units. Results Patients with sporadic CD, unaffected first degree relatives from multiplex families and ulcerative colitis patients had no greater serologic reactivity than controls. However, multiplex families' patients had twice as many positives as the other groups: for IgG 20% vs. 8%; for IgA 20% vs. 10%. Conclusions Though not attaining statistical significance, the data showed that familial CD patients had greater exposure to C. trachomatis than sporadic CD patients, supporting our earlier results from one family from the north of France. More specific serologic tests based on outer membrane proteins will need to be employed against the various Chlamydia species with zoonotic potential.

The great genetic and antigenic diversity of PRRSV, an RNA virus that causes PRRS, makes the control of this disease very difficult. To achieve broad protection to infection with this divergent virus, two T-cell mosaic sequences were designed based on 748 glycoprotein 5 (GP5) sequences using the Mosaic T-Cell Vaccine Tool Suite from the Los Alamos National Laboratory. Two DNA vaccines were constructed using the mosaic sequences thus designed. The amino acid sequences of the GP5-Mosaic vaccines were closer to strains that differ from VR2332 by at least 10%. Expression of the GP5-Mosaic vaccines was verified in E. coli BL21 (DE3) by western blot. The immunogenicity of the GP5-Mosaic vaccines was tested by vaccinating young pigs with either a gene gun (Trial 1) or by electroporation (Trial 2) followed by challenge with VR2332. Lymphocyte proliferative responses detected in virus-stimulated peripheral blood mononuclear cells (PBMC) of GP5-Mosaic-vaccinated pigs were higher than those of control pigs in both trials. In Trial 2, significantly higher levels of IFN-γ mRNA expression and lower levels of IL-10 mRNA were detected in virus-stimulated PBMC of GP5-Mosaic-vaccinated pigs as compared to control pigs. In ELISA, higher virus-specific antibodies were detected in sera from GP5-Mosaic-vaccinated animals than those detected in control animals. These antibodies were proven to be neutralizing. The viral copy numbers in serum 5 days after challenge with VR2332 decreased over 260-fold within 2 days in GP5-Mosaic-vaccinated pigs, while the viral copy numbers increased by 1.2 to 5-fold in control animals in the same period. Additionally, the viral loads in inguinal lymph nodes and spleen, and lung lesions scores of GP5-Mosaic-vaccinated animals were lower compared to those of control animals. Together the data led us to conclude that the GP5-Mosaic vaccine was immunogenic and induced partial protection in vaccinated pigs.

Background It has been recognized that the expression of type I interferon (IFN alpha/beta) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection. Objective Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFN beta) and downstream signaling events. Methods In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFN beta protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events. Results These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFN beta protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFN beta signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFN beta production. Conclusion The results of this study demonstrate that the induction of IFN beta and signaling in PAMs after PRRSV infection is variable. University of Connecticut [58-1940-2-245]; USDAUnited States Department of Agriculture (USDA) [58-1940-2-245]; Integrated Control and Elimination of PRRS [NC229]; Storrs Agricultural Experiment Station, University of Connecticut; ARS, USDAUnited States Department of Agriculture (USDA) [20043520414267] This work was supported by funds from the Specific Cooperative Agreement #58-1940-2-245 between the University of Connecticut and the USDA, ARS, USDA grant # 20043520414267, Integrated Control and Elimination of PRRS (NC229), Storrs Agricultural Experiment Station, University of Connecticut. The authors thank Drs. F. Osorio and A. Pattnaik (University of Nebraska, Lincoln, NE) for providing the chimeric viruses used in this work. The authors also thank Dr. Surya Waghela (Texas A&M University, College Station, TX) for valuable suggestions with the manuscript. Public domain – authored by a U.S. government employee

[This corrects the article DOI: 10.1371/journal.pone.0208801.].

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) infection, imposes enormous economic impact to the world pork industry. Currently there is no effective treatment to prevent PRRSV infection in swine. We report that the natural compound cryptotanshinone (Cpt) effectively inhibits the infection of various strains of PRRSV to porcine alveolar macrophages (PAMs), the primary cell target of PRRSV in vivo. Mechanistically, Cpt inhibits the activation of signal transducer and activator of transcription 3 (STAT3), and blocks the interleukin 10 (IL-10) stimulated as well as the basal level CD163 expression in PAMs. Cpt-treatment of PAMs is effective when applied either before or after PRRSV infection, with the combined pre- and post-PRRSV infection treatment resulting in the most significant, dose-dependent inhibition of PRRSV infection. Cpt inhibited both type I/II PRRSV infection in PAMs. Our study identified a new approach to prevent/treat PRRSV infection of pigs with natural compounds.

Porcine reproductive and respiratory syndrome virus (PRRSV), is a highly mutable RNA virus that affects swine worldwide and its control is very challenging due to its formidable heterogeneity in the field. In the present study, DNA vaccines constructed with PRRSV GP5-Mosaic sequences were complexed to cationic liposomes and administered to experimental pigs by intradermal and intramuscular injection, followed by three boosters 14, 28 and 42 days later. The GP5-Mosaic vaccine thus formulated was immunogenic and induced protection from challenge in vaccinated pigs comparable to that induced by a wild type (VR2332) GP5 DNA vaccine (GP5-WT). Periodic sampling of blood and testing of vaccine-induced responses followed. Interferon-γ (IFN-γ) mRNA expression by virus-stimulated peripheral blood mononuclear cells (PBMCs) of GP5-Mosaic-vaccinated pigs was significantly higher compared to pigs vaccinated with either GP5-WT or empty vector at 21, 35 and 48 days after vaccination. Cross-reactive cellular responses were also demonstrated in GP5-Mosaic vaccinated pigs after stimulation of PBMCs with divergent strains of PRRSV. Thus, significantly higher levels of IFN-γ mRNA were detected when PBMCs from GP5-Mosaic-vaccinated pigs were stimulated by four Genotype 2 strains (VR2332, NADC9, NADC30 and SDSU73), which have at least 10% difference in GP5 amino acid sequences, while such responses were recorded only upon VR2332 stimulation in GP5-WT-vaccinated pigs. In addition, the levels of virus-specific neutralizing antibodies were higher in GP5-Mosaic or GP5-WT vaccinated pigs than those in vector-control pigs. The experimental pigs vaccinated with either the GP5-Mosaic vaccine or the GP5-WT vaccine were partially protected from challenge with VR2332, as measured by significantly lower viral loads in sera and tissues and lower lung lesion scores than the vector control group. These data demonstrate that the GP5-Mosaic vaccine can induce cross-reactive cellular responses to diverse strains, neutralizing antibodies, and protection in pigs.

Highlights • We isolated rotaviruses from alpaca crias with diarrhea in cell culture. • To our knowledge this is the first isolation of rotavirus in alpacas in Peru. • The virus isolates were identified by RT-PCR. • The isolates were further identified as group A rotaviruses by VP6-specific PCRs. • The isolates identified as G3/P11 and G3/P? had partial homology to swine and human rotaviruses, Alpacas (Vicugna pacos), a species of South American camelids (SAC), suffer high morbidity and mortality from infectious diseases. Diarrhea is one of the leading causes of alpaca cria mortality in Peru and elsewhere. In order to develop appropriate control and/or treatment, it is necessary to identify infectious pathogens that cause diarrhea in crias. Rotavirus was isolated in cell culture from feces collected from crias with acute diarrhea that tested positive to rotaviral antigen by rapid immunochromatographic methods in an earlier study. The isolates were identified as rotaviruses by RT-PCR run with specific primers for human rotavirus VP7 coding sequences using total RNA extracted from cells displaying cytopathic effects as template. These alpaca isolates were further identified as group A rotaviruses by means of a VP6-specific PCR and were designated as ALRVA-K’ayra/Perú/3368-10 and ALRVA-K’ayra/Perú/3386-10. Molecular G and P typing, placed the former as G3/P11 and the latter as G3/P?. Sequence analysis of two genome segments (coding for VP4 and VP7) from the alpaca isolates revealed partial homologies to swine and human rotaviruses, respectively. These results demonstrate that rotaviruses are associated with a proportion of cases of diarrhea in crias, although prevalence and impact remain to be determined. The isolation of rotaviruses from alpaca crias with diarrhea will contribute positively to further understand the pathogen and its role in the diarrhea complex.

Chlamydia causes enteritis in young pigs and calves, and granulomas within intestinal lymphatics. Recently we called attention to the lymphangitis and lymphatic obstruction that occurs in Crohn’s disease. We searched resected tissues from patients for evidence of chlamydia and sera for evidence of previous exposure to chlamydia. Immunohistochemistry and real-time PCR were employed to seek chlamydia in preserved tissues. In the IHC, antibody to C. trachomatis served as primary antibody; in the PCR, primers specific for Chlamydiaceae were employed. Commercial ELISA kits measured anti-chlamydia IgG and IgA against C. trachomatis antigen in sera derived from a population of patients different from that which yielded the tissue specimens. IHC revealed focal positive staining for chlamydia in tissues of 5 of 19 patients. Positive reacting cells occurred within dense inflammation, in sparsely scattered macrophages in the submucosa and subserosa. Tissues from 3 of 22 control subjects were positive. Real-time PCR done on ileal, colonic, and regional lymph node tissues revealed evidence of chlamydia in 3 of 33 patients. Serology for anti-chlamydia IgG revealed 2 positive values in 24 patients, while serology for anti-chlamydia IgA revealed 4 positives among the 24 patients, and 1 positive in the 15 controls. One patient and one control had both elevated IgG and IgA titers. The 4 patients with elevated IgA titers were from a single family of 6 with Crohn’s disease, which had been previously described. Additional consideration needs to be given to the chlamydia species, including those of animal origin, which leave behind little evidence of their previous involvement.

The activation of antiviral activity induced by recombinant swine interferon beta (rswIFNβ) against PRRSV was comparatively examined in MARC-145 cells and porcine alveolar macrophages (PAMs). A dose–response analysis showed, in MARC-145 cells, that isolate Mo25544 was highly sensitive to rswIFNβ while a vaccine strain and isolate PDV130-9301 were resistant to different extents. In contrast, all three viruses were equally sensitive to rswIFNβ in PAMs even at the lowest dose of IFN utilized in the bioassays. To analyze potential differences in mechanisms of antiviral activation between these cells, treatment with 2-aminopurine (2-AP), an inhibitor of double-stranded RNA-dependent protein kinase (PKR), was performed in rswIFNβ-treated cells. Addition of 2-AP to rswIFNβ-primed MARC-145 cells restored replication of the Mo25544 isolate, and to some extent that of vaccine virus and PDV130-9301. In contrast, virus replication could not be rescued for any of the three viruses with 2-AP in rswIFNβ-treated PAMs. The differences in sensitivity of PRRSV to rswIFNβ as well as the effects of 2-AP strongly suggest that MARC-145 cells and PAMs utilize different rswIFNβ-associated antiviral pathways. Therefore, studies to understand virus-host cell interactions performed in MARC-145 cells require additional scrutiny when utilized as a host cell model for immunologic responses to PRRSV.

A 14-month-old heifer with a 17-day history of unresponsive bloody diarrhea was necropsied. There were focal, pink-red erosions of the nares and hard palate; ulcers and fissures of the tongue; and multiple ulcerative lesions of the alimentary canal. Interdigital skin of both rear limbs was ulcerated and bleeding; and the margins of the vulva contained punctiform red ulcers. The gross lesions were consistent with mucosal disease. Histopathology and laboratory testing ruled out rinderpest, foot-and-mouth disease, and vesicular stomatitis, and identified bovine virus diarrhea virus to be the cause of this disease. Lesions of the vulva similar to those seen in some stages of infectious pustular vulvovaginitis were negative for bovine herpesvirus-1 and tested positive for bovine viral diarrhea virus antigen by immunohistochemistry.

Recently we have demonstrated, with a DNA vaccine, that the immediate early protein (IE180) of pseudorabies virus provides a moderate level of protection in mice. In order to improve its immunogenicity and protective capacity, this IE180 DNA vaccine was delivered to C3H/HeJ mice either in combination with an IL-2 expressing plasmid or complexed with cationic liposomes. Co-delivery of the vaccine and IL-2 DNA by gene gun resulted in seroconversion in 5/5 of the vaccinated mice after a single administration, whereas two intramuscular (i.m.) injections were required to achieve seroconversion in all mice. Antibody and delayed-type hypersensitivity responses were augmented in mice, which received the DNA vaccine and the IL-2 gene compared to those of mice receiving the DNA vaccine alone. In addition, the time of death after challenge was significantly delayed in mice, which received the IL-2 gene. The proportion of surviving mice (40%), however, was similar to that obtained in mice which received the vaccine alone by gene gun. Liposome-mediated vaccine delivery also resulted in a higher rate of seroconversion when compared with that induced by the naked DNA vaccine. Thus, all vaccinated mice seroconverted after either two i.v. or three i.m. injections of the liposome/DNA complex, with 40 and 25% of these mice being protected against challenge, respectively. These data support that co-administration of the IE180 DNA vaccine with the IL-2 gene or delivery in liposomes are two effective approaches to increase its immunogenicity.

West Nile fever emerged in New York in the summer of 1999 when seven people, several horses and thousands of wild birds died. It was soon established that the human disease and the mortality of birds were related. Continued surveillance detected West Nile virus in mosquitoes, birds, horses, small mammals, bats and humans, and has shown its spread to several northeastern states. These events confirm the establishment of West Nile virus endemically in the United States.

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.

An earlier study in our laboratory provided evidence that pseudorabies virus (PrV) immediate early protein (IE180) may contribute to the overall immune response against PrV. To examine the response by IE180 more closely, we initiated a vaccine trial in mice with a vector DNA construct that contains the gene encoding for IE180, designated pcDNAIE180. The DNA vaccine was delivered in gold microcarriers using a Helios Gene Gun, and 70% of BALB/c mice given the DNA vaccine (2 microg/mouse) seroconverted within 2 weeks. The remaining negative mice seroconverted after a single vaccine booster. Essentially similar results were obtained on vaccination of C57BL/6 mice, whereas C3H/HeJ mice remained negative after the first vaccination, but responded after a booster. Seven months after immunization with pcDNAIE180, an overall 25% of BALB/c, C3H/HeJ, and C57BL/6 mice receiving a lethal PrV challenge were protected. In addition, a significant passive transfer of IE180-specific antibodies to the offspring from pregnant mice vaccinated with pcDNAIE180 was observed. Interestingly, a moderate level of protection (27.6%) was also observed when these offspring received a lethal PrV challenge. Moreover, an enhancement of immune responses and a twofold increase in the level of protection were observed in mice that received a second vaccine booster by gene gun 8 months after the first vaccination. Together, these data support the theory that IE180 may indeed play a role in the overall protective immunity against PrV.

Pseudorabies virus (PrV) encodes for a single and essential immediate early phosphoprotein designated IE180. In this study, IE180 was examined in lysates from various cell lines infected at high multiplicities under cycloheximide inhibition of protein synthesis and subsequent reversal. Three distinct protein patterns of IE180 which were cell-specific and dependant on the extraction procedure were revealed. Detergent lysates of PrV infected MDBK cells yielded almost exclusively wild type IE molecule (180 kDa). In contrast, SSG/94 cells, VERO or CV-1 cells did not yield 180 kDa molecules but predominantly a shorter variant of approximately 60 kDa in molecular mass. Additional bands of about 50/55 kDa were also detected in lysates of SSG/94 and VERO cells by immunoprecipitation. Lysates of CV-1 and MDBK cells also yielded a 120 kDa molecule. The smaller molecular mass bands occurred in the presence of PMSF and aprotinin however, cleavage was blocked completely by addition of Nλ- p -Tosyl- l -lysine chloromethyl ketone (TLCK) into the lysis buffer. Moreover, an ability of the shorter IE180 variants to bind heparin was also revealed in the study. These data provide useful insights on protease profiles encountered among different PrV susceptible cells and indicates the use of appropriate protease inhibitors such as TLCK to protect IE180 under these experimental conditions.

Ten muskrats (Ondatra zibethicus) each were infected with 17,000 eggs (long-term study) and eight muskrats each were infected with 8,000 eggs (short-term study) of Capillaria hepatica (Nematoda). Food intake, body weight, and selected clinicopathological parameters were measured every 2 days for 28 days in the short-term study and every 14 days for 184 days in the long-term study. Muskrats in the short-term study had moderate to severe necrotizing granulomatous hepatitis associated with mild anorexia and weight loss, varying degrees of leukocytosis with eosinophilia and elevation of serum alanine and aspartate aminotransferases. No significant changes in packed cell volume, hemoglobin, total plasma protein, albumin, blood urea nitrogen, bilirubin, lactate dehydrogenase or alkaline phosphatase were found among animals from the short-term study. Muskrats in the long-term study had severe necrotizing granulomatous hepatitis associated with marked anorexia, weight loss and 60% mortality over 39 days post-inoculation (PI); animals that survived for 184 days did not return to pre-inoculation body weights despite returning to normal food intake. Hepatic lesions at 184 days PI consisted of minimal to severe liver replacement by C. hepatica eggs. No statistically significant differences in values of clinical parameters between inoculated animals and a non-inoculated control group from the long term study were found.

Muskrat (Ondatra zibethicus) immunoglobulin fraction was separated from whole serum by Protein A Sepharose chromatography. In serum electrophoresis, this fraction had a gamma motility; when electrophoresed on a polyacrylamide gel with sodium dodecyl sulfate it resolved into two protein bands of approximately 52 and 25 kilodaltons, respectively. These bands were consistent with molecular weights of known heavy and light chains of immunoglobulin G (IgG) in other closely related species. Furthermore, the putative muskrat immunoglobulins had a strong cross-reactivity with mouse IgG1, IgG3, and kappa chain in an enzyme-linked immunosorbent assay. We propose, that the proteins bound to the Protein A Sepharose represent muskrat immunoglobulins of the IgG class.

The immediate early protein (IE180) of pseudorabies virus (PrV) was generated by cycloheximide (CHX) reversal procedures in PrV-infected swine skin cells. Using this IE180 preparation as antigen, specific proliferation was detected in mononuclear cells (PBMNCs) from swine vaccinated with a modified live virus (MLV) PrV vaccine. Two sets of data support this conclusion. (a) PBMNCs of c/cSLA inbred swine vaccinated with an MLV vaccine a year before the test exhibited significant responses against structural virion proteins and IE180. (b) Vaccination of PrV-negative swine with the same MLV vaccine induced a conversion from an unresponsive state against IE180 to one of specific antigen-driven responsiveness. The responses of vaccinated swine against IE180 were significantly higher than their preimmune responses (por = 0.003) or to the response of control swine (por = 0.045). Moreover, IE180 antigens obtained from lysates of CHX-reversed, PrV-infected cells by heparin/agarose affinity separation also stimulated specific proliferation of PBMNCs from MLV-vaccinated swine, as their proliferative responses were significantly higher than those of unvaccinated swine (por = 0.05). These data suggest that PrV IE180 contributes at least in part to the overall cellular response to PrV.

Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P<0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.

Gamma interferon produced by porcine lymphocytes (nPoIFN gamma) in response to stimulation with phorbol-myristate-acetate (PMA) and phytohemagglutinin (PHA) was monitored by a radioimmunoassay (RIA). The RIA was developed with antibodies raised in rabbits against recombinant porcine IFN gamma (rPoIFN gamma) and made monospecific on a rPoIFN gamma/Sepharose matrix. This anti-PoIFN gamma antibody was shown to bind and neutralize rPoIFN gamma specifically and to cross-react with bovine IFN gamma but not with murine IFN gamma or porcine IFN alpha. The nPoIFN gamma levels produced by lymphocytes in response to PMA and PHA were at least two-fold higher than control lymphocytes as measured by the RIA in culture fluids. These culture fluids were fractionated on Concanavalin A Sepharose (Con A/Seph) and anti-rPoIFN gamma/Seph in attempts to evaluate the induced nPoIFN gamma further. The separation was monitored by RIA and showed that nPoIFN gamma was retained on Con A/Seph suggesting the presence of sugar residues on the molecules. Pools of Con A/Seph fractions, positive in RIA, were separated further on the anti-rPoIFN gamma/Seph matrix where a total adsorption of nPoIFN gamma occurred. On a weight basis, the eluates from the anti-rPoIFN gamma/Seph had a reactivity in RIA at least four times higher than the fractions derived from Con A/Seph. This indicated that the nPoIFN gamma remained immunochemically reactive after being eluted from Con A/Seph and that the separation on anti-rPoIFN gamma/Seph chromatography was specific. Purified nPoIFN gamma exhibited a major band with the same migration characteristics of rPoIFN gamma in PAGE-SDS electrophoresis and several minor bands when reacted with 125I anti-rPoIFN gamma antibody in Western blots. A total loss of reactivity to antibody after radioiodination of nPoIFN gamma, however, prevented the confirmation of these results by immunoprecipitation. A direct, rapid and sensitive immunoassay for measurement of the relative levels of nPoIFN gamma present in biological fluids is presented and this method could be a useful complement to bioassays for IFN gamma.

La diarrea es una de las causas más importantes de mortalidad en crías de alpacas, por lo que la definición de patógenos asociados a la diarrea de crías cobra una importancia especial para formular medidas de control efectivas. Muestras de heces colectadas de crías con diarrea aguda y contenidos intestinales de crías muertas fueron examinadas por presencia de antígenos de rotavirus o coronavirus mediante pruebas inmunocromatográficas rápidas. Tres de 14 muestras de heces de alpaca fueron positivas a rotavirus mientras que los contenidos intestinales fueron negativos. Cinco de 14 muestras de heces resultaron positivas a coronavirus mientras que ningún contenido intestinal fue positivo. Estos resultados sugieren que rotavirus y coronavirus están asociados con una proporción de casos de diarrea en crías de alpacas pero no descartan la posibilidad de co-infección con otros patógenos. La prevalencia de estos virus y el impacto causado requieren investigación adicional., Diarrhea is one of the leading causes of alpaca cria mortality in Peru and elsewhere. Defining infectious pathogens involved in morbidity and mortality from diarrhea in crias is of significant importance for its appropriate control. This study reports the detection of rotavirus and coronavirus antigens in feces collected from crias with acute diarrhea and intestinal contents from dead crias. The fecal samples were screened for rotavirus or coronavirus antigens by rapid immunochromatographic methods. Three of the 14 fecal samples were positive for rotavirus antigen while the intestinal contents were all negative. Five of 14 fecal samples were positive for coronavirus and all the intestinal contents were negative. These results suggest that both rotavirus and coronavirus are associated with a proportion of cases of diarrhea in crias but do not rule out the possibility of co-infection with other pathogens. The actual prevalence and impact of these two viruses requires further investigation.

New-onset Crohn's disease and acute flares are often associated with viral infections. The aim of this study was to search for evidence of persistent or recurrent viruses in patients. Tissue blocks were obtained from surgical specimens from patients and a control population. 111 samples were tested by PCR or RT-PCR, for EBV, CMV, HSV 1, HSV 2, HHV 8, pestiviruses, and enteroviruses. Additionally, seven sets of serum samples, including pre-operative and post-operative samples, from CD patients were analyzed serologically for antibodies to EBV. The tests revealed evidence of EBV nucleic acid in tissues of 11 patients from a total of 70 tested (15.7%) and in tissues of 3 of 41 control subjects (7.3%). Evidence of pestivirus was found in one CD patient, while one patient and one control were positive for CMV. No HSV 1 or 2, HHV 8 or enteroviruses were found. The serologic tests revealed that five of seven CD patients had antibodies against the early protein, the capsid protein and the EBV nuclear antigen (EBNA). The titers were not significantly altered post-surgically. None of the patients had antibodies of the IgM isotype. Our findings vary from those of Ruther et al. who demonstrated evidence of EBV in tissues from 7 of 11 (64%) German CD patients. Antibodies to early EBV viral antigen and to nuclear antigen in five of seven Belgian patients suggest persistent active viral infection.

An indirect fluorescent antibody test (IFA) was developed to detect West Nile virus (WNV) antigens in tissues from avian species. The test samples used in the study consisted of 100 sets of tissues from dead crows that had been collected during the 2001 surveillance in Connecticut. The test tissues were punctured with a fine point Dacron cotton-tipped applicator and smeared in duplicate on 10-well diagnostic printed glass slides. Among several fixatives tested, 4% paraformaldehyde was the best. Reagent calibration for the IFA test was done in WNV-infected Vero cells and control uninfected Vero cells. Optimized antibody and fluorescent conjugate concentrations were then applied for the detection of WNV antigen on fixed tissue impression smears. Several tissues, including brain, heart, liver, kidney, and spleen were tested by the IFA test. The brain and heart seemed to be unsuitable for the test because of excessive background. Both virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) were used for validation, with the latter technique having a higher sensitivity. Therefore, IFA results were compared with RT-PCR results. The diagnostic sensitivity was 96.8% for liver, 96.4% for kidney, and 100% for spleen. The diagnostic specificity was 69% for liver, 95.3% for kidney and 95.8 for spleen. The IFA test performed best with spleen and kidney. The IFA test described here is a useful, practical, and rapid test for screening for WNV.

Swine beta interferon (swIFN-β) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-β gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-β or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-β showed no CPE. To confirm the antiviral activity of swIFN-β, culture fluids from Ad5-swIFN-β-infected cells were affinity-purified on a Sepharose–anti-swIFN-β matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-β. Additional cultures of MARC-145 cells treated with swIFN-β-containing supernatants or affinity-purified swIFN-β were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-β. swIFN-β was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-β or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-β-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-β protects both MARC-145 cells and PAMs from PRRSV infection.

West Nile fever outbreaks in the USA have caused over 700 human deaths, primarily due to neurological disease. The usual transmission route of West Nile virus (WNV) involves mosquito bites; however, alternative routes, including intrauterine infection, have also been reported. Here, the pathogenicity of WNV in mice during gestation has been investigated. An extremely high mortality rate was observed in pregnant mice (98 %, 60/61) compared with non-pregnant mice (52 %, 28/53; P

This study investigated the effect of swine interleukin 2 (IL-2) and swine interleukin 4 (IL-4) on the development of immune responses induced by a PRRSV-ORF7 DNA vaccine (phCMV-ORF7). The two cytokines were cloned separately in the eukaryotic expression vector phCMV, and delivered via gene gun as adjuvants for the DNA vaccine. Groups of 3-week-old certified PRRSV-free, castrated male, Yorkshire crossbred pigs, were vaccinated with or without the IL-2 or IL-4. The ensuing humoral and cellular immune responses were analyzed by a PRRSV-specific ELISA, and by an in vitro blastogenic response of peripheral blood mononuclear cells (PBMC) stimulated by viral antigen, respectively. The animals were boosted 21 days post-vaccination and challenged 28 days afterward. The virus loads post-challenge were measured by real time PCR. The group of swine receiving the vaccine plus IL-2 had significant virus-specific blastogenic responses 3 weeks after the vaccine-cytokine boost, when compared to those of the experimental pigs that received the vaccine plus IL-4, vaccine alone, unvaccinated controls or the pigs vaccinated with the DNA vaccine cloned in the reverse orientation (phCMV-ORF7(Rev)). None of the experimental swine had detectable specific antibodies against the virus during the vaccination phase. The virus load peak in vaccinated animals was delayed by about 72h as compared to that of the control pigs (unvaccinated and vaccinated with the phCMV-ORF7(Rev) construct). Interestingly, animals that received the phCMV-ORF7 vaccine alone consistently had low virus loads throughout the study. These results demonstrate that IL-2 has a positive inductive effect on the activation of vaccine-induced virus-specific cellular immunity, while IL-4 appeared to have a suppressive effect. Our data also suggest that ORF7 may play a role in reducing the virus load in PRRSV infected animals.

Background: Viral infections induce expression of type I interferons (IFNα/β) which result in an antiviral state within cells or animals. However, some viruses, notably influenza viruses (IV), have evolved mechanisms of evasion and blocking of IFN to survive in nature. Swine influenza virus (SIV) can inhibit IFN and as with other IV does it through its NS1 protein, identified as a virulence factor in pigs. Methods: To determine whether different SIV strains varied in their ability to induce IFNβ in cell culture and the relative sensitivity of these strains to exogenous IFNβ, three strains were tested in SJPL cells originally deposited as epithelial-like cell line of swine lung origin (ATCC, Richmond, MD) but later characterized as cells of monkey origin. Results: The SIV strains tested here induced significant synthesis of IFNβ in SJPL cells, with levels that were higher to those induced by poly I:C. The induction of IFNβ by SIV peaked at three days post-infection and was not dependent on the infectious virus dose. Interestingly, priming the cells with exogenous recombinant swine IFNβ had no significant inhibitory effect on the replication of these SIV strains. NS1antagonistic compounds did not change IFNβ induction and had variable and unexpected effects on virus replication of the three SIV strains tested. Conclusions: Our study showed that while the SIV strains tested here were capable to induce IFNβ in SJPL cells these were relatively resistant to the antiviral effects of exogenous IFNβ. NS1-antagonistic compounds did not affect IFNβ induction and had variable effects on the replication of the SIV strains tested. These seemingly contradicting results suggest virus evasion by mechanisms other than NS1.

A dual expressing (CD4(+)/CD8(+)) porcine lymphoblastoid T-cell line (pIL-2d) generated from peripheral blood mononuclear (MN) cells shown to be highly responsive to exogenous interleukin-2 (IL-2) was characterized. The swine MN cells were initially stimulated with concanavalin A (Con A), and sub-passaged using decreasing amounts of conditioned medium (CM), which was prepared from culture fluids of Con A activated porcine MN cells, until a steady growth was observed. The resulting pIL-2d cells require exogenous IL-2 from CM and are highly responsive to recombinant human IL-2 (rhIL-2). The pIL-2d cells exhibited a specific, dose-dependent proliferative response to stimulation with IL-2. The specificity of this proliferative response was confirmed to be IL-2 induced by its inhibition with an anti-swine IL-2 receptor (alpha-swIL-2R) monoclonal antibody (mAb). Furthermore, the pIL-2d cells are highly responsive to exogenous IL-2 contained in culture fluids derived from antigen-driven blastogenic tests performed with lymphocytes of vaccinated swine. This property makes the pIL-2d cells an ideal functional adjunct to immunochemical or molecular tests that are commonly used to measure total porcine IL-2. Interestingly, the phenotype of the pIL-2d cells after five or more passages was shown by flow cytometric analysis to be CD4(+)/CD8(+)/CD45RA(-)/CD25(+) and to remain unchanged thereafter. Although, the mechanism of selection and maintenance of the CD4(+)/CD8(+) DP cells developed here remains unclear, our data suggest that an oligoclonal or polyclonal expansion and maintenance of cells of this phenotype was mediated by exogenous IL-2.

West Nile (WN) virus, a mosquito-transmitted virus native to Africa, Asia, and Europe, was isolated from two species of mosquitoes, Culex pipiens and Aedes vexans , and from brain tissues of 28 American crows, Corvus brachyrhynchos , and one Cooper's hawk, Accipiter cooperii , in Connecticut. A portion of the genome of virus isolates from four different hosts was sequenced and analyzed by comparative phylogenetic analysis. Our isolates from Connecticut were similar to one another and most closely related to two WN isolates from Romania (2.8 and 3.6 percent difference). If established in North America, WN virus will likely have severe effects on human health and on the health of populations of birds.

An experimental subunit vaccine that consisted of pseudorabies virus (PRV) envelope glycoproteins enclosed into immunostimulating complexes (PRVenv/ISCOM) was constructed, and evaluated in DBA/2 mice and inbred swine of the SLA hoplotype c/c. Two to three weeks after the first vaccine dose, specific anti-PRV antibodies could be demonstrated by ELISA or virus neutralization (VN) assays. Booster PRVenv/ISCOM vaccinations resulted in rapid and significant increases in antibody titres in both mice and swine. In addition, a week after receiving the third PRVenv/ISCOM vaccine dose swine peripheral blood mononuclear cells exhibited significant proliferation in response to stimulation with PRV virion antigen. Moreover, two doses of vaccine sufficed to protect mice fully against lethal virus challenge. Therefore, the data presented here support the ISCOM as a viable antigen delivery system for subunit PRV vaccines.

West Nile virus was recovered from the brain of a red-tailed hawk that died in Westchester County, N.Y., in February 2000. Multiple foci of glial cells, lymphocytes, and a few pyknotic nuclei were observed in the brain. Three to 4 days after inoculation of Vero cells with brain homogenates, cytopathic changes were detected. The presence of West Nile virus antigen in fixed cells or cell lysates was revealed by fluorescent antibody testing or enzyme-linked immunosorbent assay, respectively. Furthermore, Reverse transcriptase-PCR with primers specific for the NS3 gene of West Nile virus resulted in an amplicon of the expected size (470 bp). Electron microscopy of thin sections of infected Vero cells revealed the presence of viral particles approximately 40 nm in diameter, within cytoplasmic vesicles. The demonstration of infection with the West Nile virus in the dead of the winter, long after mosquitoes ceased to be active, is significant in that it testifies to the survival of the virus in the region beyond mosquito season and suggests another route of transmission: in this case, prey to predator.

A series of seven neutralizing monoclonal antibodies (nMAbs) against type A12 foot-and-mouth disease virus (FMDV) was used to induce polyclonal anti-idiotypic antibodies (anti-ids) in rabbits. The anti-ids were semi-purified through isotype affinity columns and assayed by solid-phase radioimmunoassay for cross-reactivity. nMAbs which map to the same epitope on the virion appear to contain a common idiotype, and the corresponding anti-ids competitively inhibited the virus-nMAb reaction. Using a modified ELISA assay, it was possible to demonstrate binding of purified anti-ids to FMDV susceptible tissue culture cells. Such antibodies however, did not interfere with the binding of virus to cells, and the binding of anti-ids to FMDV receptor-negative cells could also be demonstrated. Mice were inoculated with purified anti-ids, and two elicited anti-viral antibodies, although these antibodies were non-neutralizing. Thus anti-ids to anti-FMDV nMAbs failed to react with cellular receptors for the virus, but were able to induce anti-viral antibody and thus should be examined as an alternative vaccine strategy for this virus.