In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

Serological survey for antibodies against pestiviruses in Wyoming domestic sheep.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle.

iPTF16geu: A multiply imaged, gravitationally lensed type Ia supernova.

We report the discovery of a multiply imaged, gravitationally lensed type Ia supernova, iPTF16geu (SN 2016geu), at redshift z = 0.409. This phenomenon was identified because the light from the stellar explosion was magnified more than 50 times by the curvature of space around matter in an intervening galaxy. We used high-spatial-resolution observations to resolve four images of the lensed supernova, approximately 0.3 arc seconds from the center of the foreground galaxy. The observations probe a physical scale of ~1 kiloparsec, smaller than is typical in other studies of extragalactic gravitational lensing. The large magnification and symmetric image configuration imply close alignment between the lines of sight to the supernova and to the lens. The relative magnifications of the four images provide evidence for substructures in the lensing galaxy.

Genomic and antigenic characterization of bovine parainfluenza-3 viruses in the United States including modified live virus vaccine (MLV) strains and field strains from cattle.

This study investigated the genetic and antigenic characterization of parainfluenza-3 virus (PI3V) of cattle. Using molecular tests including real time PCR and viral genome sequencing, PI3V strains could be separated into PI3V types, including PI3V A, PI3V B, and PI3V C. Isolates from cattle with bovine respiratory disease clinical signs and commercial vaccines in the U.S. with MLV PI3V were typed using these molecular tests. All the MLV vaccine strains tested were PI3V A. In most cases PI3V field strains from calves receiving MLV vaccines were types heterologous to the vaccine type A. Also antigenic differences were noted as PI3V C strains had lower antibody levels than PI3V A in serums from cattle receiving MLV PI3V A vaccines. This study further demonstrates there is genetic variability of U.S. PI3V strains and also antigenic variability. In addition, isolates from cattle with BRD signs and receiving MLV vaccines may have heterologous types to the vaccines, and molecular tests should be performed to differentiate field from vaccine strains. Potentially the efficacy of current PI3V A vaccines should be evaluated with other types such a PI3V B and PI3V C.

Detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease.

This study investigated viruses in bovine respiratory disease (BRD) cases in feedlots, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronaviruses (BoCV) and parainfluenza-3 virus (PI3V). Nasal swabs were collected from 114 cattle on initial BRD treatment. Processing included modified live virus (MLV) vaccination. Seven BRD necropsy cases were included for 121 total cases. Mean number of days on feed before first sample was 14.9 days. Swabs and tissue homogenates were tested by gel based PCR (G-PCR), quantitative-PCR (qPCR) and quantitative real time reverse transcriptase PCR (qRT-PCR) and viral culture. There were 87/114 (76.3%) swabs positive for at least one virus by at least one test. All necropsy cases were positive for at least one virus. Of 121 cases, positives included 18/121 (14.9%) BoHV-1; 19/121 (15.7%) BVDV; 76/121 (62.8%) BoCV; 11/121 (9.1%) BRSV; and 10/121 (8.3%) PI3V. For nasal swabs, G-PCR (5 viruses) detected 44/114 (38.6%); q-PCR and qRT-PCR (4 viruses) detected 81/114 (71.6%); and virus isolation detected 40/114 (35.1%). Most were positive for only one or two tests, but not all three tests. Necropsy cases had positives: 5/7 G-PCR, 5/7 q-PCR and qRT-PCR, and all were positive by cell culture. In some cases, G-PCR and both real time PCR were negative for BoHV-1, BVDV, and PI3V in samples positive by culture. PCR did not differentiate field from vaccines strains of BoHV-1, BVDV, and PI3V. However based on sequencing and analysis, field and vaccine strains of culture positive BoHV-1, BoCV, BVDV, and PI3V, 11/18 (61.1%) of BoHV-1 isolates, 6/17 (35.3%) BVDV isolates, and 1/10 (10.0%) PI3V identified as vaccine. BRSV was only identified by PCR testing. Interpretation of laboratory tests is appropriate as molecular based tests and virus isolation cannot separate field from vaccine strains. Additional testing using sequencing appears appropriate for identifying vaccine strains.

Comparison of 'HoBi'-like viral populations among persistent infected calves generated under experimental conditions and to inoculum virus.

Like other members from the Pestivirus genus, 'HoBi'-like pestiviruses cause economic losses for cattle producers due to both acute and persistent infections. The present study analyzed for the first time PI animals derived from a controlled infection with two different 'HoBi'-like strains where the animals were maintained under conditions where superinfection by other pestiviruses could be excluded. The sequence of the region coding for viral glycoproteins E1/E2 of variants within the swarms of viruses present in the PI calves and two viral inoculums used to generate them were compared. Differences in genetic composition of the viral swarms were observed suggesting that host factors can play a role in genetic variations among PIs. Moreover, PIs generated with the same inoculum showed amino acid substitutions in similar sites of the polyprotein, even in serum from PIs with different quasispecies composition, reinforcing that some specific sites in E2 are important for host adaptation.

Comparison of the breadth and complexity of bovine viral diarrhea (BVDV) populations circulating in 34 persistently infected cattle generated in one outbreak.

Exposure to bovine viral diarrhea viruses (BVDV) results in acute and persistent infections. Persistent infections result from in utero exposure during the first trimester of gestation. Clinical presentation, in persistently infected cattle (PI), is highly variable. The reasons for this variation is largely unknown. The BVDV circulating in PI exist as quasispecies (swarms of individual viruses). An outbreak resulting in 34 PI cattle presented an opportunity to compare a large number of PI׳s. Methods were developed to compare the circulating viral populations within PI animals. It was found that PI animals generated in the same outbreak carry circulating viral populations that differ widely in size and diversity. Further, it was demonstrated that variation in PI viral populations could be used as a quantifiable phenotype. This observation makes it possible to test the correlation of this phenotype to other phenotypes such as growth rate, congenital defects, viral shed and cytokine expression.

An ultraviolet-optical flare from the tidal disruption of a helium-rich stellar core.

The flare of radiation from the tidal disruption and accretion of a star can be used as a marker for supermassive black holes that otherwise lie dormant and undetected in the centres of distant galaxies. Previous candidate flares have had declining light curves in good agreement with expectations, but with poor constraints on the time of disruption and the type of star disrupted, because the rising emission was not observed. Recently, two 'relativistic' candidate tidal disruption events were discovered, each of whose extreme X-ray luminosity and synchrotron radio emission were interpreted as the onset of emission from a relativistic jet. Here we report a luminous ultraviolet-optical flare from the nuclear region of an inactive galaxy at a redshift of 0.1696. The observed continuum is cooler than expected for a simple accreting debris disk, but the well-sampled rise and decay of the light curve follow the predicted mass accretion rate and can be modelled to determine the time of disruption to an accuracy of two days. The black hole has a mass of about two million solar masses, modulo a factor dependent on the mass and radius of the star disrupted. On the basis of the spectroscopic signature of ionized helium from the unbound debris, we determine that the disrupted star was a helium-rich stellar core.

Bovine viral diarrhoea virus infection alters global transcription profiles in bovine endothelial cells.

Bovine viral diarrhoea viruses (BVDV) are significant pathogens of cattle worldwide. These viruses exist in both non-cytopathic and cytopathic biotypes. Non-cytopathic BVDV can establish persistent lifelong infections in cattle and are a frequent contaminant of biological reagents such as cell cultures and foetal bovine serum. We identified commercially available bovine aortic endothelial cells (BAECs) contaminated with BVDV. In this study, to determine if BVDV alters endothelial gene transcription patterns, serial analysis of gene expression (SAGE) was used to compare gene expression profiles from uninfected and BVDV contaminated BAEC. SAGE is an open ended, quantitative method for characterizing global patterns of transcription. Comparison of expression profiles of BVDV-contaminated and noninfected cells revealed significant increases in the transcription of many genes including P-selectin, tryptophan tRNA synthetase and prostaglandin D2 synthase. These changes were validated by real-time PCR. Additionally, real-time PCR demonstrated that the response to LPS and dsRNA by contaminated cells, as well as cells acutely infected with noncytopathic BVDV, is altered. The altered response may be through the high level of expression of A20 and inhibition of activation of NF-kappaB. BAECs are commonly used as a model to study endothelial cell function in many different systems. As shown here, transcriptional and probable protein changes resulting from BVDV infection significantly alter cellular responses and may have a profound impact on experimental outcome. Transcriptomic analysis provided the initial clues leading to the characterization of this altered function.

Effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (SAGE).

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide and causes considerable economic loss. The main target of infection is the porcine alveolar macrophage (PAM). Infection of PAMs by PRRSV causes significant changes in their function by mechanisms that are not understood. We have employed Serial Analysis of Gene Expression (SAGE) to examine the global expression of genes in PRRSV-infected PAMs. Total cellular RNAwas prepared from in vitro mock-infected and PRRSV strain VR-2332-infected PAMs at 0, 6, 12, 16 and 24 hours after infection, and subjected to SAGE analysis to obtain > 100,000 tags per time point. These sequences were processed to account for sequencing error before generating tag:count lists. These lists were deposited into a modified Identitag database for mapping to porcine and PRRSV genes. Identified unique mRNAtags were analyzed for their identity and relative abundance. Examination of the SAGE data indicated that there were changes in gene expression occurring in the PRRSV-infected PAMs over time post-infection. More than 400 unique tags with significantly altered expression levels were identified (p < 0.01 with Bonferroni correction). The validity and kinetics of expression of SAGE identified genes were evaluated using real-time RT-PCR.

Activation of cell signaling pathways is dependant on the biotype of bovine viral diarrhea viruses type 2.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Besides the segregation into two distinct species (BVDV1/BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are distinguished based on their behavior in epithelial cell cultures. One of the most serious forms of BVDV infection affecting immunocompetent animals of all ages is severe acute BVD (sa BVD) which is caused by highly virulent ncp BVDV2 strains. Previous studies revealed that these highly virulent ncp viruses cause cell death in a lymphoid cell line (BL3) which is not clearly associated with typical apoptotic changes (e.g. PARP cleavage) observed after infection with cp BVDV. To further characterize the underlying molecular mechanisms, we first analyzed the role of the mitochondria and caspases as key mediators of apoptosis. Compared to infection with cp BVDV2, infection with highly virulent ncp BVDV2 resulted in a delayed and less pronounced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) and a weaker activation of the caspase cascade. In contrast, infection with low virulence ncp BVDV2 showed no significant differences from the uninfected control cells. Since different pro- and anti-apoptotic cellular signaling pathways may become activated upon virus infection, we compared the effect of different BVDV2 strains on cellular signaling pathways in BL3 cells. Stress-mediated p38 MAPK phosphorylation was detected only in cells infected with cp BVDV2. Interestingly, infection with highly virulent ncp BVDV2 was found to influence the phosphoinositide 3-kinase (PI3K)-Akt pathway. This indicates that BL3 cells respond differently to infection with BVDV depending on virulence and biotype.

Detection of viruses: atomic force microscopy and surface enhanced Raman spectroscopy.

This paper demonstrates the capability of atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS) to function effectively as ultra-sensitive readout tools for chip-scale platforms designed for pathogen detection in complex biological media. AFM allows direct (i.e., label-free) visualization and quantification of nanometer-sized viruses captured on a smooth, selective surface. AFM readout led to optimization of a capture substrate for feline calicivirus (FCV), and yielded a limit of detection of 3 x 10(6) FCV/mL. SERS-based detection of FCV, carried out in a sandwich-type assay, requires labelling of the substrate-bound FCV with a selective extrinsic Raman label (ERL). These studies yielded a limit of detection of 1 x 10(6) FCV/mL. The prospects of these two readout methods as additions to the arsenal of tools in bioterrorism prevention are briefly discussed.

Lesions and tissue distribution of viral antigen in severe acute versus subclinical acute infection with BVDV2.

Differences in the distribution and spread of viral antigen, development of lesions and correlation between presence of viral antigen and lesions were compared between a low and highly virulent strain of BVDV2. Two groups of two-week- to two-month-old colostrum-deprived calves were inoculated intranasally with the naturally occurring low virulent BVDV2 strain 28508-5 or the highly virulent strain 1373. To study the sequence of virus spread and lesion development, calves were necropsied at days three, six, eight-nine and 12 to 14 post inoculation (pi). Viral antigen was detected by the indirect immunoperoxidase method in cryostat sections and lesions were evaluated in H&E-stained paraffin sections. Clinical signs and changes in lymphocyte and thrombocyte numbers confirmed the difference in virulence between the two strains. Both strains showed comparable initial infection and spread at day three pi. At day six pi, they were found widespread in lymphoid tissues and multifocally in intestinal mucosa. Lesions were very mild despite the large amount of antigen in the lymphoid tissues. After day six pi, differences between the low and highly virulent strains became more prominent. The strain of low virulence was cleared from the tissues, but there was a transient phase of depletion. The highly virulent strain continued to spread to different organs and there was severe depletion of lymphoid tissues without recovery.

Mammalian gonadotropin-releasing hormone (GnRH) receptor subtypes.

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that governs gonadotropin secretion through interaction with its seven transmembrane (7TM), G protein-coupled receptor (GPCR) expressed by anterior pituitary cells. A second decapeptide, GnRH II, originally discovered in the chicken hypothalamus was recently reported to be expressed in the mammalian hypothalamus as well. A search of the recently-sequenced human genome identified a 7TM/GPCR on chromosome 1 that exhibited a higher identity with non-mammalian vertebrate GnRH II receptors (55%) than with the human GnRH I receptor (39%). Molecular cloning and nucleotide sequencing of this putative GnRH II receptor cDNA from monkey pituitary gland revealed a 379 amino acid receptor that, unlike the GnRH I receptor, possessed a C-terminal tail. Heterologous expression and functional testing of the receptor in COS-1 cells confirmed its identity as a GnRH II receptor: measurement of 3H-inositol phosphate accumulation revealed EC(50)s for GnRH II of 0.86 nM and for GnRH I of 337 nM. Ubiquitous tissue expression of GnRH II receptor mRNA was observed using a human tissue RNA expression array and a 32P-labeled antisense riboprobe representing the 7TM region of human GnRH II receptor cDNA. As predicted by the presence of its C-terminal tail, the GnRH II receptor was desensitized by GnRH II treatment whereas the naturally tail-less GnRH I receptor was not desensitized by GnRH I. Pharmacological analysis of the GnRH II receptor revealed that GnRH I 'superagonists' were more potent than GnRH I but less potent than GnRH II. Numerous GnRH I antagonists showed neither antagonistic nor agonistic activity with the GnRH II receptor. The functions of the GnRH II receptor are unknown but may include regulation of gonadotropin secretion, female sexual behavior, or tumor cell growth.

A regulator of G protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion.

BACKGROUND: Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Galpha proteins with effectors such as phospholipase Cbeta. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. RESULTS: A truncated version of RGS3 (RGS3T = RGS3 314-519) inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqalpha than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqalpha protein. CONCLUSIONS: RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate) as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqalpha protein function. A version of RGS3 that is amino-terminally truncated is even more potent than intact RGS3 at inhibiting gonadotropin releasing hormone-stimulated inositol trisphosphate production.

Recombination with a cellular mRNA encoding a novel DnaJ protein results in biotype conversion in genotype 2 bovine viral diarrhea viruses.

Bovine viral diarrhea viruses (BVDV) exist as two biotypes, cytopathic and noncytopathic, based on their ability to produce cytopathology in cultured cells. Cytopathic viruses arise from noncytopathic viruses following genetic alteration (insertions, duplications, rearrangements) within the region encoding the NS2/3 protein. In BVDV genotype 2, the majority of biotype conversions result from the integration of a cellular mRNA into the genomic RNA of a noncytopathic virus within the NS2/3 coding region. The translation of the cellular sequences during viral replication results in the proteolytic cleavage of the nonstructural protein NS2/3 to the NS2 and NS3 proteins with the appearance of the cytopathic phenotype. Here, these cellular sequences were identified as a portion of a cellular mRNA encoding a novel DnaJ protein (bDnaJ1). The 60 amino acid J-domain was identified near the C-terminus of the protein. Potential nuclear localization, farnesylation and hydrophobic transmembrane domains were also identified. Reverse transcription-PCR analysis of the expression of bDnaJ1 in bovine tissues showed that it was expressed in all tissues examined, and additionally, there may be alternate splicing of the transcript, yielding a second form of the bDnaJ1 protein. Northern blot analysis of mock and BVDV2 infected cells indicated that infection by noncytopathic BVDV2 altered the expression level of a bDnaJ1-hybridizing transcript. The increase in expression may represent a stress response to the infection by noncytopathic BVDV.

A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates.

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that stimulates gonadotropic hormone secretion upon interaction with its membrane receptors (type I) on pituitary cells, thereby governing reproductive processes. A second releasing hormone (GnRH II) expressed in mammals was shown earlier to be expressed in nonmammals and to have its own receptor. Here we demonstrate that a second receptor (type II) gene is present in the human genome, and report the cloning and characterization of its cDNA from monkeys. The cDNA encodes a G-protein-coupled/7 transmembrane receptor having a C-terminal cytoplasmic tail; it resembles more closely the type II receptors of amphibians and fish (approximately 55% identity) than it does the type I receptor of humans (approximately 39%). The GnRH type II receptor proved to be experimentally functional with, and specific for, GnRH II. GnRH receptor type II RNA is expressed ubiquitously in human tissues. This is the first report of a GnRH type II receptor in mammals. Its identification will permit exploration of its role in regulating gonadotropin secretion, female sexual behavior, and tumor cell growth.

Phylogenetic, antigenic and clinical characterization of type 2 BVDV from North America.

Bovine viral diarrhea virus (BVDV) infection continues to have a significant impact upon US cattle producers despite the availability of more than 140 federally licensed vaccines. Detection and control is hampered by viral heterogeneity that results in differences in neutralizing epitopes, cytopathology and virulence. Recently it was found that there are two different genotypes, BVDV1 and BVDV2, among BVDV. BVDV2 isolates make up a significant proportion of the BVDV isolated in North America. Serologically BVDV2 viruses can be distinguished from BVDV1 and border disease viruses. Mab binding also distinguishes between BVDV1, BVDV2 and BDV. Like the BVDV1 viruses, BVDV2 viruses may exist as one of two biotypes, cytopathic or noncytopathic, based on their activity in cultured cells. Cytopathogenic effects on cultured cells does not correlate with virulence in vivo, as BVDV2 associated with hemorrhagic syndrome (HS) are noncytopathic. Variation among BVDV1 and BVDV2 in the 5' UTR is similar. Phylogenetic analysis and differences in virulence suggest that BVDV2 are heterogeneous. Symptoms resulting from BVDV2 infections may range from clinically inapparent to clinically severe. Recently, disease outbreaks associated with acute uncomplicated BVDV infection have been reported in the US and Canada. These outbreaks of clinically severe disease, termed HS, were all associated with viruses from the BVDV2 genotype. Not all BVDV2 isolates cause clinically severe disease. Avirulent BVDV2 isolates do exist and may predominate over virulent BVDV2 in nature. When virulent BVDV2 viruses are inoculated into calves they induce a disease characterized by fever, diarrhea, leukopenia, lymphopenia, neutropenia, thrombocytopenia, and death. Infection with avirulent BVDV2 results in a reduction of luekocytes that may be accompanied by a low-grade fever. These viruses do not cause clinical disease or a clinical leukopenia.