Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are three of the most economically important swine pathogens, causing immense economic losses to the global swine industry. Monovalent commercial vaccines against each of the three viruses are routinely used in pig farms worldwide. A trivalent vaccine against all three pathogens would greatly simplify the vaccination programme and reduce the financial burden to the swine industry. In this study, by using an attenuated strain of PRRSV (strain DS722) as a live virus vector, we generated a multi-component vaccine virus, DS722-SIV-PCV2, which expresses the protective antigens from SIV and PCV2. The DS722-SIV-PCV2 trivalent vaccine virus replicates well, and expresses PCV2 capsid and SIV HA proteins in vitro. A subsequent vaccination and challenge study in 48 pigs revealed that the DS722-SIV-PCV2-vaccinated pigs had significantly reduced lung lesions and viral RNA loads when challenged with PRRSV. Upon challenge with PCV2, the vaccinated pigs had partially reduced lymphoid lesions and viral DNA loads, and when challenged with SIV the vaccinated pigs had significantly reduced acute respiratory sign scores. The results from this study demonstrate the potential of DS722-SIV-PCV2 as a candidate trivalent vaccine, and also shed light on exploring PRRSV as a potential live virus vaccine vector.

PB1-F2 Protein Does Not Impact the Virulence of Triple-Reassortant H3N2 Swine Influenza Virus in Pigs but Alters Pathogenicity and Transmission in Turkeys.

UNLABELLED: PB1-F2 protein, the 11th influenza A virus (IAV) protein, is considered to play an important role in primary influenza virus infection and postinfluenza secondary bacterial pneumonia in mice. The functional role of PB1-F2 has been reported to be a strain-specific and host-specific phenomenon. Its precise contribution to the pathogenicity and transmission of influenza virus in mammalian host, such as swine, and avian hosts, such as turkeys, remain largely unknown. In this study, we explored the role of PB1-F2 protein of triple-reassortant (TR) H3N2 swine influenza virus (SIV) in pigs and turkeys. Using the eight-plasmid reverse genetics system, we rescued wild-type SIV A/swine/Minnesota/1145/2007 (H3N2) (SIV 1145-WT), a PB1-F2 knockout mutant (SIV 1145-KO), and its N66S variant (SIV 1145-N66S). The ablation of PB1-F2 in SIV 1145 modulated early-stage apoptosis but did not affect the viral replication in swine alveolar macrophage cells. In pigs, PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology. On the other hand, in turkeys, SIV 1145-KO infected poults, and its in-contacts developed clinical signs earlier than SIV 1145-WT groups and also displayed more extensive histopathological changes in intestine. Further, turkeys infected with SIV 1145-N66S displayed poor infectivity and transmissibility. The more extensive histopathologic changes in intestine and relative transmission advantage observed in turkeys infected with SIV 1145-KO need to be further explored. Taken together, these results emphasize the host-specific roles of PB1-F2 in the pathogenicity and transmission of IAV. IMPORTANCE: Novel triple-reassortant H3N2 swine influenza virus emerged in 1998 and spread rapidly among the North American swine population. Subsequently, it showed an increased propensity to reassort, generating a range of reassortants. Unlike classical swine influenza virus, TR SIV produces a full-length PB1-F2 protein, which is considered an important virulence marker of IAV pathogenicity. Our study demonstrated that the expression of PB1-F2 does not impact the pathogenicity of TR H3N2 SIV in pigs. On the other hand, deletion of PB1-F2 caused TR H3N2 SIV to induce clinical disease early and resulted in effective transmission among the turkey poults. Our study emphasizes the continuing need to better understand the virulence determinants for IAV in intermediate hosts, such as swine and turkeys, and highlights the host-specific role of PB1-F2 protein.

Prostate-specific antigen-retargeted recombinant newcastle disease virus for prostate cancer virotherapy.

Oncolytic virus (OV) therapies of cancer are based on the use of replication-competent, tumor-selective viruses with limited toxicity. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising OV and is inherently tumor selective and cytotoxic only to tumor cells. Replication is restricted in normal cells. Despite encouraging phase I/II clinical trials with NDV, further refinements for tumor-specific targeting are needed to enhance its therapeutic index. Systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity, and the extracellular matrix. Overcoming these hurdles is paramount to realizing the exceptional oncolytic efficacy of NDV. We engineered the F protein of NDV and generated a recombinant NDV (rNDV) whose F protein is cleavable exclusively by prostate-specific antigen (PSA). The rNDV replicated efficiently and specifically in prostate cancer (CaP) cells and 3-dimensional prostaspheres but failed to replicate in the absence of PSA. Induction of intracellular PSA production by a synthetic androgen analog (R1881) enhanced fusogenicity in androgen-responsive CaP cells. Further, PSA-cleavable rNDV caused specific lysis of androgen-independent and androgen-responsive/nonresponsive CaP cells and prostaspheres, with a half-maximal effective concentration (EC50) ranging from a multiplicity of infection of 0.01 to 0.1. PSA-retargeted NDV efficiently lysed prostasphere tumor mimics, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating no pathogenicity for chickens. Prostate-specific antigen targeting is likely to enhance the therapeutic index of rNDV owing to tumor-restricted replication and enhanced fusogenicity.

Phase I/II Trial of Urokinase Plasminogen Activator-Targeted Oncolytic Newcastle Disease Virus for Canine Intracranial Tumors.

Neurotropic oncolytic viruses are appealing agents to treat brain tumors as they penetrate the blood-brain barrier and induce preferential cytolysis of neoplastic cells. The pathobiological similarities between human and canine brain tumors make immunocompetent dogs with naturally occurring tumors attractive models for the study of oncolytic virotherapies. In this dose-escalation/expansion study, an engineered Lasota NDV strain targeting the urokinase plasminogen activator system (rLAS-uPA) was administered by repetitive intravenous infusions to 20 dogs with intracranial tumors with the objectives of characterizing toxicities, immunologic responses, and neuroradiological anti-tumor effects of the virus for up to 6 months following treatment. Dose-limiting toxicities manifested as fever, hematologic, and neurological adverse events, and the maximum tolerated dose (MTD) of rLAS-uPA was 2 × 107 pfu/mL. Mild adverse events, including transient infusion reactions, diarrhea, and fever were observed in 16/18 of dogs treated at or below MTD. No infectious virus was recoverable from body fluids. Neutralizing antibodies to rLAS-uPA were present in all dogs by 2 weeks post-treatment, and viral genetic material was detected in post-treatment tumors from six dogs. Tumor volumetric reductions occurred in 2/11 dogs receiving the MTD. Systemically administered rLAS-uPA NDV was safe and induced anti-tumor effects in canine brain tumors, although modifications to evade host anti-viral immunity are needed to optimize this novel therapy.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Complete genome sequence and pathogenicity of two swine parainfluenzavirus 3 isolates from pigs in the United States.

Two novel paramyxoviruses, 81-19252 (Texas81) and 92-7783 (ISU92), isolated from the brains of pigs in the United States in the 1980s and 1990s, were characterized. The complete genome of Texas81 virus was 15,456 nucleotides (nt) in length, that of ISU92 was 15,480 nt, and both genomes consisted of six nonoverlapping genes, predicted to encode nine proteins, with conserved and complementary 3' leader and 5' trailer regions and conserved gene starts, gene stops, and trinucleotide intergenic sequences similar to those in paramyxoviruses. The corresponding genes from these two viruses were similar in length, except for the F genes, of which the ISU92 form had an additional 24-nt U-rich 3' untranslated region. The P genes of swine viruses were predicted to produce V and D mRNAs by RNA editing (one to four G insertions in Texas81 and one to nine G insertions in ISU92) or C mRNA by alternative translation initiation. Sequence-specific features related to virus replication and host-specific amino acid signatures indicated that these viruses originated from bovine parainfluenzavirus 3 (bPIV3). Phylogenetic analysis of individual genes suggested that these viruses are novel members of the genus Respirovirus of the Paramyxovirinae subfamily and may be grouped into two subgenotypes of genotype A of bPIV3. Our comprehensive studies revealed that these swine PIV3 are variants of bPIV3 and were possibly transferred from cattle to pigs but failed to establish an active enzootic state. These two viruses were mildly pathogenic to conventionally reared pigs, and results from a limited enzyme-linked immunosorbent assay-based serosurvey of swine farms in Minnesota and Iowa in 2007 and 2008 were negative.

Type I interferon-sensitive recombinant newcastle disease virus for oncolytic virotherapy.

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic because of its potent ability to induce apoptosis. Several studies have demonstrated that NDV is selectively cytotoxic to tumor cells but not normal cells due to defects in the interferon (IFN) antiviral responses of tumor cells. Many naturally occurring strains of NDV have an intact IFN-antagonistic function and can still replicate in normal human cells. To avoid potential toxicity issues with NDV, especially in cancer patients with immunosuppression, safe NDV-oncolytic vectors are needed. We compared the cell killing abilities of (i) a recombinant NDV (rNDV) strain, Beaudette C, containing an IFN-antagonistic, wild-type V protein (rBC), (ii) an isogenic recombinant virus with a mutant V protein (rBC-Edit virus) that induces increased IFN in infected cells and whose replication is restricted in normal human cells, and (iii) a recombinant LaSota virus with a virulent F protein cleavage site that is as interferon sensitive as rBC-Edit virus (LaSota V.F. virus). Our results indicated that the tumor-selective replication of rNDV is determined by the differential regulation of IFN-alpha and downstream antiviral genes induced by IFN-alpha, especially through the IRF-7 pathway. In a nude mouse model of human fibrosarcoma, we show that the IFN-sensitive NDV variants are as effective as IFN-resistant rBC virus in clearing the tumor burden. In addition, mice treated with rNDV exhibited no signs of toxicity to the viruses. These findings indicate that augmentation of innate immune responses by NDV results in selective oncolysis and offer a novel and safe virotherapy platform.

Genetic characterization of swine influenza viruses (H3N2) isolated from Minnesota in 2006-2007.

Triple-reassortant (TR) H3N2 swine influenza viruses (SIV) are a major cause of respiratory disease in swine worldwide, causing considerable morbidity and mortality. Continuous surveillance of circulating SIV strains is imperative for effective control and prediction of new emerging strains with interspecies transmission potential. The current study characterized SIV isolates from commercial swine population in USA (2006-2007). Nine isolates were completely sequenced, and the molecular evolution of all gene segments was analyzed. Phylogenetic analysis of the nine H3N2 viruses indicated that these strains belonged to cluster-IV of the human/swine/avian TR genotype, grouping with H3N2 viruses of turkey origin, while forming a separate sub-lineage from those of human and avian origin strains. Ten amino acid changes were observed at the major antigenic sites of HA1 region compared to the cluster-III reference strain, with differences in glycosylation sites. All the nine strains were antigenically related to the cluster-IV turkey strain than the cluster-III reference strain. The results of this study suggest that contemporary TR H3N2 strains circulating in North America share the same genetic constellation, thus maintaining the gene pool without any further event of genetic reassortment unlike swine-origin pandemic strain A/California/04/2009/H1N1. These findings strongly support the need for continuous surveillance and monitoring of genetic changes in SIV, to identify evolving strains that might pose a threat to human or animal health.

Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys.

Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.

Molecular characterization of glycoprotein genes and phylogenetic analysis of two swine paramyxoviruses isolated from United States.

Two swine paramyxoviruses (SPMV)-(81-19252 (Texas-81) and 92-7783 (ISU-92)-were isolated from encephalitic pigs in the United States in 1981 and 1992. Antigenic, morphologic, and biological characteristics of these two viruses were essentially similar to members of the family Paramyxoviridae. Antigenic analysis by indirect fluorescent antibody, immunoblot, and one-way cross-neutralization tests placed these viruses along with bovine parainfluenza 3 (BPIV3) viruses. Purified virions were 50-300 nm in size and morphologically indistinguishable from other paramyxoviruses. These two viruses hemagglutinated red blood cells and had neuraminidase activity. The gene junctions of fusion (F) and hemagglutinin (HN) glycoprotein genes of these viruses contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions similar to other Paramyxoviridae. The F gene of ISU-92 was longer than Texas-81 due to insertion of a 24-nucleotide "U"-rich 3' untranslated region. Structure-based sequence alignment of glycoproteins of these two SPMVs indicated that they are essentially similar in structure and function to parainfluenzaviruses. The Texas-81 strain was closely related to BPIV3 Shipping Fever (SF) strain at nucleotide and amino acid level, while the ISU-92 strain was more closely related to BPIV3 910N strain. The envelope glycoproteins of ISU-92 had only approximately 92 and approximately 96% identity at nucleotide and amino acid levels with BPIV3-SF strain, respectively. The high sequence identities to BPIV3 indicated cross-species infection in pigs. Phylogenetic analyses based on both F protein and HN protein suggested the classification of these viruses into the subfamily Paramyxovirinae, genus Respirovirus, and genotype A of BPIV3.

Evaluation of the pathogenicity of mammalian orthoreovirus type 3 (MRV3) in germ-free gnotobiotic pigs and of the efficacy of an inactivated vaccine against MRV3 infection in neonatal conventional piglets.

A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs.

Expression and activity of the urokinase plasminogen activator system in canine primary brain tumors.

BACKGROUND: The expression of the urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-anchored protein family member, and the activity of its ligand, urokinase-type plasminogen activator (uPA), have been associated with the invasive and metastatic potentials of a variety of human brain tumors through their regulation of extracellular matrix degradation. Domesticated dogs develop naturally occurring brain tumors that share many clinical, phenotypic, molecular, and genetic features with their human counterparts, which has prompted the use of the dogs with spontaneous brain tumors as models to expedite the translation of novel brain tumor therapeutics to humans. There is currently little known regarding the role of the uPA system in canine brain tumorigenesis. The objective of this study was to characterize the expression of uPAR and the activity of uPA in canine brain tumors as justification for the development of uPAR-targeted brain tumor therapeutics in dogs. METHODS: We investigated the expression of uPAR in 37 primary canine brain tumors using immunohistochemistry, Western blotting, real-time quantitative polymerase chain reaction analyses, and by the assay of the activity of uPA using casein-plasminogen zymography. RESULTS: Expression of uPAR was observed in multiple tumoral microenvironmental niches, including neoplastic cells, stroma, and the vasculature of canine brain tumors. Relative to normal brain tissues, uPAR protein and mRNA expression were significantly greater in canine meningiomas, gliomas, and choroid plexus tumors. Increased activity of uPA was documented in all tumor types. CONCLUSIONS: uPAR is overexpressed and uPA activity increased in canine meningiomas, gliomas, and choroid plexus tumors. This study illustrates the potential of uPAR/uPA molecularly targeted approaches for canine brain tumor therapeutics and reinforces the translational significance of canines with spontaneous brain tumors as models for human disease.

Environmental role in influenza virus outbreaks.

The environmental drivers of influenza outbreaks are largely unknown. Despite more than 50 years of research, there are conflicting lines of evidence on the role of the environment in influenza A virus (IAV) survival, stability, and transmissibility. With the increasing and looming threat of pandemic influenza, it is important to understand these factors for early intervention and long-term control strategies. The factors that dictate the severity and spread of influenza would include the virus, natural and acquired hosts, virus-host interactions, environmental persistence, virus stability and transmissibility, and anthropogenic interventions. Virus persistence in different environments is subject to minor variations in temperature, humidity, pH, salinity, air pollution, and solar radiations. Seasonality of influenza is largely dictated by temperature and humidity, with cool-dry conditions enhancing IAV survival and transmissibility in temperate climates in high latitudes, whereas humid-rainy conditions favor outbreaks in low latitudes, as seen in tropical and subtropical zones. In mid-latitudes, semiannual outbreaks result from alternating cool-dry and humid-rainy conditions. The mechanism of virus survival in the cool-dry or humid-rainy conditions is largely determined by the presence of salts and proteins in the respiratory droplets. Social determinants of heath, including health equity, vaccine acceptance, and age-related illness, may play a role in influenza occurrence and spread.

A novel pathogenic Mammalian orthoreovirus from diarrheic pigs and Swine blood meal in the United States.

UNLABELLED: Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE: Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.

Pandemic H1N1 influenza A virus induces a potent innate immune response in human chorionic cells.

To understand the mechanistic basis for the reported outcomes of influenza A virus (IAV) infection during pregnancy, the effects of mouse adapted and pandemic (pdm) IAV infection in human choriocarcinoma cells were examined. Both viruses were able to infect and replicate in human placental cells, with pdm IAV being more apoptotic. A strong induction of innate signaling molecules, type I interferon and pro-inflammatory cytokine production, were associated with pdm IAV infection of human placental cells, with implications for adverse immediate and late outcomes during pregnancy.

Toxicity of engineered nanomaterials and their transformation products following wastewater treatment on A549 human lung epithelial cells.

Here we characterize the toxicity of environmentally-relevant forms of engineered nanomaterials (ENMs), which can transform during wastewater treatment and persist in aqueous effluents and biosolids. In an aerosol exposure scenario, cytotoxicity and genotoxicity of effluents and biosolids from lab-scale sequencing batch reactors (SBRs) to A549 human lung epithelial cells were examined. The SBRs were dosed with nanoAg, nano zero-valent iron (NZVI), nanoTiO2 and nanoCeO2 at sequentially increasing concentrations from 0.1 to 20 mg/L. Toxicities were compared to outputs from SBRs dosed with ionic/bulk analogs, undosed SBRs, and pristine ENMs. Pristine nanoAg and NZVI showed significant cytotoxicity to A549 cells in a dose-dependent manner from 1 to 67 µg/mL, while nanoTiO2 and nanoCeO2 only exerted cytotoxicity at 67 µg/mL. Only nanoAg induced a genotoxic response, at 9, 33 and 53 µg/mL. However, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed.

Complete genome sequence of a velogenic newcastle disease virus isolated from an apparently healthy village chicken in South India.

We report the complete genome sequence of a Newcastle disease virus (NDV) isolate, NDV-D1/1998, from an apparently healthy village chicken in South India. This class II, genotype II virus is 15,186 nucleotides in length with unique amino acid variations and was found to be a velogenic pathotype by standard pathogenicity tests.

TGF-ß-induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops.

The process of epithelial-to-mesenchymal transition (EMT) is an essential type of cellular plasticity associated with a change from epithelial cells that function as a barrier consisting of a sheet of tightly connected cells to cells with properties of mesenchyme that are not attached to their neighbors and are highly motile. This phenotypic change occurs during development and also contributes to pathological processes, such as cancer progression. The molecular mechanisms controlling the switch between the fully epithelial and fully mesenchymal phenotypes and cells that have characteristics of both (partial EMT) are controversial, and multiple theoretical models have been proposed. To test these theoretical models, we systematically measured the changes in the abundance of proteins, mRNAs, and microRNAs (miRNAs) that represent the core regulators of EMT induced by transforming growth factor-ß1 (TGF-ß1) in the human breast epithelial cell line MCF10A at the population and single-cell levels. We provide experimental confirmation for a model of cascading switches in phenotypes associated with TGF-ß1-induced EMT of MCF10A cells that involves two double-negative feedback loops: one between the transcription factor SNAIL1 and the miR-34 family and another between the transcription factor ZEB1 and the miR-200 family. Furthermore, our data showed that whereas the transition from epithelial to partial EMT was reversible for MCF10A cells, the transition from partial EMT to mesenchymal was mostly irreversible at high concentrations of TGF-ß1.

Toll-like receptor responses to Peste des petits ruminants virus in goats and water buffalo.

Ovine rinderpest or goat plague is an economically important and contagious viral disease of sheep and goats, caused by the Peste des petits ruminants virus (PPRV). Differences in susceptibility to goat plague among different breeds and water buffalo exist. The host innate immune system discriminates between pathogen associated molecular patterns and self antigens through surveillance receptors known as Toll like receptors (TLR). We investigated the role of TLR and cytokines in differential susceptibility of goat breeds and water buffalo to PPRV. We examined the replication of PPRV in peripheral blood mononuclear cells (PBMC) of Indian domestic goats and water buffalo and demonstrated that the levels of TLR3 and TLR7 and downstream signalling molecules correlation with susceptibility vs resistance. Naturally susceptible goat breeds, Barbari and Tellichery, had dampened innate immune responses to PPRV and increased viral loads with lower basal expression levels of TLR 3/7. Upon stimulation of PBMC with synthetic TLR3 and TLR7 agonists or PPRV, the levels of proinflammatory cytokines were found to be significantly higher while immunosuppressive interleukin (IL) 10 levels were lower in PPRV resistant Kanni and Salem Black breeds and water buffalo at transcriptional level, correlating with reduced viralloads in infected PBMC. Water buffalo produced higher levels of interferon (IFN) α in comparison with goats at transcriptional and translational levels. Pre-treatment of Vero cells with human IFNα resulted in reduction of PPRV replication, confirming the role of IFNα in limiting PPRV replication. Treatment with IRS66, a TLR7 antagonist, resulted in the reduction of IFNα levels, with increased PPRV replication confirming the role of TLR7. Single nucleotide polymorphism analysis of TLR7 of these goat breeds did not show any marked nucleotide differences that might account for susceptibility vs resistance to PPRV. Analyzing other host genetic factors might provide further insights on susceptibility to PPRV and genetic polymorphisms in the host.

The mechanistic influence of aligned nanofibers on cell shape, migration and blebbing dynamics of glioma cells.

Investigating the mechanistic influence of the tumor microenvironment on cancer cell migration and membrane blebbing is crucial in the understanding and eventual arrest of cancer metastasis. In this study, we investigate the effect of suspended and aligned nanofibers on the glioma cytoskeleton, cell shape, migration and plasma membrane blebbing dynamics using a non-electrospinning fiber-manufacturing platform. Cells attached in repeatable shapes of spindle on single fibers, rectangular on two parallel fibers and polygonal on intersecting fibers. Structural stiffness (N m(-1)) of aligned and suspended nanofibers (average diameter: 400 nm, length: 4, 6, and 10 mm) was found to significantly alter the migration speed with higher migration on lower stiffness fibers. For cells attached to fibers and exhibiting blebbing, an increase in cellular spread area resulted in both reduced bleb count and bleb size with an overall increase in cell migration speed. Blebs no longer appeared past a critical cellular spread area of approximately 1400 µm(2). Our results highlighting the influence of the mechanistic environment on the invasion dynamics of glioma cells add to the understanding of how biophysical components influence glioma cell migration and blebbing dynamics.