Lactate Dehydrogenase-elevating Virus Research Articles

Lactate Dehydrogenase-Elevating Virus Infection Inhibits MOG Peptide Presentation by CD11b+CD11c+ Dendritic Cells in a Mouse Model of Multiple Sclerosis.

Infections may affect the course of autoimmune inflammatory diseases of the central nervous system (CNS), such as multiple sclerosis (MS). Infections with lactate dehydrogenase-elevating virus (LDV) protected mice from developing experimental autoimmune encephalomyelitis (EAE), a mouse counterpart of MS. Uninfected C57BL/6 mice immunized with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) experienced paralysis and lost weight at a greater rate than mice who had previously been infected with LDV. LDV infection decreased the presentation of the MOG peptide by CD11b+CD11c+ dendritic cells (DC) to pathogenic T lymphocytes. When comparing non-infected mice to infected mice, the histopathological examination of the CNS showed more areas of demyelination and CD45+ and CD3+, but not Iba1+ cell infiltration. These results suggest that the protective effect of LDV infection against EAE development is mediated by a suppression of myelin antigen presentation by a specific DC subset to autoreactive T lymphocytes. Such a mechanism might contribute to the general suppressive effect of infections on autoimmune diseases known as the hygiene hypothesis.

Suppression of TRIM19 by arterivirus nonstructural protein 1 promotes viral replication

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

Comparative analysis of newly identified rodent arteriviruses and porcine reproductive and respiratory syndrome virus to characterize their evolutionary relationships.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses for the global pig industry, but its origins and evolution remain a mystery. In 2018, the genome sequences of seven arteriviruses isolated from rodents were determined, and here we publish new analysis showing that they may be ancestors of PRRSV. The sequence similarity of these viruses to PRRSV was ~60%, with shared genome organization and other characteristics, such as slippery sequences and C-rich motifs in nsp2, and a transactivated protein sequence in nsp1β. Codon usage basis analysis showed that PRRSV was closer to these rodent arteriviruses than lactate dehydrogenase-elevating virus (LDV) and they were both under pressure of natural selection. Evolutionary analysis revealed that four of the rodent arteriviruses shared the same genus with PRRSV, and were more closely related to PRRSV-2 than PRRSV-1. In addition to this, they all appeared earlier than PRRSV according to evolutionary modeling, and we speculate that they represent an intermediate step in the origin of PRRSV by arterivirus transmission from rodents to swine. Our in-depth analysis furthers our understanding of arteriviruses, and will serve as the basis for subsequent exploration of the evolution of PRRSV and other arteriviruses.

Degenerative Myelopathy and Neuropathy in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Mice Caused by Lactate Dehydrogenase-Elevating Virus (LDV).

Following implantation of patient-derived xenograft (PDX) breast carcinomas from three separate individuals, 33/51 female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice presented with progressive, unilateral to bilateral, ascending hindlimb paresis to paralysis. Mice were mildly dehydrated, in thin to poor body condition, with reduced to absent hindlimb withdrawal reflex and deep pain sensation. Microscopically, there was variable axonal swelling, vacuolation, and dilation of myelin sheaths within the ventral spinal cord and spinal nerve roots of the thoracolumbar and sacral spinal cord, as well as within corresponding sciatic nerves. Results of PCR screening of PDX samples obtained at necropsy and pooled environmental swabs from the racks housing affected animals were positive for lactate dehydrogenase-elevating virus (LDV). LDV is transmitted through animal-animal contact or commonly as a contaminant of biologic materials of mouse origin. Infection is associated with progressive degenerative myelopathy and neuropathy in strains of mice harboring endogenous retrovirus (AKR, C58), or in immunosuppressed strains (NOD-SCID, Foxn1nu), and can interfere with normal immune responses and alter engraftment and growth of xenograft tumors in immunosuppressed mice. This is the first reported series of LDV-induced poliomyelitis in NSG mice and should be recognized as a potentially significant confounder to biomedical studies utilizing immunodeficient xenograft models.

Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

Lactate dehydrogenase-elevating virus enhances natural killer cell-mediated immunosurveillance of mouse mesothelioma development

BackgroundViral infections can reduce early cancer development through enhancement of cancer immunosurveillance. This study was performed to analyse this effect of viral infection in a mouse model of solid tumor.MethodsThe experimental model used was the effect of BALB/c mouse infection by lactate dehydrogenase-elevating virus on AB1 mesothelioma cancer development.ResultsAcute infection with lactate dehydrogenase-elevating virus strongly reduced in vivo early AB1 mesothelioma growth and death resulting from cancer development. This effect was not due to a direct cytolytic effect of the virus on AB1 cells, but to an in vivo activation of natural killer cells. Gamma-interferon production rather than cytotoxic activity against AB1 cells mediated this protective effect. This gamma-interferon production by natural killer cells was dependent on interleukin-12 production.ConclusionsTogether with other reported effects of infectious agents on cancer development, this observation may support the hypothesis that enhancement of innate immunosurveillance against tumors may result from infection with common infectious agents through modulation of the host immune microenvironment.

Detection of Lactate Dehydrogenase Elevating Virus in a Mouse Vivarium Using an Exhaust Air Dust Health Monitoring Program.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.

Nuclear localization signal in TRIM22 is essential for inhibition of type 2 porcine reproductive and respiratory syndrome virus replication in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes one of the most economically important swine diseases worldwide. Tripartite motif-containing 22 (TRIM22), a TRIM family protein, has been identified as a crucial restriction factor that inhibits a group of human viruses. Currently, the role of cellular TRIM22 in PRRSV infection remains unclear. In the present study, we analyzed the effect of TRIM22 on PRRSV replication in vitro and explored the underlying mechanism. Ectopic expression of TRIM22 impaired the viral replication, while TRIM22-RNAi favored the replication of PRRSV in MARC-145 cells. Additionally, we observed that TRIM22 deletion SPRY domain or Nuclear localization signal (NLS) losses the ability to inhibit PRRSV replication. Finally, Co-IP analysis identified that TRIM22 interacts with PRRSV nucleocapsid (N) protein through the SPRY domain, while the NLS2 motif of N protein is involved in interaction with TRIM22. Although the concentration of PRRSV N protein was not altered in the presence of TRIM22, the abundance of N proteins from simian hemorrhagic fever virus (SHFV), equine arteritis virus (EAV), and murine lactate dehydrogenase-elevating virus (LDV) diminished considerably with increasing TRIM22 expression. Together, our findings uncover a previously unrecognized role for TRIM22 and extend the antiviral effects of TRIM22 to arteriviruses.

The TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-interleukin-27 antibody for maximal protection and regulatory T-cell upregulation.

In spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested a pharmacological TLR7 ligand, R848, in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T-cell allo-responsiveness and antigen presentation by CD11b+ and CD8α+ dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T-cell implantation and activation. However, it decreased interferon-gamma, tumor necrosis factor-alpha and interleukin-27 while upregulating active transforming growth factor-beta 1 plasma levels. In addition, donor and recipient Foxp3+ regulatory T-cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3+ regulatory T cells that could be further increased by inhibition of interleukin-27. The combination of anti-interleukin-27p28 mono -clonal antibody and R848 showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with upregulation of regulatory T cells. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by inhibition of interleukin-27.

Identification of the RNA Pseudoknot within the 3' End of the Porcine Reproductive and Respiratory Syndrome Virus Genome as a Pathogen-Associated Molecular Pattern To Activate Antiviral Signaling via RIG-I and Toll-Like Receptor 3.

Once infected by viruses, cells can detect pathogen-associated molecular patterns (PAMPs) on viral nucleic acid by host pattern recognition receptors (PRRs) to initiate the antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory diseases in pigs of different ages. To date, the sensing mechanism of PRRSV has not been elucidated. Here, we reported that the pseudoknot region residing in the 3' untranslated regions (UTR) of the PRRSV genome, which has been proposed to regulate RNA synthesis and virus replication, was sensed as nonself by retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3) and strongly induced type I interferons (IFNs) and interferon-stimulated genes (ISGs) in porcine alveolar macrophages (PAMs). The interaction between the two stem-loops inside the pseudoknot structure was sufficient for IFN induction, since disruption of the pseudoknot interaction powerfully dampened the IFN induction. Furthermore, transfection of the 3' UTR pseudoknot transcripts in PAMs inhibited PRRSV replication in vitro Importantly, the predicted similar structures of other arterivirus members, including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV), also displayed strong IFN induction activities. Together, in this work we identified an innate recognition mechanism by which the PRRSV 3' UTR pseudoknot region served as PAMPs of arteriviruses and activated innate immune signaling to produce IFNs that inhibit virus replication. All of these results provide novel insights into innate immune recognition during virus infection.IMPORTANCE PRRS is the most common viral disease in the pork industry. It is caused by PRRSV, a positive single-stranded RNA virus, whose infection often leads to persistent infection. To date, it is not yet clear how PRRSV is recognized by the host and what is the exact mechanism of IFN induction. Here, we investigated the nature of PAMPs on PRRSV and the associated PRRs. We found that the 3' UTR pseudoknot region of PRRSV, which has been proposed to regulate viral RNA synthesis, could act as PAMPs recognized by RIG-I and TLR3 to induce type I IFN production to suppress PRRSV infection. This report is the first detailed description of pattern recognition for PRRSV, which is important in understanding the antiviral response of arteriviruses, especially PRRSV, and extends our knowledge on virus recognition.

Differences in signal peptide processing between GP3 glycoproteins of Arteriviridae

We reported previously that carbohydrate attachment to an overlapping glycosylation site adjacent to the signal peptide of GP3 from equine arteritis virus (EAV) prevents cleavage. Here we investigated whether this unusual processing scheme is a feature of GP3s of other Arteriviridae, which all contain a glycosylation site at a similar position. Expression of GP3 from type-1 and type-2 porcine reproductive and respiratory syndrome virus (PRRSV) and from lactate dehydrogenase-elevating virus (LDV) revealed that the first glycosylation site is used, but has no effect on signal peptide cleavage. Comparison of the SDS-PAGE mobility of deglycosylated GP3 from PRRSV and LDV with mutants having or not having a signal peptide showed that GP3´s signal peptide is cleaved. Swapping the signal peptides between GP3 of EAV and PRRSV revealed that the information for co-translational processing is not encoded in the signal peptide, but in the remaining part of GP3.

Mouse nidovirus LDV infection alleviates graft versus host disease and induces type I IFN-dependent inhibition of dendritic cells and allo-responsive T cells.

IntroductionViruses have developed multiple mechanisms to alter immune reactions. In 1969, it was reported that lactate dehydrogenase‐elevating virus (LDV), a single stranded positive sense mouse nidovirus, delays skin allograft rejection and inhibits spleen alterations in graft versus host disease (GVHD). As the underlying mechanisms have remained unresolved and given the need for new therapies of this disease, we reassessed the effects of the virus on GVHD and tried to uncover its mode of action.MethodsGVHD was induced by transfer of parent (B6) spleen cells to non‐infected or LDV‐infected B6D2F1 recipients. In vitro mixed‐lymhocyte culture (MLC) reactions were used to test the effects of the virus on antigen‐presenting cells (APC) and responder T cells.ResultsLDV infection resulted in a threefold increase in survival rate with reduced weight loss and liver inflammation but with the establishment of permanent chimerism that correlated with decreased interleukine (IL)‐27 and interferon (IFN)γ plasma levels. Infected mice showed a transient elimination of splenic CD11b+ and CD8α+ conventional dendritic cells (cDCs) required for allogeneic CD4 and CD8 T cell responses in vitro. This drop of APC numbers was not observed with APCs derived from toll‐like receptor (TLR)7‐deficient mice. A second effect of the virus was a decreased T cell proliferation and IFNγ production during MLC without detectable changes in Foxp3+ regulatory T cell (Tregs) numbers. Both cDC and responder T cell inhibition were type I IFN dependent. Although the suppressive effects were very transient, the GVHD inhibition was long‐lasting.ConclusionA type I IFN‐dependent suppression of DC and T cells just after donor spleen cell transplantation induces permanent chimerism and donor cell implantation in a parent to F1 spleen cell transplantation model. If this procedure can be extended to full allogeneic bone marrow transplantation, it could open new therapeutic perspectives for hematopoietic stem cell transplantation (HSCT).

Domain Organization and Evolution of the Highly Divergent 5' Coding Region of Genomes of Arteriviruses, Including the Novel Possum Nidovirus.

In five experimentally characterized arterivirus species, the 5'-end genome coding region encodes the most divergent nonstructural proteins (nsp's), nsp1 and nsp2, which include papain-like proteases (PLPs) and other poorly characterized domains. These are involved in regulation of transcription, polyprotein processing, and virus-host interaction. Here we present results of a bioinformatics analysis of this region of 14 arterivirus species, including that of the most distantly related virus, wobbly possum disease virus (WPDV), determined by a modified 5' rapid amplification of cDNA ends (RACE) protocol. By combining profile-profile comparisons and phylogeny reconstruction, we identified an association of the four distinct domain layouts of nsp1-nsp2 with major phylogenetic lineages, implicating domain gain, including duplication, and loss in the early nsp1 evolution. Specifically, WPDV encodes highly divergent homologs of PLP1a, PLP1b, PLP1c, and PLP2, with PLP1a lacking the catalytic Cys residue, but does not encode nsp1 Zn finger (ZnF) and "nuclease" domains, which are conserved in other arteriviruses. Unexpectedly, our analysis revealed that the only catalytically active nsp1 PLP of equine arteritis virus (EAV), known as PLP1b, is most similar to PLP1c and thus is likely to be a PLP1b paralog. In all non-WPDV arteriviruses, PLP1b/c and PLP1a show contrasting patterns of conservation, with the N- and C-terminal subdomains, respectively, being enriched with conserved residues, which is indicative of different functional specializations. The least conserved domain of nsp2, the hypervariable region (HVR), has its size varied 5-fold and includes up to four copies of a novel PxPxPR motif that is potentially recognized by SH3 domain-containing proteins. Apparently, only EAV lacks the signal that directs -2 ribosomal frameshifting in the nsp2 coding region.IMPORTANCE Arteriviruses comprise a family of mammalian enveloped positive-strand RNA viruses that include some of the most economically important pathogens of swine. Most of our knowledge about this family has been obtained through characterization of viruses from five species: Equine arteritis virus, Simian hemorrhagic fever virus, Lactate dehydrogenase-elevating virus, Porcine respiratory and reproductive syndrome virus 1, and Porcine respiratory and reproductive syndrome virus 2 Here we present the results of comparative genomics analyses of viruses from all known 14 arterivirus species, including the most distantly related virus, WPDV, whose genome sequence was completed in this study. Our analysis focused on the multifunctional 5'-end genome coding region that encodes multidomain nonstructural proteins 1 and 2. Using diverse bioinformatics techniques, we identified many patterns of evolutionary conservation that are specific to members of distinct arterivirus species, both characterized and novel, or their groups. They are likely associated with structural and functional determinants important for virus replication and virus-host interaction.

Energy parasites trigger oncogene mutation

Purpose: Cancer initialization can be explained as a result of parasitic virus energy consumption leading to randomized genome chemical bonding.Materials and methods: Analysis of experimental data on cell-mediated immunity (CMI) containing about 12,000 cases of healthy humans, cancer patients and patients with precancerous cervical lesions disclosed that the specific cancer and the non-specific lactate dehydrogenase-elevating (LDH) virus antigen elicit similar responses. The specific antigen is effective only in cancer type of its origin but the non-specific antigen in all examined cancers. CMI results of CIN patients display both healthy and cancer state. The ribonucleic acid (RNA) of the LDH virus parasitizing on energy reduces the ratio of coherent/random oscillations. Decreased effect of coherent cellular electromagnetic field on bonding electrons in biological macromolecules leads to elevating probability of random genome reactions.Results: Overlapping of wave functions in biological macromolecules depends on energy of the cellular electromagnetic field which supplies energy to bonding electrons for selective chemical bonds. CMI responses of cancer and LDH virus antigens in all examined healthy, precancerous and cancer cases point to energy mechanism in cancer initiation.Conclusions: Dependence of the rate of biochemical reactions on biological electromagnetic field explains yet unknown mechanism of genome mutation.

Abstract 1469: The inflammatory pre-metastatic niche in reliably metastatic models of triple negative breast cancer

Abstract The purpose of this study was to evaluate Triple Negative Breast Cancer (TNBC) murine models to use for investigations of the pre-metastatic niche. We selected two murine TNBC based on published gene expression studies which demonstrated molecular profiles that mirrored human breast tumor subtypes. The 2225L murine tumor has similar gene expression patterns to human tumors of the basal-like phenotype. When 2225L was implanted in the mammary fat pad or sc flank of syngeneic naïve Balb/c female mice, no metastases were observed. Tumors were subsequently resected at approximately 600-800 mm3 to promote the growth of seeded metastases. To select for a consistently metastatic 2225L variant, primary tumors or lung metastases from the most consistently metastatic 2225L tumors were serially passaged sc. The resulting variant (2225LM) is highly metastatic, with lung metastases in 90% (43/48) of mice from the most recent 4 cohorts compared to 27% (47/176) of mice in 66 previous passages. Metastases were not observed at any other site. We also studied the T11 murine tumor, which is representative of the claudin-low phenotype. However, when tested for mouse pathogens prior to implantation, T11 was positive for lactate dehydrogenase elevating virus (LDEV). To eliminate LDEV, T11 tumors were serially passaged into athymic nude rats. The resulting LDEV-free T11 tumor was used for in vivo studies. After sc implantation of T11 in Balb/c mice, consistent lung metastases were observed (89%; 24/27 mice). Tumor-infiltrating immune cell subsets were assessed using flow cytometry. CD11b+ Ly6G- and CD11b+ Ly6G+ cells comprised the majority of tumor-infiltrating immune cells and both subsets were more prevalent in 2225LM than T11. Infiltrating lymphoid (CD3+) cells were a minor component of both tumors. In preliminary studies to quantify lung-infiltrating immune cells, levels of CD11b+ Ly6G+ cells were 1.5 fold higher in 2225LM and T11 tumor-bearing mice compared to controls. Prior to the development of frank metastases (12-30 days post-2225L implant), lung analysis by ELISA showed a steady increase in the neutrophil chemoattractant KC and the pro-tumor M2/N2 chemokine marker MCP-1. Analysis of lungs with visible 2225LM metastases detected increased levels of KC (6-27 fold) and MCP-1 (11-23 fold), when compared to naïve mice. These chemokines were not elevated at non-metastatic sites (i.e. kidney). To conclude, our lab has selected, developed and characterized two TNBC mouse models. Both tumor cell lines are reliable metastatic models of TNBC and will be used to further characterize the inflammatory microenvironment of the pre-metastatic niche, including the continuum of gene expression patterns and changing cellular components. Citation Format: Kassondra Balestrieri, H Keith Pittman, Matthew Britt, Mohamed Ramez, Nasreen Vohra, Kathryn Verbanac. The inflammatory pre-metastatic niche in reliably metastatic models of triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1469.

Novel highly specific anti-periostin antibodies uncover the functional importance of the fascilin 1-1 domain and highlight preferential expression of periostin in aggressive breast cancer.

Periostin (POSTN), a secreted homodimeric protein that binds integrins αvβ3, αvβ5, and α6β4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over-expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop six mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvβ3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN-induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136-51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1-1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136-151 peptide to inhibit integrin-mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status.

Reorganization and expansion of the nidoviral family Arteriviridae.

The family Arteriviridae presently includes a single genus Arterivirus. This genus includes four species as the taxonomic homes for equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), porcine respiratory and reproductive syndrome virus (PRRSV), and simian hemorrhagic fever virus (SHFV), respectively. A revision of this classification is urgently needed to accommodate the recent description of eleven highly divergent simian arteriviruses in diverse African nonhuman primates, one novel arterivirus in an African forest giant pouched rat, and a novel arterivirus in common brushtails in New Zealand. In addition, the current arterivirus nomenclature is not in accordance with the most recent version of the International Code of Virus Classification and Nomenclature. Here we outline an updated, amended, and improved arterivirus taxonomy based on current data. Taxon-specific sequence cut-offs are established relying on a newly established open reading frame 1b phylogeny and pairwise sequence comparison (PASC) of coding-complete arterivirus genomes. As a result, the current genus Arterivirus is replaced by five genera: Equartevirus (for EAV), Rodartevirus (LDV + PRRSV), Simartevirus (SHFV + simian arteriviruses), Nesartevirus (for the arterivirus from forest giant pouched rats), and Dipartevirus (common brushtail arterivirus). The current species Porcine reproductive and respiratory syndrome virus is divided into two species to accommodate the clear divergence of the European and American “types” of PRRSV, both of which now receive virus status. The current species Simian hemorrhagic fever virus is divided into nine species to accommodate the twelve known simian arteriviruses. Non-Latinized binomial species names are introduced to replace all current species names to clearly differentiate them from virus names, which remain largely unchanged.

Involvement of Fcα/μ Receptor in IgM Anti-Platelet, but Not Anti-Red Blood Cell Autoantibody Pathogenicity in Mice.

IgM anti-mouse platelet autoantibodies cause thrombocytopenia by mediating uptake of opsonized thrombocytes, whereas IgM anti-erythrocyte autoantibodies induce anemia through a phagocytosis-independent cell destruction. In this article, we show that infection with lactate dehydrogenase-elevating virus, a benign mouse arterivirus, exacerbates the pathogenicity of IgM anti-platelet, but not anti-erythrocyte autoantibodies. To define the role of Fcα/μ receptor (Fcα/μR) in IgM-mediated thrombocytopenia and anemia, we generated mice deficient for this receptor. These animals were resistant to IgM autoantibody-mediated thrombocytopenia, but not anemia. However, the lactate dehydrogenase-elevating virus-induced exacerbation of thrombocytopenia was not associated with enhanced Fcα/μR expression on macrophages. These results indicate that Fcα/μR is required for the pathogenicity of IgM anti-platelet autoantibodies but is not sufficient to explain the full extent of the disease in virally infected animals.

ORF5a Protein of Porcine Reproductive and Respiratory Syndrome Virus is Indispensable for Virus Replication

Porcine reproductive and respiratory syndrome (PRRS) was first recognized in 1987 in the United States and shortly thereafter in Europe [12, 35]. The disease has since continued to plague nearly all pig-producing countries, causing severe economic losses in the global swine industry [1, 26]. The etiological agent of PRRS, the PRRS virus (PRRSV), was isolated almost simultaneously in Europe and North America in the early 1990s [3, 6, 36]. PRRSV is a member of the family Arteriviridae including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus of mice, and simian hemorrhagic fever virus, which forms the order Nidovirales along with the Coronaviridae family [5, 19, 31]. Since the emergence, PRRSV has evolved divergently on the two continents and consequently, consists of two major genotypes, European (type 1) and North American (type 2) [9, 10, 23, 28]. The two genotypes exhibits antigenic and genetic variations, sharing only about 60% sequence identity at the genome level [24, 18]. PRRSV is a small enveloped virus with a singlestranded, positive-sense RNA genome of ~15 kb in size. The PRRSV genome possesses the 5' cap structure and 3' polyadenylated tail and constitutes the 5' untranslated region (UTR), ten open reading frames (ORF1a, ORF1b, ORF2a, ORF2b, and ORFs 3 through 7 including ORF5a), and the 3' UTR [8, 11, 22, 30, 36]. The two large ORF1a and 1b occupying the 5' two-third of the genome encode the ORF1a and ORF1ab polyproteins by a ribosome frameshifting mechanism that are translated directly from the genomic RNA. The polyproteins are then autocleaved into 14 protease and replicase-associated nonstructural proteins In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7 codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.