Antiangiogenic Vascular Endothelial Growth Factor-Blocking Peptides Displayed on the Capsid of an Infectious Oncolytic Parvovirus: Assembly and Immune Interactions.

As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies / ELISA indirecto (iELISA) para la detección de rutina de anticuerpos contra el virus diminuto del ratón (MVM) en colonias de ratones

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.

Se desarrolló un ELISA indirecto para detectar anticuerpos contra el virus diminuto del ratón (Mice minute virus -.#91;MVM-.#93;), utilizando un antígeno producido a partir de células BHK-21 infectadas con la cepa prototipo del virus. Se establecieron las diluciones óptimas de antígeno y el suero a utilizar. Para analizar la variabilidad en el laboratorio, se determinaron la reproducibilidad y la repetibilidad dentro de una placa y entre placas. Luego se analizaron 460 sueros provenientes de bioterios convencionales y clasificados previamente como positivos o negativos por inmunofluorescencia indirecta. El valor de corte se determinó mediante una curva ROC. Los resultados se compararon con los obtenidos con la prueba de inmunofluorescencia indirecta. El ELISA mostró 100% de sensibilidad y un 99% de especificidad. Esta técnica demostró ser una herramienta útil para desarrollar en laboratorios de virología estándar y puede utilizarse como prueba tamiz para seleccionar animales de manera más rápida que con la tradicional prueba de inmunofluorescencia indirecta.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies.

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN® GS-/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-ß-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc.

In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models.

UNLABELLED: APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo IMPORTANCE: It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.

Induction of an embryonic mouse innate immune response following inoculation in utero with minute virus of mice.

UNLABELLED: We used an embryonic-infection model system to show that MVMp, the prototypic minute virus of mice (MVM) serotype and a member of the genus Protoparvovirus, triggers a comprehensive innate immune response in the developing mouse embryo. Direct inoculation of the midtrimester embryo in utero with MVMp results in a widespread, productive infection. During a 96-h infection course, embryonic beta interferon (IFN-ß) and IFN-γ transcription were induced 90- and 60-fold, respectively. IFN-ß levels correlated with the embryo viral burden, while IFN-γ levels first increased and then decreased. Production of proinflammatory cytokines, interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α), also increased, but by smaller amounts, approximately 7-fold each. We observed increased levels of downstream antiviral effector molecules, PKR and phosphorylated STAT2. Finally, we showed that there is an immune cell response to the virus infection. Infected tissues in the embryo exhibited an increased density of mature leukocytes compared to the same tissues in uninfected embryos. The responses we observed were almost completely restricted to the infected embryos. Uninfected littermates routinely exhibited small increases in innate immune components that rarely reached statistical significance compared to negative controls. Similarly, the placentae of infected embryos did not show any significant increase in transcription of innate immune cytokines. Since the placenta has both embryonic and maternal components, we suggest there is minimal involvement of the dam in the response to infection. IMPORTANCE: Interaction between the small single-stranded vertebrate DNA viruses, the protoparvoviruses, and the host innate immune system has been unclear. The issue is important practically given the potential use of these viruses as oncotherapeutic agents. The data reported here stand in contrast to studies of innate immune response during protoparvovirus infection of adult hosts, which invariably reported no or minimal and sporadic induction of an interferon response during infection. We conclude that under conditions of robust and productive MVM infection, a normal murine host is able to mount a significant and broad innate immune response.

Parvovirus evades interferon-dependent viral control in primary mouse embryonic fibroblasts.

Engagement of innate viral sensors elicits a robust antiviral program via the induction of type I interferons (IFNs). Innate defense mechanisms against ssDNA viruses are not well defined. Here, we examine type I IFN induction and effectiveness in controlling a ssDNA virus. Using mouse embryonic fibroblasts (MEFs), we found that a murine parvovirus, minute virus of mice (MVMp), induced a delayed but significant IFN response. MEFs deficient in mitochondrial antiviral signaling protein (MAVS) mounted a wild-type IFN response to MVMp infection, indicating that RIG-I-dependent RNA intermediate recognition is not required for innate sensing of this virus. However, MVMp-induced IFNs, as well recombinant type I IFNs, were unable to inhibit viral replication. Finally, MVMp infected cells became unresponsive to Poly (I:C) stimulation. Together, these data suggest that the MVMp efficiently evades antiviral immune mechanisms imposed by type I IFNs, which may in part explain their efficient transmission between mice.

Activation of a glioma-specific immune response by oncolytic parvovirus Minute Virus of Mice infection.

Rodent autonomous parvoviruses (PVs) are endowed with oncotropic properties and represent virotherapeutics with inherent oncolytic features. This work aimed to evaluate the capacity of Minute Virus of Mice (MVMp) to act as an adjuvant stimulating a mouse glioblastoma-specific immune response. MVMp was shown to induce cell death through apoptosis in glioma GL261 cells. Antigen-presenting cells (APCs) provide the initial cue for innate and adaptive immune responses, and thus MVMp-infected GL261 cells were tested for their ability to activate dendritic cells (DCs) and microglia (MG), two distinct cell types that are able to act as APCs. MG and discrete DC subsets were activated after co-culture with MVMp-infected glioma GL261 cells, as evidenced by upregulation of specific activation markers (CD80, CD86) and release of proinflammatory cytokines (tumor necrosis factor-α and interleukin-6). The in vivo analysis of immunodeficient and immunocompetent mice revealed a clear difference in their susceptibility to MVMp-mediated tumor suppression. Immunocompetent mice were fully protected from tumor outgrowth of GL261 cells infected ex vivo with MVMp. In contrast, immunodeficient animals were less competent for MVMp-dependent tumor inhibition, with only 20% of the recipients being protected, arguing for an additional immune component to allow full tumor suppression. In keeping with this conclusion, immunocompetent mice engrafted with MVMp-infected glioma cells developed a level of anti-tumor immunity with isolated splenocytes producing elevated levels of interferon-γ. In rechallenge experiments using uninfected GL261 cells, we could show complete protection against the tumor, arguing for the induction of a T-cell-mediated, tumor-specific, long-term memory response. These findings indicate that the anticancer effect of PVs can be traced back not only for their direct oncolytic effect, but also to their ability to break tumor tolerance.

Coping with parvovirus infections in mice: health surveillance and control.

Parvoviruses of mice, minute virus of mice (MVM) and mouse parvovirus (MPV), are challenging pathogens to eradicate from laboratory animal facilities. Due to the impediment on rodent-based research, recent studies have focused on the assessment of re-derivation techniques and parvoviral potential to induce persistent infections. Summarizing recent data, this review gives an overview on studies associated with parvoviral impact on research, diagnostic methods, parvoviral persistence and re-derivation techniques, demonstrating the complex nature of parvovirus infection in mice and unfolding the challenge of controlling parvovirus infections in laboratory animal facilities.

Presence of Minute virus of mice in immunocompetent mice despite the onset of host immunity.

Infections with the autonomous parvovirus Minute virus of mice (MVM) are generally characterized as acute and self-limiting. However, MVM remains with considerably high prevalence rates in laboratory mouse colonies impeding rodent based research. The objective of this study was to assess whether the immunosuppressive variant of MVM (MVMi) establishes a persistent infection in immunocompetent adult mice. Therefore, we approached the question whether replicating and/or infectious virus is present in mice after the decline of viral shedding and whether immunosuppression might modify the infection. Dissection or induction of immunosuppression of individually housed mice was performed at 8 weeks post inoculation after fecal samples tested negative for viral DNA for at least 2 subsequent weeks as determined by weekly PCR analyses. MVMi mRNA was detected by both, RT-PCR and in situ RT-PCR in spleens at 8 weeks post inoculation with positive cells resembling lymphocytes and macrophages. These findings and the use of explant cultures strongly indicated the presence of replicating virus in spleens at 8 weeks post inoculation. Following immunosuppression (by irradiation), an induction of viral shedding was observed. Additionally, an increase in the amount of viral DNA was detected by real-time qPCR in mesenteric lymph nodes after irradiation. In summary, our data support the notion that MVMi persists in lymphoid tissue of immunocompetent adult mice despite the onset of host immunity.

Translation control by protein kinase R restricts minute virus of mice infection: role in parvovirus oncolysis.

The relevance of translational control in the gene expression and oncotropism of the autonomous parvoviruses was investigated with MVMp, the prototype strain of minute virus of mice (MVM), infecting normal and transformed rodent and human cells of different tissue origins. Mouse embryo fibroblasts (MEFs) and NIH 3T3 fibroblasts were resistant to MVMp infection, but 3T3 fibroblasts derived from double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) knockout mice (PKR(o/o)) behaved in a manner that was highly permissive to productive MVMp replication. NIH 3T3 resistance correlated with significant phosphorylation of eukaryotic translation initiation factor 2 (eIF2) occurring at early time points after infection. Permissive PKR(o/o) cells were converted to MVMp-restrictive cells after reintroduction of the PKR gene by transfection. Conversely, regulated expression of the vaccinia virus E3 protein, a PKR inhibitor, in MEFs prevented eIF2alpha phosphorylation and increased MVMp protein synthesis. In vitro-synthesized genome-length R1 mRNA of MVMp was a potent activator of PKR. Virus-resistant primary MEFs and NIH 3T3 cells responded to MVMp infection with significant increases in eIF2alpha phosphorylation. In contrast, virus-permissive mouse (PKR(o/o), BHK21, and A9) and human transformed (NB324K fibroblast, U373 glioma, and HepG2 hepatoma) cells consistently showed no significant increase in the level of eIF2alpha phosphorylation following MVMp infection. The synthesis of the viral NS1 protein was inversely correlated with the steady-state PKR levels. Our results show that the PKR-mediated antiviral response is an important mechanism for control of productive MVMp infection, and its impairment in human transformed cells allowed efficient MVMp gene expression. PKR translational control may therefore contribute to the oncolysis of MVMp and other autonomous parvoviruses.

Activation of an antiviral response in normal but not transformed mouse cells: a new determinant of minute virus of mice oncotropism.

Parvovirus minute virus of mice (MVMp) is endowed with oncotropic properties so far ascribed only to the dependency of the virus life cycle on cellular factors expressed during S phase and/or modulated by malignant transformation. For other viruses oncotropism relies on their inability to circumvent type I interferon (IFN)-induced innate antiviral mechanisms, the first line of defense triggered by normal cells against viral infections. These agents propagate, therefore, preferentially in transformed/tumor cells, which often lack functional antiviral mechanisms. The present study aimed at investigating whether antiviral processes also contribute to MVMp oncotropism. Our results demonstrate that in contrast to MVMp-permissive transformed mouse A9 fibroblasts, freshly isolated normal counterparts (mouse embryonic fibroblasts [MEFs]) mount, through production and release of type I IFNs upon their infection, an antiviral response against MVMp lytic multiplication. Pretreatment of MEFs with a type I IFN-beta-neutralizing antibody, prior to MVMp infection, inhibits the virus-triggered antiviral response and improves the fulfillment of the MVMp life cycle. Our results also show that part of the A9 permissiveness to MVMp relies on the inability to produce type I IFNs upon parvovirus infection, a feature related either to an A9 intrinsic deficiency of this process or to an MVMp-triggered inhibitory mechanism, since stimulation of these cells by exogenous IFN-beta strongly inhibits the parvovirus life cycle. Taken together, our results demonstrate for the first time that parvovirus infection triggers an innate antiviral response in normal cells and suggest that the MVMp oncotropism depends at least in part on the failure of infected transformed cells to mount such a response.

PEG enhances viral clearance on ceramic hydroxyapatite.

Viral clearance across ceramic hydroxyapatite (CHT) was examined in two elution systems: sodium chloride and sodium chloride plus poly(ethylene glycol) (PEG). In both cases clearance of xenotropic murine leukemia virus was significant (3-4 log) while that of minute virus of mice varied between 1.7 and 2.7 log; in addition, the addition of PEG to the elution buffer enhanced viral clearance. The data are in agreement with the previous results and demonstrate that additional clearance can be obtained by adding PEG to a ceramic hydroxyapatite buffer system.

Embryo transfer rederivation of C.B-17/Icr-Prkdc(scid) mice experimentally infected with mouse parvovirus 1.

We determined whether embryos derived from C.B-17/Icr-Prkdc(scid) (SCID) mice infected with mouse parvovirus (MPV) 1b and mated to MPV-naive B6C3F1 mice would transmit virus to naive recipient female mice and rederived progeny. Viral DNA was detected by quantitative PCR (qPCR) in lymphoid tissues, gonad, sperm, and feces of all MPV1b-inoculated SCID mice. Viral DNA was detected in 1 of 16 aliquots of embryos from infected male SCID mice and in 12 of 18 aliquots of embryos from infected female SCID mice. All recipient female mice implanted with embryos from infected SCID male mice and their progeny were negative by serology and qPCR. In contrast, 3 of 5 recipient female mice implanted with embryos from infected SCID female mice and 14 of 15 progeny mice from these recipients were seropositive by multiplex fluorescent immunoassay (MFI) for MPV capsid antigen (rVP2). All of these mice were negative by MFI for parvovirus nonstructural protein antigen (rNS1) and by qPCR, with the exception of 1 recipient female mouse that displayed weak rNS1 seroreactivity and low levels of MPV DNA in lymphoid tissues. Seroreactivity to rVP2 declined over time in all progeny mice from infected SCID female mice until all were seronegative by 20 wk of age, consistent with maternal antibody transfer. Given that the high levels of MPV contamination detected in our experimentally infected SCID mice are unlikely in naturally infected immunocompetent mice, these data indicate that embryo transfer rederivation is effective for the eradication of MPV from infected colonies.

Minute virus of mice: antibody response, viral shedding, and persistence of viral DNA in multiple strains of mice.

Minute virus of mice (MVM) is a major concern for laboratory animal facilities because it remains with considerably high prevalence despite strict barrier systems. The aim of this study was to elucidate potential risks associated with MVM infection by investigating the role of the genetic background on antibody production and persistence as well as viral shedding. Mice of various strains and stocks were inoculated oronasally with the immunosuppressive strain MVMi; in addition, natural infection was modeled through contact exposure. As determined by serology, seroconversion and serum levels of IgG differed considerably among strains and stocks, especially in the contact-exposed group. For example, C57BL/6J mice responded well to exposure in contrast to FVB/N, NMRI, ICR, and C3H/HeN mice. Titration studies indicated that the viral dose necessary to induce seroconversion was strain-dependent. Experiments to dissect the role of the major histocompatibility complex haplotype in the response to MVMi gave inconclusive results. To detect viral persistence, spleens and feces were analyzed by PCR at 16 wk after exposure, and the infectivity of PCR-positive spleens was investigated by IP and oronasal inoculation of naive mice. Although DNA was detected in the spleens of some mice, feces remained negative, and naive mice were not infected by inoculation. In addition, viral shedding declined rapidly after day 20 postinoculation. In summary, the data show that seroconversion and antibody response to MVMi infection depend on the genetic background of mice, with the infective dose being a critical factor. The role of viral DNA in chronically infected mice will require further elucidation.

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody.

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.

Gender influences infectivity in C57BL/6 mice exposed to mouse minute virus.

Two natural outbreaks of mouse minute virus (MMV) are described. Observations during management of the naturally infected colonies led to a study in which 4-wk-old C57BL/6NCr and C57BL/6Tac mice were inoculated oronasally with an immunosuppressive variant of MMV (MMVi), as were adult C57BL/6NCr lactating dams or their pups (age, 10 d). By day 28 postinoculation, 100% of the 4-wk-old male C57BL/6NCr and C57BL/6Tac mice, 56.2% of 4-wk-old C57BL/6NCr female and 62.5% of 4-wk-old C57BL/6Tac female mice, 100% of adult lactating C57BL/6NCr dams, and 100% of inoculated pups (10 d) had seroconverted. Serologically positive nursing dams did not infect their nursing pups. In contrast, when nursing pups were inoculated, 100% of their dams seroconverted by 28 d postinoculation. Only 1 of 4 facility sentinels (Tac:SW female mice) seroconverted to MMVi and none of the 4 research sentinels (Tac:SW female mice) seroconverted under a once-weekly bedding transfer program. Consequently, 4 new research Tac:SW sentinels of each gender (n = 8) were placed in known-positive cages at cage-change; 100% of the male mice but 0% of the females seroconverted by day 48. Study results suggest gender influences both infectivity and the ability to detect subclinical infections of MMVi. Other factors that may influence detection of MMV include mouse strain or stock, short shedding period, and prolonged time between cage changes. In light of the data from both the natural infections and the experimental cases, cessation of breeding likely will be beneficial when trying to eradicate this virus.

Humoral immune responses against minute virus of mice vectors.

BACKGROUND: Owing to their oncolytic properties, autonomous rodent parvoviruses and derived vectors constitute potential anti-tumor agents. METHODS: Humoral immune responses to minute virus of mice (MVMp) were characterized. In particular, the generation of neutralizing antibodies on subsequent therapeutic virus applications was evaluated in a mouse melanoma model. Mice bearing subcutaneous melanomas were injected intratumorally with virus and re-injected 10 days later in a second tumor on the other flank. Four days after the first or second injection, the tumors and lymph nodes were analyzed by RT-PCR for gene expression. RESULTS: Injection of MVMp in tumor-bearing B6 mice resulted in viral gene expression in tumors and draining lymph nodes. A repeated virus administration did not lead to detectable viral transcription if it was preceded by a virus infection 10 days earlier. This protection correlated with the induction of virus-neutralizing antibodies following the first virus application. The restrictions on viral gene expression after a consecutive MVMp injection could be alleviated in subsequent applications by the use of viruses consisting of MVMp genomes packaged into capsids of a related parvovirus. Neutralizing antibody induction was irrespective of the route of administration and of the presence of a tumor and persisted at significant levels at least up to 26 weeks after the viral infection. MVMp infection of B6 mice stimulated the generation of IgM and IgG anti-viral antibodies, the latter mainly of the T-helper (Th) 1-dependent IgG2, and the T-cell-independent IgG3 subclasses. CONCLUSIONS: Neutralizing antibodies impede the effectiveness of a subsequent virus administration, but can be overcome by pseudotyping.

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.

NS1 interaction with CKII alpha: novel protein complex mediating parvovirus-induced cytotoxicity.

During a productive infection, the prototype strain of the parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations in permissive A9 fibroblasts, culminating in cell lysis at the end of infection. These cytopathic effects (CPE) result from rearrangements and destruction of the cytoskeletal micro- and intermediate filaments, while other structures such as the nuclear lamina and particularly the microtubule network remain protected throughout the infection (J. P. F. Nüesch et al., Virology 331:159-174, 2005). In order to unravel the mechanism(s) by which parvoviruses trigger CPE, we searched for NS1 interaction partners by differential affinity chromatography, using distinct NS1 mutants debilitated specifically for this function. Thereby, we isolated an NS1 partner polypeptide, whose interaction with NS1 correlated with the competence of the viral product for CPE induction, and further identified it by tandem mass spectrometry and Western blotting analyses to consist of the catalytic subunit of casein kinase II, CKIIalpha. This interaction of NS1 with CKIIalpha suggested interference by the viral protein with intracellular signaling. Using permanent cell lines expressing dominant-negative CKIIalpha mutants, we were able to show that this kinase activity was indeed specifically involved in parvoviral CPE and progeny particle release. Furthermore, the NS1/CKIIalpha complex proved to be able to specifically phosphorylate viral capsids, indicating a mediator function of NS1 for CKII activity and specificity, at least in vitro. Altogether our data suggest that parvovirus-induced CPE is mediated by NS1 interference with intracellular CKII signaling.