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Minute Virus of Mice (MVM) is an autonomous parvovirus of the Parvoviridae family that replicates in mouse cells and transformed human cells. MVM genomes localize to cellular sites of DNA damage with the help of their essential non-structural phosphoprotein NS1 to establish viral replication centers. MVM replication induces a cellular DNA damage response that is mediated by signaling through the ATM kinase pathway, while inhibiting induction of the ATR kinase signaling pathway. However, the cellular signals regulating virus localization to cellular DNA damage response sites has remained unknown. Using chemical inhibitors to DNA damage response proteins, we have discovered that NS1 localization to cellular DDR sites is independent of ATM or DNA-PK signaling but is dependent on ATR signaling. Pulsing cells with an ATR inhibitor after S-phase entry leads to attenuated MVM replication. These observations suggest that the initial localization of MVM to cellular DDR sites depends on ATR signaling before it is inactivated by vigorous virus replication. [ABSTRACT FROM AUTHOR]

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25–45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to "switch on/off" the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection. [ABSTRACT FROM AUTHOR]

Being nonpathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses with great potential as anti-cancer agents. As these viruses replicate in the host cell nucleus, they must gain access to the nucleus during infection. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the nuclear envelope (NE) and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. In this study, we used MVM as a model to investigate the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays, mutagenesis, and pharmacological inhibition, we identified a functional myristoylation site at the sequence 78GGKVGH83 of the unique portion of the capsid protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this myristoylation site or with purified virions, we found tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying this cleavage exposes G (glycine) 78 at the N-terminus of VP1u for myristoylation. Subsequent experiments using inhibitors of myristoylation and cellular proteases with MVM-infected cells, or an imaging-based quantitative NE permeabilization assay, further indicate protein myristoylation and a chymotrypsin-like activity are essential for MVM to locally disrupt the NE during viral nuclear entry. We thus propose a model for the nuclear entry of MVM in which NE disruption is mediated by VP1u myristoylation after the intact capsid undergoes proteolytic processing to expose the required N-terminal G for myristoylation. [ABSTRACT FROM AUTHOR]

This study aimed to propose a methodology for developing a mechanistic model for viral clearance of the minute virus of mice (MVM) on flow-through anion exchange (AEX) chromatography. Protein surface analysis was applied to investigate the possibility of molecular interaction between the recombinant biotherapeutic and MVM. The protein product-free Tris buffers were spiked with MVM, and the MVM elution profile from AEX chromatography was quantitatively analyzed using quantitative polymerase chain reaction (qPCR) for pooled fractions. GoSilico™ Chromatography Modeling Software was applied to develop the mechanistic models for MVM species. For evaluating the visual fit of the developed model, the R 2 of intact MVM virions and uncoated capsids between the simulated and measured amount in each fraction are 0.880 and 0.948, respectively. Response surface plots of logarithmic reduction values (LRV) against pH and conductivity in loaded sample were generated to show the range for suitable loaded sample conditions for achieving a good LRV. To evaluate the applicability of the developed MVM elution model to a recombinant biotherapeutic, two demonstrations of AEX chromatography purification were performed with a loaded sample of a model monoclonal antibody. The peaks of the MVM species in the elution step of both runs were accurately simulated by the developed model. In addition, to assess the possibility of molecular interaction between the virus and the target protein significantly affecting the MVM elution behavior, the antibody's surface was evaluated in terms of hydrophobicity/hydrophilicity using surface analysis., (© 2024 American Institute of Chemical Engineers.)

Abstract There is a growing need to provide effective adventitious agent mitigation for high risk upstream cell culture raw materials used for the production of biologics. It is also highly important in the growing fields of cell and gene therapies. Glucose is a critical raw material necessary for effective cell growth and productivity; however, glucose is the highest risk animal‐origin‐free raw material for viral contamination, and often the highest risk raw material in the upstream process as more companies move to chemically defined media. This study examines the efficacy of utilizing High Temperature Short Time (HTST) pasteurization for inactivation of physiochemically resistant, worst‐case parvovirus using a bench‐scale HTST system. We demonstrated approximately six log inactivation of Minute Virus of Mice (MVM) in concentrated glucose feeds without impacting the subsequent performance of the glucose in a Chinese Hamster Ovary (CHO) expression system.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.

Minute Virus of Mice (MVM) is an autonomous parvovirus of the Parvoviridae family that replicates in mouse cells and transformed human cells. MVM genomes localize to cellular sites of DNA damage with the help of their essential non-structural phosphoprotein NS1 to establish viral replication centers. MVM replication induces a cellular DNA damage response that is mediated by signaling through the ATM kinase pathway, while inhibiting induction of the ATR kinase signaling pathway. However, the cellular signals regulating virus localization to cellular DNA damage response sites has remained unknown. Using chemical inhibitors to DNA damage response proteins, we have discovered that NS1 localization to cellular DDR sites is independent of ATM or DNA-PK signaling but is dependent on ATR signaling. Pulsing cells with an ATR inhibitor after S-phase entry leads to attenuated MVM replication. These observations suggest that the initial localization of MVM to cellular DDR sites depends on ATR signaling before it is inactivated by vigorous virus replication.

In virus clearance study (VCS) design, the amount of virus loaded onto the virus filters (VF) must be carefully controlled. A large amount of virus is required to demonstrate sufficient virus removal capability; however, too high a viral load causes virus breakthrough and reduces log reduction values. We have seen marked variation in the virus removal performance for VFs even with identical VCS design. Understanding how identical virus infectivity, materials and operating conditions can yield such different results is key to optimizing VCS design. The present study developed a particle number‐based method for VCS and investigated the effects on VF performance of discrepancies between apparent virus amount and total particle number of minute virus of mice. Co‐spiking of empty and genome‐containing particles resulted in a decrease in the virus removal performance proportional to the co‐spike ratio. This suggests that empty particles are captured in the same way as genome‐containing particles, competing for retention capacity. In addition, between virus titration methods with about 2.0 Log10 difference in particle‐to‐infectivity ratios, there was a 20‐fold decrease in virus retention capacity limiting the throughput that maintains the required LRV (e.g., 4.0), calculated using infectivity titers. These findings suggest that ignoring virus particle number in VCS design can cause virus overloading and accelerate filter breakthrough. This article asserts the importance of focusing on virus particle number and discusses optimization of VCS design that is unaffected by virological characteristics of evaluation systems and adequately reflect the VF retention capacity. [ABSTRACT FROM AUTHOR]

There is a growing need to provide effective adventitious agent mitigation for high risk upstream cell culture raw materials used for the production of biologics. It is also highly important in the growing fields of cell and gene therapies. Glucose is a critical raw material necessary for effective cell growth and productivity; however, glucose is the highest risk animal‐origin‐free raw material for viral contamination, and often the highest risk raw material in the upstream process as more companies move to chemically defined media. This study examines the efficacy of utilizing High Temperature Short Time (HTST) pasteurization for inactivation of physiochemically resistant, worst‐case parvovirus using a bench‐scale HTST system. We demonstrated approximately six log inactivation of Minute Virus of Mice (MVM) in concentrated glucose feeds without impacting the subsequent performance of the glucose in a Chinese Hamster Ovary (CHO) expression system. [ABSTRACT FROM AUTHOR]

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X) identified in a previous glycan microarray screen.

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Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A minimal coarse-grained model for T=1 viral capsids assembled from 20 protein rigid trimers has been designed by extending a previously proposed form of the interaction energy written as a sum of anisotropic pairwise interactions between the trimeric capsomers. The extension of the model has been performed to properly account for the coupling between two internal coordinates: the one that measures the intercapsomer distance and the other that gives the intercapsomer dihedral angle. The model has been able to fit with less than a 10% error the atomic force microscopy (AFM) indentation experimental data for the empty capsid of the minute virus of mice (MVM), providing in this way an admissible picture of the main mechanisms behind the capsid deformations. In this scenario, the bending of the intercapsomer dihedral angle is the angular internal coordinate that can support larger deformations away from its equilibrium values, determining important features of the AFM indentation experiments as the elastic constants along the three symmetry axes of the capsid and the critical indentations. From the value of one of the parameters of our model, we conclude that trimers in the MVM must be quite oblate tops, in excellent agreement with their known structure. The transition from the linear to the nonlinear regimes sampled in the indentation process appears to be an interesting topic for future research in physical virology.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work., (© 2023 Takeda Manufacturing Austria AG and The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.

Viruses can arise during the manufacture of biopharmaceuticals through contamination or endogenous expression of viral sequences. Regulatory agencies require "viral clearance" validation studies for each biopharmaceutical prior to approval. These studies aim to demonstrate the ability of the manufacturing process at removing or inactivating virus and are conducted by challenging scaled‐down manufacturing steps with a "spike" of live virus. Due to the infectious nature of these live viruses, "spiking studies" are typically conducted in specialized biological safety level‐2 facilities. The costs and logistics associated with these studies limit viral clearance analysis during process development and characterization. In this study, a noninfectious Minute Virus of Mice‐Mock Virus Particle (MVM‐MVP) was generated for use as an economical small virus spiking surrogate. An immunoglobin G containing solution was spiked with live MVM or MVM‐MVP and processed through Planova nanofiltration units. Flux decay data was collected and particle reduction values were calculated from TCID50 and Immuno‐qPCR analysis. The data indicated comparable filtration performance and particle reduction between infectious MVM and noninfectious surrogate, MVM‐MVP. This proof of concept study suggests the feasibility of utilizing MVPs for predictive size‐based viral clearance assessments during process development and characterization as an alternative to homologous infectious virus. [ABSTRACT FROM AUTHOR]

The T1 parvovirus Minute Virus of Mice (MVM) was used to study the roles that phosphorylation and N-terminal domains (Nt) configuration of capsid subunits may play in icosahedral nuclear viruses assembly. In synchronous MVM infection, capsid subunits newly assembled as two types of cytoplasmic trimeric intermediates (3VP2, and 1VP1:2VP2) harbored a VP1 phosphorylation level fivefold higher than that of VP2, and hidden Nt. Upon nuclear translocation at S phase, VP1-Nt became exposed in the heterotrimer and subsequent subviral assembly intermediates. Empty capsid subunits showed a phosphorylation level restored to VP1:VP2 stoichiometry, and the Nt concealed in their interior. However ssDNA-filled virus maturing at S/G2 lacked VP1 phosphorylation and one major VP2 phosphopeptide, and exposed VP2-Nt. Endosomal VP2-Nt cleavage resulted in VP3 subunits devoid of any phospholabel, implying that incoming viral particles specifically harbor a low phosphorylation status. Phosphorylation provides a mechanistic coupling of parvovirus nuclear assembly to the cell cycle. [ABSTRACT FROM AUTHOR]

Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus ( H. hepaticus) and Salmonella typhimurium (S. typhimurium) . The detection limit of the standard was 10 copies/μL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV. [ABSTRACT FROM AUTHOR]

In minute virus of mice (MVM) capsids, icosahedral five-fold channels serve as portals mediating genome packaging, genome release, and the phased extrusion of viral peptides. Previous studies suggest that residues L172 and V40 are essential for channel function. The structures of MVMi wildtype, and mutant L172T and V40A virus-like particles (VLPs) were solved from cryo-EM data. Two constriction points, termed the mid-gate and inner-gate, were observed in the channels of wildtype particles, involving residues L172 and V40 respectively. While the mid-gate of V40A VLPs appeared normal, in L172T adjacent channel walls were altered, and in both mutants there was major disruption of the inner-gate, demonstrating that direct L172:V40 bonding is essential for its structural integrity. In wildtype particles, residues from the N-termini of VP2 map into claw-like densities positioned below the channel opening, which become disordered in the mutants, implicating both L172 and V40 in the organization of VP2 N-termini. [ABSTRACT FROM AUTHOR]

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral 'spiking' studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies. [ABSTRACT FROM AUTHOR]

Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova™ BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0-49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1294-1302, 2017 [ABSTRACT FROM AUTHOR]

Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVM-induced DDR is the establishment of a potent premitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others reported previously, depends upon a significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function, thus preventing mitotic entry. We show here that while the stability of cyclin B1 RNA was not affected by MVM infection, the production of nascent cyclin B1 RNA was substantially diminished at late times postinfection. Ectopic expression of NS1 alone did not reduce cyclin B1 expression. MVM infection also reduced the levels of cyclin B1 protein, and RNA levels normally increased in response to DNA-damaging reagents. We demonstrated that at times of reduced cyclin B1 expression during infection, there was a significantly reduced occupancy of RNA polymerase II and the essential mitotic transcription factor FoxM1 on the cyclin B1 gene promoter. Additionally, while total FoxM1 levels remained constant, there was a significant decrease of the phosphorylated, likely active, forms of FoxM1. Targeting of a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via clustered regularly interspaced short palindromic repeats (CRISPR)-enzymatically inactive Cas9 in MVM-infected cells increased both cyclin B1 protein and RNA levels, implicating FoxM1 as a critical target for cyclin B1 inhibition during MVM infection. [ABSTRACT FROM AUTHOR]

Parvoviruses are single-stranded DNA viruses that utilize host proteins to vigorously replicate in the nuclei of host cells, leading to cell cycle arrest. The autonomous parvovirus, minute virus of mice (MVM), forms viral replication centers in the nucleus which are adjacent to cellular DNA damage response (DDR) sites, many of which are fragile genomic regions prone to undergoing DDR during the S phase. Since the cellular DDR machinery has evolved to transcriptionally suppress the host epigenome to maintain genomic fidelity, the successful expression and replication of MVM genomes at these cellular sites suggest that MVM interacts with DDR machinery distinctly. Here, we show that efficient replication of MVM requires binding of the host DNA repair protein MRE11 in a manner that is independent of the MRE11-RAD50-NBS1 (MRN) complex. MRE11 binds to the replicating MVM genome at the P4 promoter, remaining distinct from RAD50 and NBS1, which associate with cellular DNA break sites to generate DDR signals in the host genome. Ectopic expression of wild-type MRE11 in CRISPR knockout cells rescues virus replication, revealing a dependence on MRE11 for efficient MVM replication. Our findings suggest a new model utilized by autonomous parvoviruses to usurp local DDR proteins that are crucial for viral pathogenesis and distinct from those of dependoparvoviruses, like adeno-associated virus (AAV), which require a coinfected helper virus to inactivate the local host DDR. IMPORTANCE The cellular DNA damage response (DDR) machinery protects the host genome from the deleterious consequences of DNA breaks and recognizes invading viral pathogens. DNA viruses that replicate in the nucleus have evolved distinct strategies to evade or usurp these DDR proteins. We have discovered that the autonomous parvovirus, MVM, which is used to target cancer cells as an oncolytic agent, depends on the initial DDR sensor protein MRE11 to express and replicate efficiently in host cells. Our studies reveal that the host DDR interacts with replicating MVM molecules in ways that are distinct from viral genomes being recognized as simple broken DNA molecules. These findings suggest that autonomous parvoviruses have evolved distinct mechanisms to usurp DDR proteins, which can be used to design potent DDR-dependent oncolytic agents., Competing Interests: The authors declare no conflict of interest.

The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers.

Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. [ABSTRACT FROM AUTHOR]

Minute virus of mice (MVM) is one of the most prevalent infectious agents in laboratory mouse colonies. In this study, we optimized a loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue (HNB) for rapid and visual detection of MVM. The reaction, which entailed addition of HNB dye prior to amplification, was performed in one step in a single tube at 62 °C for 45 min. The limit of detection of the assay was 104 copies, which was 100-fold lower than that of conventional PCR. The assay specifically amplified MVM DNA and did not cross-react with other viruses. To validate the established LAMP system, we applied it 287 samples and detected 19 positives. In conclusion, LAMP with HNB is a sensitive, and simple assay for rapid detection of MVM infections in laboratory animals, thus offers a platform for quality monitoring. • A LAMP assay for MVM detection using HNB was developed and reported for the first time. • The LAMP assay was more convenient and 100 times more sensitive than the corresponding PCR assay for MVM detection. • The optimized LAMP method is not only a simple and rapid test, but also a valuable tool for diagnosis of MVM. [ABSTRACT FROM AUTHOR]

The efficacy of gaseous disinfection is critical for prevention and treatment of microbial contamination in biotechnological facilities. For an evaluation of gaseous disinfection efficacy, a down-scaled laboratory model was established, using currently available carrier tests and a custom-made dry fog box. A mixture of peroxyacetic acid and hydrogen peroxide (PAA/HP) was investigated as example, at concentrations between 0.4 and 2.9 mL/m 3 for up to 3 h for inactivation of a panel of lipid-enveloped and non-lipid-enveloped viruses. The influenza viruses were most sensitive to PAA/HP treatment and minute virus of mice was most resistant. Bovine viral diarrhea virus and reovirus III showed intermediate stability and similar inactivation kinetics. Use of the dry fog box circumvents dedicating an entire lab for the investigation, which renders the generation of data more cost-effective and allows for production of highly reproducible kinetic data. [ABSTRACT FROM AUTHOR]