Mouse Minute Virus Research Articles

Electrophoretically Snagging Viral Genomes in Wormlike Micelle Networks Using Peptide Nucleic Acid Amphiphiles and dsDNA Oligomers.

We demonstrate that the attachment of 30-170 bp dsDNA oligomers to ssDNA viral genomes gives a significant additional mobility shift in micelle-tagging electrophoresis (MTE). In MTE, a modified peptide nucleic acid amphiphile is attached to the viral genome to bind drag-inducing micelles present in capillary electrophoresis running buffers. Further attachment of 30-170 bp dsDNA oligomers drastically shifts the mobility of the 5.1 kB ssDNA genome of mouse minute virus (MMV), providing a new mechanism to improve resolution in CE-based analysis of kilobase nucleic acids. A model based on biased-reptation electrophoresis, end-labeled free-solution electrophoresis, and Ferguson gel-filtration theory is presented to describe the observed mobility shifts.

Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing.

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.

Revisiting mouse minute virusinactivation by high temperature short time treatment.

In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.

Non-Invasive Fecal Based PCR Assays for Detection of Mouse Parvo Virus and Minute Virus of Mice in Laboratory Mice

Background: Murine parvoviruses are a few of the most common pathogens of laboratory mice posing potential threats to mice colonies and experiments conducted in mice. We initiated this study to develop the fecal based PCR assays as an alternative to serology and to check its feasibility to detect the infections in mice caused by Mouse Parvo Virus (MPV) and Minute Virus of mice (MVM).Methods: Primers targeting the VP2 gene of MPV and MVM were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse fecal DNA. Selected thirty-seven mice at the age of 6 to 18 weeks randomly and collected blood samples for serology and fecal samples for PCR assays. Result: PCR assays of MPV and MVM detected as low as 0.4 fg of the target plasmid DNA. PCR assays in fecal samples of mice detected the presence of natural infections of MPV and MVM and their respective prevalence was 24% and 30%. Diagnostic sensitivity of MPV and MVM were 74% and 76% respectively. Our findings indicate that the fecal-PCR assay may be a useful, non-invasive and sensitive diagnostic tool in the ante-mortem detection of MPV and MVM in the health monitoring program.

Novel spiking methods developed for anion exchange chromatography operating in a continuous process.

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a nonhomogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in-line spiking. This is challenging to be implemented at the contract research organization performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow-through anion exchange chromatography step in a monoclonal antibody (mAb) manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of anion exchange chromatography unit operating in continuous processing.

Adventitious agent contamination risk mitigation: engineering MMV virus resistance into CHO cells

Background The introduction of animal origin free (AOF) media has significantly reduced the incidence of adventitious virus contamination in biological production systems. Nevertheless, contamination by the parvovirus Mouse Minute Virus (MMV) remains a continuing challenge. Although infrequent, infection of a fermenter can be catastrophic for a manufacturer, and can also have a potential impact on drug supply, patient safety and have regulatory implications. In this work, we evaluated engineering Chinese Hamster Ovary (CHO) cell lines to create a new host cell line that

Mouse Minute Virus (MMV) Contamination--A Case Study: Detection, Root Cause Determination, and Corrective Actions

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) The production of biologic drugs using mammalian cell production systems offers the benefits of high yield, proper protein folding, and faithful post-translational modifications. However, mammalian cell culture is vulnerable to contamination with adventitious agents, including mouse minute virus (MMV). The case study presented here demonstrates that MMV is a ubiquitous threat to CHO (Chinese hamster ovary) cell-based production of biologic drugs and that animal-free media components can be a contamination source. Compounding the risk posed by MMV, the contamination may be "silent," with no impact on cell viability and product titers. Furthermore, contamination may not be detected using in vitro virus assays, and assays based on PCR (polymerase chain reaction) are required for reliable detection. The development of effective corrective and preventative action (CAPA) was greatly aided by the identification of the source of the contamination as an animal-free recombinant media additive. The execution of a CAPA that included disposal of contaminated materials, decontamination of the facility, and replacement of the contaminated raw material allowed the resumption of MMV-free production.

Effectiveness of mouse minute virus inactivation by high temperature short time treatment technology: A statistical assessment

Viral contamination of mammalian cell cultures in GMP manufacturing facility represents a serious safety threat to biopharmaceutical industry. Such adverse events usually require facility shutdown for cleaning/decontamination, and thus result in significant loss of production and/or delay of product development. High temperature short time (HTST) treatment of culture media has been considered as an effective method to protect GMP facilities from viral contaminations. Log reduction factor (LRF) has been commonly used to measure the effectiveness of HTST treatment for viral inactivation. However, in order to prevent viral contaminations, HTST treatment must inactivate all infectious viruses (100%) in the medium batch since a single virus is sufficient to cause contamination. Therefore, LRF may not be the most appropriate indicator for measuring the effectiveness of HTST in preventing viral contaminations. We report here the use of the probability to achieve complete (100%) virus inactivation to assess the effectiveness of HTST treatment. By using mouse minute virus (MMV) as a model virus, we have demonstrated that the effectiveness of HTST treatment highly depends upon the level of viral contaminants in addition to treatment temperature and duration. We believe that the statistical method described in this report can provide more accurate information about the power and potential limitation of technologies such as HTST in our shared quest to mitigate the risk of viral contamination in manufacturing facilities.

Lack of Transmission of Mouse Minute Virus (MMV) from In Vitro-Produced Embryos to Recipients and Pups Due to the Presence of Cumulus Cells During the In Vitro Fertilization Process

The risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of cumulus-enclosed oocytes (CEOs) or without cumulus cells (CDOs) in the presence of MMV-exposed (10(4) TCID(50) [mean tissue culture infective dose]/ml MMVp [prototype strain of MMV]) spermatozoa was evaluated. Also, the time after embryo transfer to detection of MMV antibody and the presence of MMV DNA in the mesenteric lymph nodes of recipients and pups were investigated. All mice were MMV free, but two seropositive recipients and four seropositive pups were found in the group with CDOs. With regard to the CEOs, two of 11 holding drops and five of 11 groups of embryos were MMV positive using PCR, while neither holding drops nor embryos carried infectious MMVp, as evidenced by the in vitro infectivity assay. From IVF with CDOs, five of 14 holding drops and four of nine groups of embryos were MMV positive, while one of 14 holding drops and no embryos carried infectious MMVp. When 10(5) cumulus cells were analyzed 5 h after exposure to 10(4) TCID(50)/ml MMVp, cells had an average titer of 10(4) TCID(50)/ml MMVp. The present data show that, in contrast to CDOs, 2-cell embryos from CEOs did not transmit infectious MMVp to the holding drops and to recipients. This observation is due to the presence of cumulus cells during the IVF process that reduce entry of MMV into the zona pellucida and absorb some of the virus. These data further confirm the efficacy of the IVF procedure in producing embryos that are free of infectious virus, leading to virus-free seronegative recipients and rederived pups.

Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types

Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus-spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry.

First Trial in the Developmental Phase of the "Performance Evaluation Program" Based on the ICLAS Animal Quality Network Program: Self-Assessment of Microbiological Monitoring Methods Using Test Samples Supplied by ICLAS

The first trial in the developmental phase of the "Performance Evaluation Program" based on the new programs of the International Council for Laboratory Animal Science (ICLAS) was carried out. ICLAS supplied test samples to each diagnostic laboratory for self-assessment of microbiological monitoring methods. We found that 1 mouse serum sample was positive for mouse minute virus and another was positive for Mycoplasma pulmonis antibodies, and 1 rat serum sample was positive for Sendai virus antibody. Mouse parvovirus was detected from mouse spleen and mesenteric lymph node homogenate, and Helicobacter spp. were detected from mouse feces. Corynebacterium bovis was isolated from a mouse skin sample. These results were in agreement with those notified by the ICLAS after the trial. After this trial, the program will eventually be made available to all diagnostic laboratories willing to participate in it.

Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 100 TCID50 MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10−5 TCID50 MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.

Production of Virus-Free Seronegative Pups from Murine Embryos Arising from In Vitro Fertilization with Mouse Minute Virus-Exposed Spermatozoa

In the present study, the risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of oocytes with MMV-exposed spermatozoa and to resulting pups was evaluated. Also, the time of seroconversion of recipients and pups was investigated. To achieve this goal, IVF of oocytes with cryopreserved spermatozoa from the inbred C3HeB/FeJ mouse strain was performed, and the resulting embryos were transferred to suitable Swiss recipients. Three groups were investigated: 1) oocytes or the developing embryos were continuously exposed to 10(4) TCID(50) MMVp per milliliter in the fertilization (human tubal fluid [HTF]), culture (KSOM), and embryo transfer (M2) media (positive control); 2) oocytes and spermatozoa were exposed to MMVp in the HTF medium only and transferred after a standard washing procedure with 10 washing steps in virus-free KSOM and M2; and 3) oocytes and spermatozoa were exposed to virus-free HTF, KSOM, and M2 (negative control). To detect antibodies to MMV in recipients and progeny, serological analyses were performed by ELISA on Days 14, 21, 28, and 42, and on Days 42 and 63, respectively, after embryo transfer. The presence of MMV in the washing drops was analyzed by PCR and an in vitro infectivity assay, while organs of some recipients and pups were analyzed by PCR. Using 10(4) of the tissue culture infective dose of MMVp per millilitre in the fertilization medium only, the present results demonstrate that 10 washing steps in the IVF-ET procedure are sufficient to remove the virus to a noninfectious dose, producing MMV-free seronegative recipients and pups. As such, there is minimal risk of transmission of MMV to recipients and pups if spermatozoa become contaminated with such viral loads.

Transmission of Mouse Minute Virus (MMV) but Not Mouse Hepatitis Virus (MHV) Following Embryo Transfer with Experimentally Exposed In Vivo-Derived Embryos1

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 105/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 107 mean tissue culture infective dose (TCID50)/ml of MHV-A59 and 102 TCID50/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.

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.

Simultaneous Serodetection of 10 Highly Prevalent Mouse Infectious Pathogens in a Single Reaction by Multiplex Analysis

Under current practices of mouse colony maintenance, sera from mice are analyzed for antibodies against several widespread infectious pathogens by conventional immunoassays, generally enzyme-linked immunosorbent assay (ELISA). To test for multiple agents, these methods consume large volumes of mouse serum and are laborious and time-consuming. More efficient immunoassays, using small amounts of sample, are therefore needed. Accordingly, we have developed a novel multiplex diagnostic system that employs fluorescent microbeads, coated with purified antigens, for simultaneous serodetection of 10 mouse infectious agents. Individually identifiable, fluorescent microbeads were coated with antigens from Sendai virus, mouse hepatitis virus, Theiler's mouse encephalomyelitis virus/GDVII strain, mouse minute virus, mouse cytomegalovirus, respiratory enteric orphan virus (Reo-3 virus), mouse parvovirus, calf rotavirus for epizootic diarrhea virus of infant mice, vaccinia virus for ectromelia virus, and Mycoplasma pulmonis. Standard sera, singly positive for antibodies to individual infectious agents, were generated by inoculation of BALB/cj and C57BL/6j mice. Sera from these experimentally infected mice, as well as sera from naturally infected mice, were analyzed using a mixture of microbeads coated with antigens of the 10 infectious agents listed above. Results demonstrated that the multiplex assay was at least as sensitive and specific as ELISA for serodetection. Importantly, the multiplex assay required only 1 microliter of serum for simultaneous serodetection of the 10 mouse infectious agents in one reaction vessel. Thus, this multiplex microbead assay is a reliable, efficient, and cost-effective diagnostic modality that will impact serosurveillance of mice used in research.

179IMPORTANCE OF EMBRYO TRANSFERS IN TRANSGENIC MOUSE FACILITIES

With the increasing demand for and production of transgenic and mutant mice for biomedical research, embryo transfer plays a paramount role. The purpose of performing embryo transfer in this species is to generate transgenic mice via blastocyst injection of embryonic stem cells or pronuclear injection of DNA constructs, to revitalize cryopreserved sperm and embryos, and to generate mouse lines that meet specific pathogen-free health standards for breeding in barrier areas (rederivation). We present results from two years of carrying out embryo transfers for rederivation purposes in the large mouse breeding facility of the GSF—National Research Center for Environment and Health, Neuherberg, Germany. Pathogens to be eradicated from inbred transgenic (C57BL/6 background) and mutant (C3H background) mouse lines included mouse hepatitis virus, mouse minute virus, and mouse parvovirus. In vitro- and in vivo-produced two-cell embryos were washed 3 times in M2 medium. A total of 20 embryos each were transferred to the oviduct of 8- to 12-week-old specific pathogen-free pseudopregnant (Day 0.5) Swiss recipients under aseptic conditions. Mice were then kept singly in individually ventilated cages and manipulated in a Class II laminar flow hood. From each transfer to one to five recipients with embryos originating from the same mouse line, one recipient was tested for the presence of microorganisms 6 to 12 weeks after embryo transfer, i.e. at 0 to 6 weeks after weaning, according to the FELASA (Federation of European Laboratory Animal Science Associations) Guidelines. A total of 290 embryo transfers were performed for revitalization of cryopreserved sperm from 52 mouse lines, cryopreserved two-cell embryos from 18 mouse lines and rederivation of 12 mouse lines using freshly collected two-cell embryos. From these 290 embryo transfers, 59 mouse lines were re-established (40 from cryopreserved sperm, 7 from cryopreserved embryos and 12 from in vivo-produced embryos). Health monitoring of 54 recipients showed that all mouse lines generated were free of all pathogens stated in the FELASA list. The results presented here show that all 12 (100%) mouse lines were re-established after transfer of freshly collected two-cell embryos whereas 77% and 39% success rates were observed for revitalization of cryopreserved sperm and embryos, respectively. The success of embryo transfer in eradicating pathogens depends on the inability of these pathogens to transverse the zona pellucida and enter and/or infect embryonic cells. In our mouse facility, embryo transfer provided an efficient method to successfully revitalize cells of the mouse germ line as well as to eradicate prevalent murine pathogens. Furthermore, the results demonstrate the efficiency of transferring embryos of different origins and thereby obtaining and maintaining specific pathogen-free health standards in our mouse colonies.