Propagation and Purification of Ectromelia Virus.

Ectromelia virus (ECTV) is an orthopoxvirus that causes mousepox in mice. Members of the genus orthopoxvirus are closely related and include variola (the causative agent of smallpox in humans), monkeypox, and vaccinia. Common features of variola virus and ECTV further include a restricted host range and similar disease progression in their respective hosts. Mousepox makes an excellent small animal model for smallpox to investigate pathogenesis, vaccine and antiviral agent testing, host-virus interactions, and immune and inflammatory responses. The availability of a wide variety of inbred, congenic, and gene-knockout mice allows detailed analyses of the host response. ECTV mutant viruses lacking one or more genes encoding immunomodulatory proteins are being used in numerous studies in conjunction with wild-type or gene-knockout mice to study the functions of these genes in host-virus interactions. The methods used for propagation of ECTV in cell culture, purification, and quantification of infectious particles through viral plaque assay are described. © 2018 by John Wiley & Sons, Inc.

Development of a SYBR Green I real-time PCR for detection and quantitation of orthopoxvirus by using Ectromelia virus.

Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed. This assay was able to detect a minimum of 10 copies of standard DNA and 5 TCID50 units of ECTV. In addition, no cross-reactions were observed with two DNA viruses, such as herpes simplex virus and swine pseudorabies virus, and one RNA virus, vesicular stomatitis virus. Furthermore, intra- and inter-assay variability data showed that this method had a highly reproducibility and reliability. Moreover, the current assay was faster and had a higher sensitivity for detection of ECTV genomic DNA in cell cultured and clinical test samples. Therefore, the high sensitivity and reproducibility of this SYBR Green real-time PCR approach is a more effective method than the conventional PCR for ECTV diagnosis and quantitation.

Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures.

In 2007, the United States- Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.

The genome sequence of ectromelia virus Naval and Cornell isolates from outbreaks in North America.

Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection.

Mousepox, a small animal model of smallpox.

Ectromelia virus infections in the laboratory mouse have emerged as a valuable model to investigate human orthopoxvirus infections to understand the progression of disease, to discover and characterize antiviral treatments, and to study the host-pathogen relationship as it relates to pathogenesis and the immune response. Here we describe how to safely work with the virus and protocols for common procedures for the study of ectromelia virus in the laboratory mouse including the preparation of virus stocks, the use of various routes of inoculation, and collection of blood and tissue from infected animals. In addition, several procedures are described for assessing the host response to infection: for example, measurement of virus-specific CD8 T cells and the use of ELISA and neutralization assays to measure orthopoxvirus-specific antibody titers.

Studying NK cell responses to ectromelia virus infections in mice.

Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell proliferation by bromodeoxyuridine loading or by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE).

The orthopoxvirus type I IFN binding protein is essential for virulence and an effective target for vaccination.

Nonliving antiviral vaccines traditionally target proteins expressed at the surface of the virion with the hope of inducing neutralizing antibodies. Orthopoxviruses (OPVs), such as the human smallpox virus and the mouse-equivalent ectromelia virus (ECTV; an agent of mousepox), encode immune response modifiers (IRMs) that can increase virulence by decreasing the host immune response. We show that one of these IRMs, the type I interferon (IFN) binding protein (bp) of ECTV, is essential for ECTV virulence and is a natural target of the antibody response. More strikingly, we demonstrate that immunization with recombinant type I IFN bp protects mice from lethal mousepox. Collectively, our experiments have important implications for our understanding of the role of IRMs in OPV virulence and of type I IFNs in OPV infections. Furthermore, our work provides proof of concept that effective antiviral vaccines can be made to prevent disease by targeting virulence factors as an alternative to the traditional approach that attempts to prevent infection by virus neutralization.

An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge.

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

The multidrug resistance gene mdr1a influences resistance to ectromelia virus infection by mechanisms other than conventional immunity.

P-glycoprotein (P-gp), an ATP-dependent membrane pump encoded by mdr, plays, in addition to its ability to efflux toxins, a role in the resistance to pathogens. We employed mdr1a gene knock out (mdr1a-/-) mice and ectromelia virus (EV) to elucidate the role of P-gp in resistance to EV. Mdr1a-/- mice are more susceptible to EV infection than wild type (wt) mice, showing increased mortality and morbidity. Unexpectedly, virus titres in liver, and in vitro in macrophages and splenocytes were significantly lower in the more susceptible mdr1a-/- mice than wt littermates. Analysis of immunological mechanisms known to influence resistance to EV infection, such as NK and cytotoxic T cell responses, EV specific antibody and cytokine levels did not reveal significant differences between the two strains of mice. Only dendritic cells from mdr1a-/- mice showed impaired migration to the draining lymph nodes compared to wt mice. Our data show that P-gp plays an important role in EV infection by as yet undefined mechanisms.

Inhibition of interferons by ectromelia virus.

Ectromelia virus (EV) is an orthopoxvirus (OPV) that causes mousepox, a severe disease of laboratory mice. Mousepox is a useful model of OPV infection because EV is likely to be a natural mouse pathogen, unlike its close relatives vaccinia virus (VV) and variola virus. Several studies have highlighted the importance of mouse interferons (IFNs) in resistance to and recovery from EV infection, but little is known of the anti-IFN strategies encoded by the virus itself. We have determined that 12 distinct strains and isolates of EV encode soluble, secreted receptors for IFN-gamma (vIFN-gammaR) and IFN-alpha/beta (vIFN-alpha/betaR) that are homologous to those identified in other OPVs. We demonstrate for the first time that the EV vIFN-gammaR has the unique ability to inhibit the biological activity of mouse IFN-gamma. The EV vIFN-alpha/betaR was a potent inhibitor of human and mouse IFN-alpha and human IFN-beta but, surprisingly, was unable to inhibit mouse IFN-beta. The replication of all of the EVs included in our study and of cowpox virus was more resistant than VV to the antiviral effects induced in mouse L-929 cells by IFN-alpha/beta and IFN-gamma. Sequencing studies showed that this EV resistance is likely to be partly mediated by the double-stranded-RNA-binding protein encoded by an intact EV homolog of the VV E3L gene. The absence of a functional K3L gene, which encodes a viral eIF-2alpha homolog, in EV suggests that the virus encodes a novel mechanism to counteract the IFN response. These findings will facilitate future studies of the role of viral anti-IFN strategies in mousepox pathogenesis. Their significance in the light of earlier data on the role of IFNs in mousepox is discussed.

Mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum.

Mousepox was identified in a single mouse-holding room in early 1999 after a group of 20 CAF1/Hsd mice were inoculated SC with a killed murine spindle cell tumor line, S1509A. The cell line had been used without complications multiple times and was determined to be free of viral contamination on the basis of results of mouse antibody production testing. Of the 20 mice inoculated, 12 mice died by postinoculation day 8. Severe lymphoid and hepatic necrosis was observed in select mice subjected to histologic examination. Ballooning degeneration of epithelial cells with intracytoplasmic eosinophilic inclusion bodies was observed in the skin overlying the inoculation site of the single mouse from which this tissue site was evaluated. Presence of ectromelia virus was confirmed by use of immunohistochemical and polymerase chain reaction analyses, and the virus was isolated after serum, pooled from 5 of the index cases, was inoculated into an immune-naive mouse. Investigation into the source of virus contamination included inoculating mice with aliquots of various S1509A freeze dates; chemically defined media and supplements, including fetal bovine serum; and two lots of pooled commercial mouse sera, after heat inactivation at 56 degrees C for 30 minutes used as a medium supplement. One lot of pooled commercial mouse serum was identified as the source of ectromelia virus. This lot of serum was inadvertently used to feed S1509A cells that were subsequently inoculated into mice. We determined that the contaminated serum, which was purchased in late 1998, originated from China. The serum was imported into the United States as a batch of 43 L in early 1995. The serum was blended into a single lot and filtered (0.2 microm) before distribution to major suppliers throughout the country. The serum was sold or further processed to obtain a variety of serum-derived products. Because murine serum is generally sold in small aliquots (10 to 50 ml), we speculate that several thousand aliquots may have been derived from this batch of serum and, if inoculated into mice, would likely result in additional mousepox outbreaks.

Homing studies on distribution of ectromelia (mousepox) virus-specific T cells adoptively transferred into syngeneic H-2d mice: paradigm of lymphocyte migration.

Mousepox (infectious ectromelia) may be used as a model for studies on the cellular immune response and pathogenesis of generalized viral infections. Ectromelia virus (EV) initially replicates in the footpad (f.p.) skin at the site of infection, next in draining lymph nodes, and then in the spleen and liver where the virus may induce extensive necrotic process with inflammatory reaction. We show in this study that after recipient BALB/c mice (H-2d) f.p. infection with EV prior to the adoptive transfer of syngeneic donor EV-specific cytotoxic T lymphocytes interferon-gamma-positive (IFN-gamma-+), interleukin-2-positive (IL-2+), and IL-4+ of both phenotypes, CD8+ approximately 70%, and CD4+ approximately 30%) preferentially migrated to the inguinal and auxiliary lymph nodes, spleen, liver, and skin at the site of infection (f.p.). Many particles of EV with the morphology characteristic for orthopoxviruses and virus-specific immunofluorescence within the cells of inguinal and auxiliary lymph nodes, liver, spleen, and skin have been observed using high-resolution transmission electron microscopy and fluorescence antibody technique, respectively. Results presented in this article support the concept that immune T cells adoptively transferred into infected recipient mice are able not only to specific migration in the host and homing in the sites of virus replication, but also to develop immunoprotection in the transferred animals.

Specific detection of mousepox virus by polymerase chain reaction.

Polymerase chain reaction was applied to the rapid identification and detection of mousepox virus. This was accomplished by selection of primers targeting the A-type inclusion body protein gene. By investigating 20 strains belonging to five different species of the genus Orthopoxvirus, amplification was achieved only with the seven mousepox virus strains examined. The size of the resulting DNA fragment accounted for 116 base pairs and contained a recognition site for the restriction enzyme HindII, thus confirming its viral origin. Amplification of mousepox virus specific sequences was also possible from infected mouse lung tissue and serum.

Mousepox outbreak in a laboratory mouse colony.

Mousepox was diagnosed in and eradicated from a laboratory mouse colony at the Naval Medical Research Institute. The outbreak began with increased mortality in a single room; subsequently, small numbers of animals in separate cages in other rooms were involved. Signs of disease were often mild, and overall mortality was low; BALB/cByJ mice were more severely affected, and many of them died spontaneously. Conjunctivitis was the most common clinical sign of disease in addition to occasional small, crusty scabs on sparsely haired or hairless areas of skin. Necropsy findings included conjunctivitis, enlarged spleen, and pale liver. Hemorrhage into the pyloric region of the stomach and proximal portion of the small intestine was observed in experimentally infected animals. In immune competent and immune deficient mice, the most common histologic finding was multifocal to coalescing splenic necrosis; necrosis was seen less frequently in liver, lymph nodes, and Peyer's patches. Necrosis was rarely observed in ovary, vagina, uterus, colon, or lung. Splenic necrosis often involved over 50% of the examined tissue, including white and red pulp. Hepatic necrosis was evident as either large, well-demarcated areas of coagulative necrosis or as multiple, random, interlacing bands of necrosis. Intracytoplasmic eosinophilic inclusion bodies were seen in conjunctival mucosae and haired palpebra. Ectromelia virus was confirmed as the causative agent of the epizootic by electron microscopy, immunohistochemistry, animal inoculations, serologic testing, virus isolation, and polymerase chain reaction. Serologic testing was of little value in the initial stages of the outbreak, although 6 weeks later, orthopoxvirus-specific antibody was detected in colony mice by indirect fluorescent antibody and enzyme-linked immunosorbent assay procedures. The outbreak originated from injection of mice with a contaminated, commercially produced, pooled mouse serum. The most relevant concern may be the unknown location of the source of the virus and the presence of a reservoir for this virus within the United States.

Electron microscopy, plaque assay and preliminary serological characterization of three ectromelia virus strains isolated in Poland in the period 1986-1988.

Three strains of viruses have been identified as ectromedia virus (EV) based on their origin, clinical features, morphology, size of virions (140 x 220 nm), replicative ability and specific cytoplasmatic fluorescence. The mean diameter of plaques produced by EV strains was 0.76 mm (range 0.5-1.0 mm). The neutralizing properties of the tested sera were evaluated by seroneutralization (SN) and hemagglutination inhibition (HAI) tests.

Evidence that NK cells and interferon are required for genetic resistance to lethal infection with ectromelia virus.

C 57 BL/6 mice developed resistance to lethal intravenous challenge with virulent (Moscow strain) ectromelia virus between 2 and 3 weeks of age. The fraction of C57 BL/6 mice in which virus was detected in spleen was significantly lower than for DBA/2 mice by day 3. Thereafter, C 57 BL/6 mice had significantly reduced virus titers in spleen compared with those of DBA/2 mice. Resistance was abrogated by treatment with anti-asialo GM1 gammaglobulin, which blocks NK cell activity, or with anti-interferon (IFN) alpha, beta. C 57 BL/6 mice carrying the bg/bg mutation, associated with a deficiency of NK cells, were highly susceptible to lethal infection as were athymic mice derived from a resistant genetic background. Virus titers in spleens of C 57 BL/6 mice treated with anti-asialo GM1 or anti-IFN alpha, beta were significantly higher 4 days after virus challenge than were titers in C 57 BL/6 mice treated with normal rabbit serum. The results strongly suggest that genetic resistance to lethal ectromelia virus infection requires non-specific host defenses such as NK cells and IFN alpha, beta that are activated during the first 3 to 4 days of infection.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. IV. Studies with the Moscow strain.

The pathogenesis and transmission of infection with the Moscow strain of ectromelia virus were studied in inbred mice. BALB/cAnNcr had high morbidity and mortality and C57BL/6Ncr (B6) mice had high morbidity and low mortality. Virus was detected in B6 mice for 2 weeks after subcutaneous (s.c.) inoculation and infected mice developed lesions compatible with acute mousepox. B6 inoculated by footpad transmitted infection to cagemates for up to five weeks and soiled cages that had housed infected mice were infectious for three weeks. S.c.-inoculated B6 mice also transmitted by contact for 2 weeks. Transmission was attributed to oronasal excretion of virus. Airborne transmission of infection between adjacent cages occurred at a low rate. Ectromelia virus-free progeny were derived from previously infected dams. These studies indicate that the highly virulent and infectious Moscow strain of ectromelia virus caused self-limiting infection in inbred mice and that direct contact is the most efficient means of transmission. These findings support the concept that mousepox can be contained by husbandry practices that minimize or eliminate the spread of infection by direct contact or fomites.