Vaccinia Virus Dissemination Research Articles

Daily ingestion of the probiotic Lactobacillus paracasei ST11 decreases Vaccinia virus dissemination and lethality in a mouse model.

Vaccinia virus (VACV) is an important pathogen. Although studies have shown relationships between probiotics and viruses, the effect of probiotics on VACV infection is unknown. Therefore, this work aims to investigate the probiotics effects on VACV infection. Mice were divided into four groups, two non-infected groups, one receiving the probiotic, the other one not receiving it, and two groups infected intranasally with VACV Western Reserve (VACV-WR) receiving or not receiving the probiotic. Viral titres in organs and cytokine production in the lungs were analysed. Lung samples were also subjected to histological analysis. The intake of probiotic results in reduction in viral spread with a significant decrease of VACV titer on lung, liver and brain of treated group. In addition,treatment with the probiotic results in attenuated mice lung inflammation showing fewer lesions on histological findings and decreased lethality in mice infected with VACV. The ingestion of Lactobacillus paracasei ST11 (LPST11) after VACV infection resulted in 2/9 animal lethality compared with 4/9 in the VACV group. This is the first study on probiotics and VACV interactions, providing not only information about this interaction, but also proposing a model for future studies involving probiotics and other poxvirus.

Vaccinia virus dissemination requires p21-activated kinase 1.

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

Filaggrin deficiency promotes the dissemination of cutaneously inoculated vaccinia virus

Eczema vaccinatum is a life-threatening complication of smallpox vaccination in patients with atopic dermatitis (AD) characterized by dissemination of vaccinia virus (VV) in the skin and internal organs. Mutations in the filaggrin (FLG) gene, the most common genetic risk factor for AD, confer a greater risk for eczema herpeticum in patients with AD, suggesting that it impairs the response to cutaneous viral infections. We sought to determine the effects of FLG deficiency on the response of mice to cutaneous VV inoculation. VV was inoculated by means of scarification of unsensitized skin or skin topically sensitized with ovalbumin in FLG-deficient flaky tail (ft/ft) mice or wild-type (WT) control mice. The sizes of primary and satellite skin lesions were measured, and hematoxylin and eosin staining was performed. VV genome copy numbers and cytokine mRNA levels were measured by using quantitative PCR. VV inoculation in unsensitized skin of ft/ft mice, independent of the matted hair mutation, resulted in larger primary lesions, more abundant satellite lesions, heavier viral loads in internal organs, greater epidermal thickness, dermal cellular infiltration, and higher local Il17a, Il4, Il13, and Ifng mRNA levels than in WT control mice. VV inoculation at sites of topical ovalbumin application amplified all of these features in ft/ft mice but had no detectable effect in WT control mice. The number of satellite lesions and the viral loads in internal organs after cutaneous VV inoculation were significantly reduced in both unsensitized and topically sensitized ft/ftxIl17a(-/-) mice. FLG deficiency predisposes to eczema vaccinatum. This is mediated primarily through production of IL-17A.

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin–based motility

Vaccinia virus dissemination relies on the N-WASP-ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP-ARP2/3 and Rac1-FHOD1 pathways.

Effects of Postchallenge Administration of ST-246 on Dissemination of IHD-J-Luc Vaccinia Virus in Normal Mice and in Immune-Deficient Mice Reconstituted with T Cells

Whole-body bioimaging was used to study dissemination of vaccinia virus (VACV) in normal and in immune deficient (nu(-)/nu(-)) mice protected from lethality by postchallenge administration of ST-246. Total fluxes were recorded in the liver, spleen, lungs, and nasal cavities of live mice after intranasal infection with a recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve were calculated for individual mice to assess viral loads. Treatment for 2 to 5 days of normal BALB/c mice with ST-246 at 100 mg/kg starting 24 h postchallenge conferred 100% protection and reduced viral loads in four organs compared to control mice. Mice also survived after 5 days of treatment with ST-246 at 30 mg/kg, and yet the viral loads and poxes were higher in these mice compared to 100-mg/kg treatment group. Nude mice were not protected by ST-246 alone or by 10 million adoptively transferred T cells. In contrast, nude mice that received T cells and 7-day treatment with ST-246 survived infection and exhibited reduced viral loads compared to nonreconstituted and ST-246-treated mice after ST-246 was stopped. Similar protection of nude mice was achieved using adoptively transferred 1.0 and 0.1 million, but not 0.01 million, purified T cells or CD4(+) or CD8(+) T cells in conjunction with ST-246 treatment. These data suggest that ST-246 protects immunocompetent mice from lethality and reduces viral dissemination in internal organs and poxvirus lesions. Furthermore, immune-deficient animals with partial T cell reconstitution can control virus replication after a course of ST-246 and survive lethal vaccinia virus challenge.

Filaggrin Deficiency Impairs Viral Containment in Mice Cutaneously Inoculated with Vaccinia Virus (VV)

Eczema Vaccinatum (EV) is a life threatening complication of exposure to smallpox vaccination in patients with atopic dermatitis (AD) characterized by dissemination of VV in skin and internal organs. Filaggrin deficiency is present in up to 48% of patients with AD, and filaggrin null mutations are associated with eczema herpeticum. We examined the effects of filaggrin deficiency on the response of mice to cutaneous VV inoculation.

Quantitative bioimaging of WRvFire and of IHDJ-Luc vaccinia virus dissemination in mice and the effects of prophylactic and therapeutic treatments with IgG and antiviral. (45.18)

Abstract Whole body bioluminescence imaging was employed to test the effects of IgG-based therapies and Cidofovir on lethality and virus dissemination in BALB/c mice challenged intranasally with 10^5 pfu of recombinant vaccinia viruses (VV) expressing luciferase, WRvFire or IHDJ-Luc. Both viruses induced lethality within 6-8 days, with slightly different kinetics of replication in internal organs. Using statistical analysis of 80 animals/group we determined that replication of IHDJ-Luc (increased Extracellular Enveloped Virion compared to WR) rose faster and peaked earlier than WRvFire (predominantly Intracellular Mature Virion) in the liver and spleen. In the nasal cavity, WRvFire persisted at significantly higher levels than IHDJ-Luc. Vaccinia immunoglobulin for intravenous injection (VIGIV) administered at day -2 protected 100% of animals from WRvFire and IHDJ-Luc infections. Sym002 (26 anti-vaccinia human mAbs) completely protected mice from both challenges at 300-fold lower dose than VIGIV. A single treatment of Cidofovir (100 mg/kg) at day 1, 2, or 3 p.i. fully protected mice from WRvFire challenge. Complete protection conferred by treatments correlated with significant reductions in bioluminescence in nasal cavity or lungs and in both the liver and spleen between days 1-5. Our data demonstrated that despite some differences observed in the kinetics of VV dissemination, infections with these viruses were similarly susceptible to prophylactic and therapeutic treatments.

ST-246 Inhibits In Vivo Poxvirus Dissemination, Virus Shedding, and Systemic Disease Manifestation

Orthopoxvirus infections, such as smallpox, can lead to severe systemic disease and result in considerable morbidity and mortality in immunologically naïve individuals. Treatment with ST-246, a small-molecule inhibitor of virus egress, has been shown to provide protection against severe disease and death induced by several members of the poxvirus family, including vaccinia, variola, and monkeypox viruses. Here, we show that ST-246 treatment not only results in the significant inhibition of vaccinia virus dissemination from the site of inoculation to distal organs, such as the spleen and liver, but also reduces the viral load in organs targeted by the dissemination. In mice intranasally infected with vaccinia virus, virus shedding from the nasal and lung mucosa was significantly lower (approximately 22- and 528-fold, respectively) upon ST-246 treatment. Consequently, virus dissemination from the nasal site of replication to the lung also was dramatically reduced, as evidenced by a 179-fold difference in virus levels in nasal versus bronchoalveolar lavage. Furthermore, in ACAM2000-immunized mice, vaccination site swabs showed that ST-246 treatment results in a major (approximately 3,900-fold by day 21) reduction in virus detected at the outside surfaces of lesions. Taken together, these data suggest that ST-246 would play a dual protective role if used during a smallpox bioterrorist attack. First, ST-246 would provide therapeutic benefit by reducing the disease burden and lethality in infected individuals. Second, by reducing virus shedding from those prophylactically immunized with a smallpox vaccine or harboring variola virus infection, ST-246 could reduce the risk of virus transmission to susceptible contacts.

Quantitative bioimaging of the WR and IHDJ vaccinia virus dissemination and the effects of prophylactic immunoglobulin treatments (128.23)

Abstract Protection from lethal challenge with recombinant WR-Luciferase (TK+) and IHDJ-Luciferase (TK+) vaccinia viruses (VV) was studied by detecting firefly luciferase activity in live Balb/c mice using IVIS technology. High correlation (R2=0.8-0.9) was observed between viral loads in the organs measured by plaque assay and bioluminescence. WR- and IHDJ VV that generate predominantly IMV and EEV forms, respectively, induced lethality within 8-10 days after intranasal challenge with 10^5 pfu yet their dissemination and susceptibility to therapies differed. WR persisted at significantly higher levels than IHDJ at the site of challenge, while IHDJ replicated better in the liver in agreement with the roles of EEV and IMV in host dissemination and inter host transmission. Vaccinia immunoglobulin (VIGIV) at Day -2 completely protected from the WR but not from IHDJ challenge. Sym002, a mixture of 26 human anti-vaccinia mAbs (Symphogen, Denmark), completely protected from WR challenge at 1000 lower dose than VIGIV, but only partially protected from IHDJ. Protection by Sym002 correlated with a significantly reduced flux at the nasopharyngeal area and in the liver. Importantly, our study detected significantly higher bioluminescence at the nasopharyngeal area and in the lungs in non-survivors than in survivors suggesting that bioimaging can substitute for lethality endpoint for evaluation of anti-smallpox treatments.

Role for CCR5 in Dissemination of Vaccinia Virus In Vivo

In an earlier report, we provided evidence that expression of CCR5 by primary human T cells renders them permissive for vaccinia virus (VACV) replication. This may represent a mechanism for dissemination throughout the lymphatic system. To test this hypothesis, wild-type CCR5(+/+) and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies using different infective doses of VACV, we identified viral replication in the lungs of both CCR5(+/+) and CCR5(-/-) mice, yet there were diminished viral loads in the spleens and brains of CCR5(-/-) mice compared with CCR5(+/+) mice. Moreover, in association with VACV infection, we provide evidence for CD4+ and CD8+ T-cell as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5(+/+) but not CCR5(-/-) mice, and we show that the CCR5-expressing T cells harbor virus. We demonstrate that this CCR5 dependence is VACV specific, since CCR5(-/-) mice are as susceptible to intranasal influenza virus (A/WSN/33) infection as CCR5(+/+) mice. In a final series of experiments, we provide evidence that adoptive transfer of CCR5(+/+) bone marrow leukocytes into CCR5(-/-) mice restores VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data suggest a novel role for CCR5 in VACV dissemination in vivo.

250 A role for CCR5 in dissemination of vaccinia virus in vivo

250 A role for CCR5 in dissemination of vaccinia virus in vivo

Lack of Transmission of Vaccinia Virus

Lack of Transmission of Vaccinia Virus

The microepidemiology of vaccinial infection as studied in HeLa cell stationary cultures

The dissemination of vaccinia virus in HeLa cell cultures grown on glass was studied by two different experimental approaches. The addition of antiserum immediately after the adsorption of 30–200 PFU of virus per culture failed to prevent the formation of foci of infection. Antibody, however, restricted the rate of progression of the infection. Infected cells were found to be capable of initiating foci of infection in the continued presence of antibody, even in cell sheets previously treated with antiserum. Virus release into the medium was very slow and scanty, since plaque counts in cultures receiving antiserum as late as 30 hours after the termination of the virus adsorption period were only slightly higher than those in cultures receiving antiserum immediately after virus adsorption. Infectivity assays of the fluid and cellular phases of monolayer cultures which had adsorbed 40–200 PFU of virus before the addition of an antibody-free maintenance medium showed a rise in the number of infected cells as early as 7 hours postadsorption, and confirmed the slow and limited release of newly formed virus into the medium. The rate at which the number of infected cells increased following inoculation of cultures with virus particles or infected cells was found to be dependent on the density of the cell population. It is concluded that passage of virus from an infected cell to immediately adjoining cells is predominantly responsible for the progress of vaccinial infection in HeLa cell monolayer cultures. The possible routes involved in this passage are discussed.