John W. Huggins

A Mouse Model for Evaluation of Prophylaxis and Therapy of Ebola Hemorrhagic Fever

The Zaire subtype of Ebola virus (EBO-Z) is lethal for newborn mice, but adult mice are resistant to the virus, which prevents their use as an animal model of lethal Ebola infection. We serially passed EBO-Z virus in progressively older suckling mice, eventually obtaining a plaque-purified virus that was lethal for mature, immunocompetent BALB/c and C57BL/6 inbred and ICR (CD-1) outbred mice. Pathologic changes in the liver and spleen of infected mice resembled those in EBO-Z-infected primates. Virus titers in these tissues reached 10(9) pfu/g. The LD50 of mouse-adapted EBO-Z virus inoculated into the peritoneal cavity was approximately 1 virion. Mice were resistant to large doses of the same virus inoculated subcutaneously, intradermally, or intramuscularly. Mice injected peripherally with mouse-adapted or intraperitoneally with non-adapted EBO-Z virus resisted subsequent challenge with mouse-adapted virus.

Cidofovir Protects Mice against Lethal Aerosol or Intranasal Cowpox Virus Challenge

The efficacy of cidofovir for treatment of cowpox virus infection in BALB/c mice was investigated in an effort to evaluate new therapies for virulent orthopoxvirus infections of the respiratory tract in a small animal model. Exposure to 2(-5)x10(6) pfu of cowpox virus by aerosol or intranasally (inl) was lethal in 3- to 7-week-old animals. One inoculation of 100 mg/kg cidofovir on day 0, 2, or 4, with respect to aerosol infection, resulted in 90%-100% survival. Treatment on day 0 reduced peak pulmonary virus titers 10- to 100-fold, reduced the severity of viral pneumonitis, and prevented pulmonary hemorrhage. The same dose on day -6 to 2 protected 80%-100% of inl infected mice, whereas 1 inoculation on day -16 to -8 or day 3 to 6 was partially protective. Cidofovir delayed but did not prevent the death of inl infected mice with severe combined immunodeficiency. Treatment at the time of tail scarification with vaccinia virus did not block vaccination efficacy.

Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.

We assessed the activities of 24 different antiviral compounds against smallpox (two strains of variola major and one of variola minor), monkeypox, vaccinia and cowpox viruses by a neutral red uptake assay. To establish assay parameters, we examined viral replication and its inhibition at various times postinfection and at several multiplicities of infection. Drugs were selected to target a range of functions involved in viral replication. Eight compounds (cidofovir, cyclic HPMPC (cHPMPC), HPMPA, ribavirin, tiazofurin, carbocyclic 3-deazaadenosine, 3-deazaneplanocin A and DFBA (1-(2,4-difluorobenzyloxy)adenosine perchlorate)-a derivative of adenosine N1-oxide) inhibited the replication of all three variola strains and the other orthopoxviruses at drug concentrations within a pharmacologically achievable range. Two others (methisazone and bis-POM-PMEA) showed a lesser degree of antiviral effect, while the remainder were inactive. To examine possible naturally occurring drug resistance among a large number of variola isolates obtained from different geographical regions and at different times, we examined the sensitivity of 35 different strains of variola as well as other orthopoxviruses to a subset of three of the most active compounds: cidofovir, cHPMPC, and ribavirin. Preliminary data indicate that nearly all isolates appear to have similar drug sensitivities. These findings are currently being verified and expanded.

Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

ABSTRACT We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However, all five assays had nearly 100% sensitivity on both machines with samples above the LOD (>12 gene copies). These real-time PCR assays represent a battery of tests to screen for and confirm the presence of variola virus DNA. The early detection of a smallpox outbreak is crucial whether the incident is an act of bioterrorism or an accidental occurrence.

Characterization of Wild-Type and Cidofovir-Resistant Strains of Camelpox, Cowpox, Monkeypox, and Vaccinia Viruses

ABSTRACT Cidofovir {[(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] [HPMPC]}-resistant forms of camelpox, cowpox, monkeypox, and vaccinia viruses were developed by prolonged passage in Vero 76 cells in the presence of drug. Eight- to 27-fold-higher concentrations of cidofovir were required to inhibit the resistant viruses than were needed to inhibit the wild-type (WT) viruses. Resistant viruses were characterized by determining their cross-resistance to other antiviral compounds, examining their different replication abilities in two cell lines, studying the biochemical basis of their drug resistance, and assessing the degrees of their virulence in mice. These viruses were cross resistant to cyclic HPMPC and, with the exception of vaccinia virus, to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. Three of the four resistant cowpox and monkeypox viruses exhibited reduced abilities to infect and replicate in 3T3 cells compared to their abilities in Vero 76 cells. Compared to the WT virus polymers the resistant cowpox virus DNA polymerase was 8.5-fold less sensitive to inhibition by cidofovir diphosphate, the active form of the drug. Intracellular phosphorylation of [3H]cidofovir was not stimulated or inhibited by infection with resistant cowpox virus. In intranasally infected BALB/c mice, WT cowpox virus was 80-fold more virulent than the resistant virus. Cidofovir treatment (100 mg/kg of body weight, given one time only as early as 5 min after virus challenge) of a resistant cowpox virus infection could not protect mice from mortality. However, the drug prevented mortality in 80 to 100% of the mice treated with a single 100-mg/kg dose at 1, 2, 3, or 4 days after WT virus challenge. By application of these results to human orthopoxvirus infections, it is anticipated that resistant viruses may be untreatable with cidofovir but their virulence may be attenuated. Studies will need to be conducted with cidofovir-resistant monkeypox virus in monkeys to further support these hypotheses.

Antiviral Drug Therapy of Filovirus Infections: S-Adenosylhomocysteine Hydrolase Inhibitors Inhibit Ebola Virus In Vitro and in a Lethal Mouse Model

Ebola (subtype Zaire) viral replication was inhibited in vitro by a series of nine nucleoside analogue inhibitors of S-adenosylhomocysteine hydrolase, an important target for antiviral drug development. Adult BALB/c mice lethally infected with mouse-adapted Ebola virus die 5-7 days after infection. Treatment initiated on day 0 or 1 resulted in dose-dependent protection, with mortality completely prevented at doses > or =0.7 mg/kg every 8 h. There was significant protection (90%) when treatment was begun on day 2, at which time, the liver had an average titer of 3 x 10(5) pfu/g virus and the spleen had 2 x 10(6) pfu/g. Treatment with 2.2 mg/kg initiated on day 3, when the liver had an average titer of 2 x 10(7) pfu/g virus and the spleen had 2 x 10(8) pfu/g, resulted in 40% survival. As reported here, Carbocyclic 3-deazaadenosine is the first compound demonstrated to cure animals from this otherwise lethal Ebola virus infection.

Synergistic antiviral effects of ribavirin and the C-nucleoside analogs tiazofurin and selenazofurin against togaviruses, bunyaviruses, and arenaviruses.

Binary combinations of the N-nucleoside ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and the C-nucleoside analog selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) or tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) were tested in vitro for activity against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, Rift Valley fever, Korean hemorrhagic fever, and Pichinde viruses. The 50% effective dose for each compound alone or in a series of combinations was determined with a plaque reduction assay. Combinations of ribavirin and selenazofurin were synergistic against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, and Pichinde viruses, with fractional inhibitory concentrations of 0.1, 0.2, 0.4, 0.4, respectively, but showed additive effects against Korean hemorrhagic fever and Rift Valley fever viruses. Combinations of ribavirin and tiazofurin were synergistic against yellow fever and Japanese encephalitis (fractional inhibitory concentrations, 0.41 and 0.48, respectively) but showed additive effects against Korean hemorrhagic fever virus. Combinations of selenazofurin and tiazofurin had additive effects against Japanese encephalitis, yellow fever, and Korean hemorrhagic fever viruses. The effect of combinations on cell toxicity was additive, both in monolayers of nondividing cells incubated under agar for the same period as the plaque assay and for rapidly dividing cells given short-term exposure (4 h), followed by determination of the proportion of surviving cells with a colony forming assay.

Experience with intravenous ribavirin in the treatment of hemorrhagic fever with renal syndrome in Korea

Abstract Results of a clinical study using intravenous (IV) ribavirin for treating Department of Defense personnel with hemorrhagic fever with renal syndrome (HFRS) acquired in Korea from 1987 to 2005 were reviewed to determine the clinical course of HFRS treated with IV ribavirin. A total of 38 individuals enrolled in the study had subsequent serological confirmation of HFRS. Four of the 38 individuals received three or fewer doses of ribavirin and were excluded from treatment analysis. Of the remaining 34 individuals, oliguria was present in one individual at treatment initiation; none of the remaining 33 subjects developed oliguria or required dialysis. The mean peak serum creatinine was 3.46mg/dl and occurred on day 2 of ribavirin therapy. Both the peak serum creatinine and the onset of polyuria occurred on mean day 6.8 of illness. Reversible hemolytic anemia was the main adverse event of ribavirin, with a ≥25% decrease in hematocrit observed in 26/34 (76.5%) individuals. While inability to adjust for all baseline variables prevents comparison to historical cohorts in Korea where oliguria has been reported in 39–69% cases and dialysis required in approximately 40% HFRS cases caused by Hantaan virus, the occurrence of 3% oliguria and 0% dialysis requirement in the treatment cohort is supportive of a previous placebo-controlled HFRS trial in China where IV ribavirin given early resulted in decreased occurrence of oliguria and decreased severity of renal insufficiency.

Smallpox Vaccine Does Not Protect Macaques with AIDS from a Lethal Monkeypox Virus Challenge

It is unknown whether smallpox vaccination would protect human immunodeficiency virus type 1 (HIV-1)-infected individuals, because helper CD4(+) cells, the targets of HIV-1 infection, are necessary for the induction of both adaptive CD8(+) cell and B cell responses. We have addressed this question in macaques and have demonstrated that, although smallpox vaccination is safe in immunodeficient macaques when it is preceded by immunization with highly attenuated vaccinia strains, the macaques were not protected against lethal monkeypox virus challenge if their CD4(+) cell count was <300 cells/mm(3). The lack of protection appeared to be associated with a defect in vaccinia-specific immunoglobulin (Ig) switching from IgM to IgG. Thus, vaccination strategies that bypass CD4(+) cell help are needed to elicit IgG antibodies with high affinity and adequate tissue distribution and to restore protection against smallpox in severely immunocompromised individuals.

Antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro

Two inhibitors of cellular inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and ribavirin, were evaluated for inhibitory activity against orthopoxviruses. Unrelated antipoxvirus agents tested for comparison included 6-azauridine, cidofovir (HPMPC) and cyclic HPMPC. MPA inhibited camelpox, cowpox, monkeypox and vaccinia viruses by 50% in plaque reduction assays at 0.2–3 μM in African green monkey kidney (Vero 76) and mouse 3T3 cells. Ribavirin was considerably more active in 3T3 cells (50% inhibition at 2-l2 μM) than in Vero 76 cells (inhibitory at 30–250 μM) against these viruses. In cytotoxicity assays, MPA and ribavirin were more toxic to replicating cells than to stationary cell monolayers, with greater toxicity seen in 3T3 than in Vero 76 cells. The superior antiviral potency and increased toxicity of ribavirin in 3T3 cells was related to greater accumulation of mono-, di- and triphosphate forms of the drug compared with Vero 76 cells. For both MPA and ribavirin, virus inhibition was closely correlated to the extent of suppression of intracellular guanosine triphosphate (GTP) pools. Treatment with extracellular guanosine (which restored intracellular GTP levels) did not lead to complete reversal of the anticowpox virus activity of ribavirin. This suggests that other modes of virus inhibition also appear to contribute to the anti-orthopoxvirus activity of ribavirin. Biological differences in mode of action and immunosuppressive potential between ribavirin and MPA may account for why the former compound is active against orthopoxvirus infections in animals and the latter inhibitor is not.