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Enterovirus D68 (EV-D68) is a non-polio enterovirus that affects the respiratory system and can cause serious complications, especially in children and older people with weakened immune systems. As an emerging virus, there are no current antiviral therapies or vaccines available. Our goal was to develop a mouse model of human EV-D68 infection that mimicked the disease observed in humans and could be used for evaluation of experimental therapeutics. This is the first report of a respiratory disease model for EV-D68 infection in mice. We adapted the virus by 30 serial passages in AG129 mice, which are deficient in IFN- α/β and –γ receptors. Despite a lack of weight loss or mortality in mice, lung function measured by plethysmography, showed an increase in enhanced pause (Penh) on days 6 and 7 post-infection. In addition, as virus adapted to mice, virus titer in the lungs increased 320-fold, and the pro-inflammatory cytokines MCP-1 and RANTES increased 15-fold and 2-fold in the lung, respectively. In addition, a time course of mouse-adapted EV-D68 infection was determined in lung, blood, liver, kidney, spleen, leg muscle, spinal cord and brain. Virus in the lung replicated rapidly after intranasal inoculation of adapted virus, 10(6) CCID(50)/mL by 4 hours and 10(8.3) CCID(50)/mL by 24 hours. Virus then spread to the blood and other tissues, including spinal cord and brain. This mouse model for EV-D68 infection includes enhanced pause (Penh) as an indicator of morbidity, and viremia, virus titers and proinflammatory cytokines in the lung, and lung histopathology as indicators of disease. Our mouse-adapted virus has a similar antiviral profile to the original isolate as well as another respiratory picornavirus, rhinovirus-14. This model will be valuable in evaluating experimental therapies in the future.

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9μM) and MPA (0.8-2.5μM). 6-Azauridine toxicity was 6.1-17.5μM with NR, VTG, and WST-1, compared to 48-92μM with ALB-A and ALB-F (P

Compounds were evaluated for antiviral activity in rhabdomyosarcoma (RD) cells against a recent 2014 clinical isolate of enterovirus D68 (EV-D68), a 1962 strain of EV-68D, rhinovirus 87 (RV-87, serologically the same as EV-D68), and enterovirus 71 (EV-71). Test substances included known-active antipicornavirus agents (enviroxime, guanidine HCl, pirodavir, pleconaril, and rupintrivir), nucleobase/nucleoside analogs (3-deazaguanine and ribavirin), and three novel epidithiodiketopiperazines (KCN-2,2′-epi-19, KCN-19, and KCN-21). Of these, rupintrivir was the most potent, with 50% inhibition of viral cytopathic effect (EC 50 ) and 90% inhibition (EC 90 ) of virus yield at 0.0022–0.0053 μM against EV-D68. Enviroxime, pleconaril and the KCN compounds showed efficacy at 0.01–0.3 μM; 3-deazaguanine and pirodavir inhibited EV-D68 at 7–13 μM, and guanidine HCl and ribavirin were inhibitory at 80–135 μM. Pirodavir was active against EV-71 (EC 50 of 0.78 μM) but not against RV-87 or EV-D68, and all other compounds were less effective against EV-71 than against RV-87 and EV-D68. The most promising compound inhibiting both virus infections at low concentrations was rupintrivir. Antiviral activity was confirmed for the ten compounds in virus yield reduction (VYR) assays in RD cells, and for enviroxime, guanidine HCl, and pirodavir by cytopathic effect (CPE) assays in A549, HeLa-Ohio-1, and RD cells. These studies may serve as a basis for further pre-clinical discovery of anti-enterovirus inhibitors. Furthermore, the antiviral profiles and growth characteristics observed herein support the assertion that EV-D68 should be classified together with RV-87.

Background: The discovery of novel influenza virus inhibitors remains an important priority in light of the emergence of drug-resistant viruses. Toward this end, a library of over 6,000 compounds was tested for antiviral activity. Methods: Strains of influenza virus were evaluated by cytopathic effect (CPE) inhibition and virus yield reduction assays. Intracellular nucleoside triphosphate pools were analysed by strong anion exchange HPLC. Dihydroorotate dehydrogenase inhibition assays were conducted. Influenza virus-infected mice were treated for 5 days with D282. Results: A non-nucleoside, 4-[(4-butylphenyl)amino]-2-methylene-4-oxo-butanoic acid (D282), was discovered that inhibited influenza A and B virus CPE by 50% at 6–31 μM (giving selectivity indices of >13 to >67, based on cytotoxicity of >400 μM in stationary cell cultures). Ribavirin (positive control) was active at 14–44 μM (yielding selectivity indices of >9 to >29, with >400 μM toxicity). D282 and ribavirin inhibited virus yield by 90% at 9.5 ±3.3 and 10.8 ±3.2 μM, respectively. The antiviral activity of D282 in vitro was reversed by addition of uridine, cytidine and orotic acid. D282 exhibited an uncompetitive inhibition of mouse liver dihydroorotate dehydrogenase (inhibitor constant [Ki] of 2.3 ±0.9 μM, Michaelis constant [Km] of 150 ±16 μM). Because cellular pyrimidine biosynthesis was inhibited, D282-treated cells had decreased uridine triphosphate and cytidine triphosphate levels. D282 (≤100 mg/kg/day) failed to prevent death of mice infected with influenza. Conclusions: D282 was active against influenza A and B viruses by inhibiting de novo pyrimidine biosynthesis. Although effective in vitro, the compound, like others in its class, was devoid of antiviral activity in infected mice.

Enantiomerically pure cyclopentyl cytosine [( − )-carbodine 1 ] was synthesized from d -ribose and evaluated for its anti-influenza activity in vitro in comparison to the (+)-carbodine, (±)-carbodine and ribavirin. ( − )-Carbodine 1 exhibited potent antiviral activity against various strains of influenza A and B viruses.

Phenothiazine and derivatives were tested for inhibition of SARS-CoV replication. Phenothiazine slightly inhibited SARS-CoV replication in a neutral red (NR) uptake assay. Adding a propylamino group to give promazine reduced virus yields (VYR assay) with an EC(90)=8.3+/-2.8 microM, but without selectivity. Various substitutions in the basic phenothiazine structure did not promote efficacy. Phenazine ethosulfate was the most potent compound by VYR assay (EC(90)=6.1+/-4.3 microM). All compounds were toxic (IC(50)=6.6-74.5 microM) except for phenoxathiin (IC(50)=858+/-208 microM) and 10-(alpha-diethylamino-propionyl) phenothiazine.HCl (IC(50)=195+/-71.2 microM). Consequently, none were selective inhibitors of SARS-CoV replication (SI values

The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.

Avian influenza H5N1 infections can cause severe, lethal human infections. Whether influenza A virus treatments effectively ameliorate avian influenza H5N1 human infections is uncertain. The research objective was to evaluate the efficacy of novel zinc and other metallo-ion formulations in two influenza A mouse models. Mice infected with influenza A/Duck/MN/1525/81 (H5N1) virus were treated orally 48 h before virus exposure and then twice daily for 13 days with ZnAL42. The optimal dosing regimen for ZnAL42 was achieved at 17.28 mg/kg 48 h prior to virus exposure, twice daily for 7 days. The survival rate was 80% compared with 10% in the untreated control group and a 100% survival rate with ribavirin (75 mg/kg/day, twice a day for 5 days, beginning 4 h before virus exposure). ZnAL42 treatment significantly lessened the decline in arterial oxygen saturation (SaO2; P2. The data suggest that the prophylactic use of ZnAL42 is effective against avian influenza H5N1 or H1N1 virus infection in mice and should be further explored as an option for treating human influenza virus infections.

Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 microg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies. Studies were conducted in cell culture and in rodent animal models to determine the efficacy of interferon-alpha (IFN-α), interferon (IFN) inducers and ribavirin, alone or in combination with IFN, in treating WNV. Intraperitoneal injection of IFN-α B/D (qd for 7 days), polyIpolyC(12)U [Ampligen (every other day for 7 days)] and topically applied imiquimod (qd for 7 days), administered from 1 day before viral challenge, were effective in protecting, respectively, 100%, 100% and 70% of BALB/c mice from mortality induced by subcutaneous injection of WNV. When IFN-α B/D or Ampligen treatments were delayed to 4–6 h before viral challenge in mice, efficacy was greatly diminished. Infected Syrian golden hamsters treated with interferon alphacon-1 (Infergen) and Ampligen 4–6 h before viral challenge gained more weight and had a greater survival than saline-treated animals. A combination study of subcutaneously administered Infergen (5 to 0.05 μg/kg/day) and ribavirin (75 to 7.5 mg/kg/day) in >7 week old hamsters demonstrated that Infergen was slightly efficacious in reducing mortality and disease signs; however, it was not synergistic in its antiviral effects when combined with ribavirin. Ribavirin treatment alone increased mortality of infected hamsters. The reduced mortality correlated with reduced plasma viraemia. Since WNV-infected patients have already been treated with IFN and ribavirin and future clinical trials have been suggested, this first report of IFN alone or in combination with ribavirin in WNV-infected animal models might provide useful information for subsequent treatment of patients.

Iminosugars that are competitive inhibitors of endoplasmic reticulum (ER) α-glucosidases have been demonstrated to have antiviral activity against a diverse set of viruses. A novel iminosugar, UV-4B, has recently been shown to provide protection against lethal infections with dengue and influenza A (H1N1) viruses in mice. In the current study, the breadth of activity of UV-4B against influenza was examined ex vivo and in vivo. Efficacy of UV-4B against influenza A and B viruses was shown in primary human bronchial epithelial cells, a principal target tissue for influenza. Efficacy of UV-4B against influenza A (H1N1 and H3N2 subtypes) and influenza B was demonstrated using multiple lethal mouse models with readouts including mortality and weight loss. Clinical trials are ongoing to demonstrate safety of UV-4B and future studies to evaluate antiviral activity against influenza in humans are planned.

Background: Amantadine constitutes an interesting, diamond crystal lattice-shaped, antivirally active amine with an inhibitory effect on influenza A viruses causing common ‘flu’ in humans. Unfortunately, amantadine forfeited most of its therapeutic potential because of resistance development in recent influenza A virus isolates. The antiviral efficacy of amantadine congeners can be chemically modified, resulting in re-constitution, improvement and/or extension of antiviral activities mediated by amino-adamantyls. Methods: Newly synthesized compounds were evaluated towards HIV type-1 (HIV-1) replication in primary human lymphocytes. One N-phenacyl amantadine derivative was investigated for inhibiting the in vitro replication of respiratory viruses (influenza A viruses, influenza B virus, human parainfluenza virus type 3 and severe acute respiratory syndrome coronavirus). Results: Two ketone-stabilized 1-adamantyl singlet nitrenes were discovered serendipitously. To our best knowledge these are the first persistently stable nitrenes to be reported. Their structure was proved by determining the X-ray single crystal structure of one hydrolytic elaboration product. This salt adduct revealed an incommensurately modulated crystal structure, which was solved by extensive computational refinement. We could show that ketone-stabilized 1-adamantyl singlet nitrenes are versatile synthons for the synthesis of antiviral drug candidates. An amantadine–folate conjugate was inhibitory on HIV-1 replication in primary human lymphocytes, and one N-phenacyl amantadine derivative was inhibitory towards low pathogenic avian influenza A virus (H5N1) replication in vitro. Conclusions: These results indicate that the aromatic-aliphatic ketone-stabilized 1-adamantyl singlet nitrenes, beyond being of fundamental interest in organic chemistry, represent versatile synthons for the synthesis of new amantadine-related potentially antiviral drugs.

Various fluorodeoxyribonucleosides were evaluated for their antiviral activities against influenza virus infections in vitro and in vivo . Among the most potent inhibitors was 2′-deoxy-2′-fluorocytidine (2′-FdC). It inhibited various strains of low and highly pathogenic avian influenza H5N1 viruses, pandemic H1N1 viruses, an oseltamivir-resistant pandemic H1N1 virus, and seasonal influenza viruses (H3N2, H1N1, influenza B) in MDCK cells, with the 90% inhibitory concentrations ranging from 0.13 to 4.6 μM, as determined by a virus yield reduction assay. 2′-FdC was then tested for efficacy in BALB/c mice infected with a lethal dose of highly pathogenic influenza A/Vietnam/1203/2004 H5N1 virus. 2′FdC (60 mg/kg/d) administered intraperitoneally (i.p.) twice a day beginning 24 h after virus exposure significantly promoted survival (80% survival) of infected mice ( p = 0.0001). Equally efficacious were the treatment regimens in which mice were treated with 2′-FdC at 30 or 60 mg/kg/day (bid X 8) beginning 24 h before virus exposure. At these doses, 70–80% of the mice were protected from death due to virus infection ( p = 0.0005, p = 0.0001; respectively). The lungs harvested from treated mice at day four of the infection displayed little surface pathology or histopathology, lung weights were lower, and the 60 mg/kg dose reduced lung virus titers, although not significantly compared to the placebo controls. All doses were well tolerated in uninfected mice. 2′-FdC could also be administered as late as 72 h post virus exposure and still significantly protect 60% mice from the lethal effects of the H5N1 virus infection ( p = 0.019). Other fluorodeoxyribonucleosides tested in the H5N1 mouse model, 2′-deoxy-5-fluorocytidine and 2′-deoxy-2′,2′-difluorocytidine, were very toxic at higher doses and not inhibitory at lower doses. Finally, 2′-FdC, which was active in the H5N1 mouse model, was also active in a pandemic H1N1 influenza A infection model in mice. When given at 30 mg/kg/d (bid X 5) beginning 24 h before virus exposure), 2′-FdC also significantly enhanced survival of H1N1-infected mice (50%, p = 0.038) similar to the results obtained in the H5N1 infection model using a similar dosing regimen (50%, p in vitro and in vivo inhibition of avian influenza virus replication, 2′FdC may qualify as a lead compound for the development of agents treating influenza virus infections.

The enantiomerically pure cyclopentyl cytosine, (-)-carbodine (VI), is synthesized and evaluated for its in vitro antiinfluenza activity in comparison to its racemate and (+)-enantiomer.

Severe acute respiratory syndrome (SARS) is a highly lethal emerging disease caused by coronavirus SARS-CoV. New lethal animal models for SARS were needed to facilitate antiviral research. We adapted and characterized a new strain of SARS-CoV (strain v2163) that was highly lethal in 5- to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. Strain v2163 increased IL-1α, IL-6, MIP-1α, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. The infection largely mimicked human disease, but lung pathology lacked hyaline membrane formation. In vitro efficacy against v2163 was shown with known inhibitors of SARS-CoV replication. In v2163-infected mice, Ampligen™ was fully protective, stinging nettle lectin (UDA) was partially protective, ribavirin was disputable and possibly exacerbated disease, and EP128533 was inactive. Ribavirin, UDA, and Ampligen™ decreased IL-6 expression. Strain v2163 provided a valuable model for anti-SARS research.

Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the d-(-) enantiomer of carbodine [(-)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC50) of 0.2 microg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the l-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (-)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/(kgd) resulted in significant improvement in survival, virus load in the brain, weight change, and mean day-to-death in a mouse model of TC-83 VEEV disease. A single dose of (-)-carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (-)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (-)-carbodine is effective when initiated after the establishment of brain infection.

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 μM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO2), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO2. Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.

Compounds approved for therapeutic use and in vitro inhibitors of severe acute respiratory syndrome coronavirus (SARS-CoV) were evaluated for inhibition in the mouse SARS-CoV replication model. A hybrid interferon, interferon alpha (IFN-α) B/D, and a mismatched double-stranded (ds) RNA interferon (IFN) inducer, Ampligen® (poly I:poly C124), were the only compounds that potently inhibited virus titres in the lungs of infected mice as assessed by CPE titration assays. When mice were dosed intraperitoneally (i.p.) with IFN-α B/D once daily for 3 days beginning 4 h after virus exposure, SARS-CoV replication in the lungs of infected mice was reduced by 1 log10 at 10,000 and 32,000 IU; at the highest dose of 100,000 IU, virus lung titres were below detectable limits. Ampligen® used i.p. at 10 mg/kg 4 h prior to virus exposure also reduced virus lung titres to below detectable limits. Nelfinavir, β-D-N4-hydroxycytidine, calpain inhibitor VI, 3-deazaneplanocin A and Alferon® (human leukocyte IFN-α-n3) did not significantly reduce lung virus titres in mice. Anti-inflammatory agents, chloroquine, amodiaquin and pentoxifylline, were also inactive in vivo, suggesting that although they may be useful in ameliorating the hyperinflammatory response induced by the virus infection, they will not significantly reduce the replication of the virus, the inducer of inflammatory response. Thus, anti-inflammatory agents may only be useful in treating virus lung infections if used in combination with agents that inhibit virus replication. In summary, the data suggest that induction of IFN by mismatched dsRNA or actual treatment with exogenous IFN-α can inhibit SARS-CoV replication in the lungs of mice.

The objectives of this study were to determine if injection of West Nile virus (WNV) into timed-pregnant mice would result in fetal infection and if administration of WNV-reactive immunoglobulin would increase dam survival and reduce fetal viral titers. Dams injected on 7.5 days post-coitus (dpc) had detectable viral titers in the placenta 10.5 dpc with a mean titer of 10 4.9 50% cell-culture infectious doses per gram of tissue (CCID 50 /g tissue). The mean placental titer increased to 10 8.6 CCID 50 /g tissue at 12.5 dpc. Infectious virus was detectable 12.5 dpc in 10 of 10 fetuses with a mean titer of 10 7.5 CCID 50 /g tissue. Treatment of dams (challenged with WNV on 7.5 dpc) with WNV-reactive human immunoglobulin (Ig) on 8.5 and 9.5 dpc resulted in a significant reduction of virus in fetuses as compared with non-reactive human Ig-treated females on 12.5 dpc ( P ≤ 0.001). Treatment also resulted in survival of dams to term. Treatment of dams with WNV-reactive human Ig on 12.5 and 13.5 dpc also resulted in reduction of viral titer on 14.5 dpc, indicating that later treatment may also be efficacious. This suggests that Ig treatment may be useful in treating fetal WNV infection in women.