A series of pyrazinecarboxamide derivatives T-705 (favipiravir), T-1105 and T-1106 were discovered to be candidate antiviral drugs. These compounds have demonstrated good activity in treating viral infections in laboratory animals caused by various RNA viruses, including influenza virus, arenaviruses, bunyaviruses, West Nile virus (WNV), yellow fever virus (YFV), and foot-and-mouth disease virus (FMDV). Treatment has in some cases been effective when initiated up to 5-7 days after virus infection, when the animals already showed signs of illness. Studies on the mechanism of action of T-705 have shown that this compound is converted to the ribofuranosyltriphosphate derivative by host enzymes, and this metabolite selectively inhibits the influenza viral RNA-dependent RNA polymerase without cytotoxicity to mammalian cells. Interestingly, these compounds do not inhibit host DNA and RNA synthesis and inosine 5'-monophosphate dehydrogenase (IMPDH) activity. From in vivo studies using several animal models, the pyrazinecarboxamide derivatives were found to be effective in protecting animals from death, reducing viral burden, and limiting disease manifestations, even when treatment was initiated after virus inoculation. Importantly, T-705 imparts its beneficial antiviral effects without significant toxicity to the host. Prompt development of these compounds is expected to provide effective countermeasures against pandemic influenza virus and several bioweapon threats, all of which are of great global public health concern given the current paucity of highly effective broad-spectrum drugs.

West Nile virus (WNV) has emerged as a significant cause of epidemic viral encephalitis and flaccid limb paralysis, yet the mechanism by which it enters the CNS remains uncertain. We used compartmentalized neuron cultures to demonstrate that WNV spreads in both retrograde and anterograde directions via axonal transport. Transneuronal spread of WNV required axonal release of viral particles and was blocked by addition of a therapeutic neutralizing antibody. To test the physiologic significance of axonal transport in vivo, we directly inoculated the sciatic nerve of hamsters with WNV. Intrasciatic infection resulted in paralysis of the hind limb ipsilateral but not contralateral to the injection site. Limb paralysis was blocked either by surgical transection of the sciatic nerve or treatment with the therapeutic neutralizing antibody. Collectively, these studies establish that WNV undergoes bidirectional spread in neurons and that axonal transport promotes viral entry into the CNS and acute limb paralysis. Moreover, antibody therapeutics directly inhibit transneuronal spread of WNV infection and prevent the development of paralysis in vivo.flavivirus ͉ neuron ͉ retrograde ͉ transneuronal spread W est Nile virus (WNV) is a neurotropic member of the Flaviviridae family of RNA viruses and is related to other important arthropod-borne human pathogens. WNV is maintained in an enzootic cycle between mosquitoes and birds and has become an important global cause of epidemic encephalitis. Since its emergence in the United States in 1999, Ϸ26,000 cases of symptomatic WNV infection have been confirmed (www.cdc. gov/ncidod/dvbid/westnile/surv&control.htm#maps), and seroprevalence studies suggest that several million people have been infected (1).Rodent models have provided insight into the mechanisms of WNV spread to the CNS. After s.c. inoculation, WNV-infected dendritic cells traffic to the draining lymph node, resulting in a primary viremia and infection of peripheral tissues. Within 6 days, WNV is cleared from the serum and peripheral organs and enters the CNS and induces neurological disease (reviewed in ref.2). Nonetheless, the specific mechanisms by which WNV or other neurotropic flaviviruses enter into the CNS are largely unknown. CNS infection may occur in part via hematogenous spread, as increased viremia in immunodeficient mice (2) and TNF-␣-mediated changes in blood-brain-barrier permeability correlate with earlier CNS entry (3).Axonal transport from infected peripheral neurons mediates CNS entry and pathogenesis of viruses in the Herpesviridae, Rhabdoviridae, and Picornaviridae families (4-6). Viral spread in neurons is generally mediated by fast axonal transport, a microtubule-associated, anterograde and retrograde transport system. In classical studies, CNS infection of rabies virus or poliovirus was prevented by axonal ligation or degeneration (7,8). Insights into the biology of axonal spread have been facilitated by the development of compartmentalized, or Campenot, chambers for culturing neurons (9). These s...

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3 ؊/؊ mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3 ؊/؊ mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.

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 5–6 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 inhihibitors 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.

We evaluated two types of compounds for efficacy in inhibiting SARSCoV replication in vitro: calpain inhibitors (a class of cellular cysteine proteinases) and a number of nucleoside analogues. Cytopathic effect reduction assays visually determined with spectrophotometric verification by neutral red (NR) uptake assay were used to evaluate cytotoxicity and antiviral potency of the compounds. Significantly inhibitory compounds were then evaluated in virus yield reduction assays. Two calpain inhibitors, Val-Leu-CHO (calpain inhibitor VI) and Z-Val-Phe-Ala-CHO (calpain inhibitor III), were the most potent inhibitors of SARSCoV. By virus yield reduction assay, calpain inhibitor VI had a 90% effective concentration (EC90) of 3 μM and calpain inhibitor III had an EC90 of 15 μM. β-D-N4-hydroxycytidine was the most selective nucleoside analogue inhibitor with an EC90 of 6 μM by virus yield reduction assay. These compounds or analogues warrant further evaluation as potential therapies for treating SARS or could be used as lead compounds for discovery of more potent SARSCoV inhibitors.