Neuronal damage during acute viral encephalomyelitis can result directly from virus infection or indirectly from the host immune response to infection. In neurodegenerative diseases and stroke, neuronal death also can result from excess release of excitatory amino acid neurotransmitters, such as glutamate. To determine the role of glutamate excitotoxicity in fatal alphavirus-induced paralytic encephalomyelitis, we treated mice infected with neuroadapted Sindbis virus (NSV) with antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtypes of glutamate receptors. Both apoptotic and necrotic neurons in the hippocampus were decreased in animals treated with MK-801, an NMDA receptor antagonist, or GYKI-52466, an AMPA receptor antagonist. However, only AMPA receptor blockade prevented damage to spinal cord motor neurons and protected mice from paralysis and death due to NSV infection. Protection was not caused by altered virus replication because treatment did not affect virus distribution and actually delayed virus clearance. These results provide evidence that NSV infection activates neurotoxic pathways that result in aberrant glutamate receptor stimulation and neuronal damage. Furthermore, AMPA receptor-mediated motor neuron death is an important contributor to paralysis and mortality in acute alphavirus-induced encephalomyelitis.

Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells.Sindbis virus (SV) is an alphavirus, a member of the togavirus family. It is transmitted naturally to a variety of hosts by insect vectors and was originally isolated in 1952 from mosquitoes in Egypt (21). In the laboratory, SV causes an acute encephalomyelitis when inoculated into mice, leading to prominent neuronal infection. A strain of SV more neurovirulent for mice was developed by serial passage of the original isolate in mouse brain (6). Following intracerebral inoculation, this neuroadapted strain (NSV) causes a severe, often fatal, encephalomyelitis accompanied by prominent hindlimb paralysis in adult mice (7,8). Paralysis develops as a result of infection that spreads to motor neurons of the lumbosacral spinal cord which innervate the hindlimb musculature (7). The pathogenesis of this hindlimb paralysis is not completely understood; while some motor neuron degeneration has been observed in NSV-infected mice (8), other animals have been reported to recover neurologic function following infection (4, 7, 13). Our present study was carried out to determine whether paralysis results from the loss of function or the actual degeneration of infected lumbar motor neurons.We find that NSV-induced paralysis typically begins in C57BL/6 mice inoculated with 10 3 PFU of virus after 4 days of infection, and all mice show some degree of paralysis by day 5. Paralysis is initially mild with decreased hindlimb movement, but as the disease worsens, all animals become severely paralyzed and unable to move their hindlimbs in response to a pain stimulus. After 10 days, more than 90% of infected mice had died.To determine how lumbar motor neurons in NSV-infected mice were affected in comparison to the kinetics with which hindlimb paralysis developed, we examined 2-m-thick plasticembedded sections of spinal cord by light microscopy. The cell bodies of motor neurons in uninfected mice exhibited a characteristic morphology, with large nuclei, dispersed chromatin, and prominent nucleoli and Nissl substance (Fig. 1A). At day 3 of infection, most motor neurons had an overall morphology that was similar to that of uninfected cells. However, subtle pathologic changes, including mild swelling with an increase in cytoplasmic vacuolation, were noted in some of these cells (Fig. 1B). These changes progressed to severe abnormalities over several days and correlated with the onset and increasing severi...

Clearance of hepatitis C virus (HCV) infection in humans andHepatitis C virus (HCV) belongs to the genus Hepacivirus of the family Flaviviridae. Worldwide, an estimated 170 million people are infected with HCV, and it is the most common reason for liver transplantation (1). The most likely outcome of infection is chronicity, which is thought to be attributable to the ability of the virus to rapidly mutate and outpace the host immune response. HCV exists as six different genotypes and more than 30 subtypes and is highly heterogeneous between and within isolates, thus hampering studies on sequence-function relationships.The outcome of infection with HCV is thought to be determined by the initial character and vigor of the host immune response to the infection, which in turn may be determined by factors such as viral species, viral load, and route of entry. HCV clearance in humans is associated with an early, strong cellular immune response against multiple viral epitopes, and in particular against NS3 (6, 7, 27). After clearance, both CD4 ϩ and CD8 ϩ responses are maintained (26). Loss of a CD4 ϩ response can result in recurrence of HCV infection (10). A nonsustained and/or dysfunctional HCV-specific CD8 ϩ response has been implicated in HCV persistence (11,19).Because the majority of patients do not present with acute HCV infection, the early cytotoxic-T-lymphocyte (CTL) response in humans has been difficult to characterize fully. At present, the only animal model for HCV is the chimpanzee, although it has been possible to infect mice harboring chimeric mouse-human livers (23). In chimpanzees, viral clearance has been observed in Ͼ60% of infections (2). Like humans, clearance was not associated with a humoral response against the envelope proteins. Other studies have shown clearance in chimpanzees to be associated with a relatively high number of intrahepatic CTL specificities occurring synchronously and early in infection (5). The failure to clear HCV infection may be determined in part through the acquisition of mutations in epitopes recognized by CTLs (8). Such mutations may appear early in infection and persist for years in the viral population.In order to fully characterize the host response to HCV infection and relate it to the sequence of the viral population, several studies have developed full-length HCV clones, mostly of genotypes 1a and 1b, whose RNA transcripts have demonstrated infectivity after intrahepatic inoculation into chimpanzees (3,15,16,18,22,(28)(29)(30)(31). Such studies have been useful for examining the long-term immune response of the host from the initial point of infection and for examining the molecular evolution of HCV preparations with a known genomic sequence. However, despite these studies, the precise mechanisms of HCV persistence and clearance in the chimpanzee model are still unknown. We previously reported the construction of an infectious HCV genotype 1b clone and showed that it caused persistent infection in two chimpanzees (X0142 and X0234) (28). In this report, we show th...

A characteristic of all hepadnaviruses is the relaxed-circular conformation of the DNA genome within an infectious virion. Synthesis of the relaxed-circular genome by reverse transcription requires three template switches. These template switches, as for the template switches or strand transfers of other reverse-transcribing genetic elements, require repeated sequences (the donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. The mechanism for each of the template switches in hepadnaviruses is poorly understood. To determine whether sequences other than the donor and acceptor sites are involved in the template switches of duck hepatitis B virus (DHBV), a series of molecular clones which express viral genomes bearing deletion mutations were analyzed. We found that three regions of the DHBV genome, which are distinct from the donor and acceptor sites, are required for the synthesis of relaxed-circular DNA. One region, located near the 3 end of the minus-strand template, is required for the template switch that circularizes the genome. The other two regions, located in the middle of the genome and near DR2, appear to be required for plus-strand primer translocation. We speculate that these cis-acting sequences may play a role in the organization of the minus-strand DNA template within the capsid particle so that it supports efficient template switching during plus-strand DNA synthesis.

The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5r and 3r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5 end, the middle, and the 3 end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3 of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/ utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism.Hepadnaviruses are a family of DNA viruses whose primary site of replication is the liver (for reviews, see references 3 and 13). Each family member displays a narrow host range. Infections can be either acute or chronic. Liver disease is a common, but not obligatory, consequence of infection. It is thought that the host's immune response to the infection is a major contributor to the development of liver disease. Humans that are chronically infected with hepatitis B virus (HBV) are at an increased risk for the development of primary liver cancer, making HBV one of the leading causes of this malignancy worldwide.Hepadnaviruses replicate their genomes through reverse transcription of an RNA precursor (16). DNA replication occurs within the viral capsid in the cytoplasm of the infected cell. Only after synthesis of a significant portion of plus-strand DNA are capsids able efficiently to leave the cell as enveloped virions. Capsids with immature genomes inefficiently exit the cell as enveloped virions. Therefore, virus production is dependent on correct and efficient execution of each of the steps of reverse transcription within the infected cell. As with other reverse transcription schemes, template switching is integral to hepadnavirus DNA synthesis (5, 17). Template switching is the process in which the DNA strand undergoing synthesi...