Glycoprotein B is a specific determinant of herpes simplex virus type 1 neuroinvasiveness.

Herpes simplex virus type 1 strains ANG and KOS lack neuroinvasiveness when inoculated on the footpads of mice, and because the strains are able to complement each other, the genes associated with this phenotype differ. In this study, we used marker rescue techniques to show that at least two genes cloned from ANG are required to restore neuroinvasiveness to KOS. One of the two fragments required is the 6.3-kb BamHI-A/EcoRI-D fragment (0.15 to 0.19 map units). The second has been identified as the sequence encoding glycoprotein B (gB) (UL27). Analysis of ANG and KOS DNA sequences in the relevant region of the gB gene revealed two nucleotide differences which result in amino acid differences in the gB protein. One appears to be unique to the strain of KOS used in our laboratory. The second, at codon 523 of the mature gB protein, encodes a valine in KOS and an alanine in ANG. Recombinant KOS viruses which contained ANG sequences in this region were constructed, and two independently selected recombinants demonstrated increased neuroinvasiveness in mice. From these results, we conclude that gB significantly influences neuroinvasiveness. Mechanisms by which this might occur are discussed.     

Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2.

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.     

Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS.

It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness.     

Neuroadapted yellow fever virus 17D: determinants in the envelope protein govern neuroinvasiveness for SCID mice

A molecular clone of mouse-neuroadapted yellow fever 17D virus (SPYF-MN) was used to identify critical determinants of viral neuroinvasiveness in a SCID mouse model. Virus derived from this clone differs from nonneuroinvasive YF5.2iv virus at 29 nucleotide positions, encoding 13 predicted amino acid substitutions and 2 substitutions in the 3' untranslated region (UTR). The virulence determinants of SPYF-MN for SCID mice were identified by constructing and characterizing intratypic viruses in which the E protein of SPYF-MN was expressed in the YF5.2iv background (SPYF-E) or the E protein of YF5.2iv was expressed in the SPYF-MN background (YF5.2-E). SPYF-E caused lethal encephalitis in young adult SCID mice after intraperitoneal inoculation, with average survival times and tissue virus burdens resembling those of mice inoculated with the parental SPYF-MN virus. To define which domains of the E protein are involved in neuroinvasiveness, two viruses were tested in which the amino acid substitutions in domains I-II and III were segregated. This revealed that substitutions in domain III (residues 305, 326, and 380) were critical for the neuroinvasive phenotype, based on average survival times and tissue burdens of infectious virus. Comparison of growth properties of the various intratypic viruses in cell culture indicated that no inherent defects in replication efficiency were likely to account for the biological differences observed in these experiments. These findings demonstrate that the E protein is a critical factor for yellow fever virus neuropathogenesis in the SCID mouse model and that the neuroinvasive properties depend principally on functions contributed by domain III of this protein. To assess whether critical determinants for neuroinvasion of normal ICR mice by SPYF virus were also in the E protein, sequences of viruses recovered from brains of ICR mice succumbing to encephalitis with the parental SPYF virus were derived. No differences were found in the E protein; however, two substitutions were present in the 3' UTR compared to that of SPYF-MN, one of which is predicted to alter RNA secondary structure in this region. These findings suggest that the 3' UTR may also affect neuroinvasiveness of SPYF virus in the mouse model.     

Non-invasive bioluminescence imaging for monitoring herpes simplex virus type 1 hematogenous infection

Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the replication and tropism of KOS strain HSV-1 viruses expressing the firefly luciferase reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro light emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection.     

Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34.5 gene, glycoprotein processing, and neuroinvasiveness

Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease.     

Identification, transfer, and characterization of cloned herpes simplex virus invasiveness regions.

Following peripheral inoculation of experimental animals, herpes simplex virus type 2 (HSV-2) strains are more virulent than HSV-1 strains, and clinical studies suggest that they possess enhanced virulence in humans. One dramatic type-specific difference in virulence is observed following inoculation of the chorioallantoic membrane (CAM) of the chicken embryo: HSV-2, but not HSV-1, makes large pocks on the CAM, invades the mesoderm, generalizes in the embryo, and kills the chicken. These properties have been believed to be specific for HSV-2, and their molecular basis is unknown. We now report that an HSV-1 strain, ANG, behaves even more efficiently than HSV-2. In addition, we have transferred restriction fragments of ANG DNA to another HSV-1 strain, 17 syn+, conferring the CAM virulence phenotype on the normally CAM-avirulent 17 syn+. Like ANG, these recombinant viruses are 10(6)-fold more virulent (PFU/50%) lethal dose [LD50] ratio, less than or equal to 10(2)) than the parental 17 syn+ strain (PFU/LD50 ratio, greater than or equal to 10(8)). A molecularly cloned library of ANG DNA was used to identify two distinct regions containing the virulence functions. Transfer of sequences contained in either cloned ANG EcoRI fragment A (0.49 to 0.64 map units) or F (0.32 to 0.42 map units) DNA to 17 syn+ confers CAM virulence, whereas other cloned regions of the ANG genome do not. Using cloned DNA, we derived and plaque purified several virulent recombinant viruses with inserts from either the ANG EcoRI fragment A (INV-I) or F (INV-II) areas. In each instance, the transfer of the cloned INV-I or INV-II sequences enhanced virulence for the chicken embryo 10(6)-fold (PFU/LD50 ratio, less than or equal to 10(2]. In addition, the transfer of the cloned ANG EcoRI-F INV-II sequences resulted in a 10(3)-fold enhancement of neuroinvasiveness and virulence for mice. Following footpad inoculation, these recombinants kill mice with a PFU/LD50 ratio of approximately 10(3) (similar to HSV-2 strains) compared with 10(6) for 17 syn+. Thus, we have identified, cloned, and transferred two DNA regions from HSV-1 ANG which contain virulence genes (INV-I and INV-II) important in mesodermal invasiveness on the CAM and, in the case of INV-II, neuroinvasiveness in the mouse. In each instance, the recombinant HSV-1 viruses have attained enhanced virulence beyond that described for HSV-1 strains and similar to that seen with HSV-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complementing defective viruses that express separate paramyxovirus glycoproteins provide a new vaccine vector approach

Replication-defective vaccine vectors based on vesicular stomatitis virus (VSV) lacking its envelope glycoprotein gene (G) are highly effective in animal models. However, such ΔG vectors are difficult to grow because they require complementation with the VSV G protein. In addition, the complementing G protein induces neutralizing antibodies in animals and thus limits multiple vector applications. In the process of generating an experimental Nipah virus (a paramyxovirus) vaccine, we generated two defective VSVΔG vectors, each expressing one of the two Nipah virus (NiV) glycoproteins (G and F) that are both required for virus entry to host cells. These replication-defective VSV vectors were effective at generating NiV neutralizing antibody in mice. Most interestingly, we found that these two defective viruses could be grown together and passaged in tissue culture cells in the absence of VSV G complementation. This mixture of complementing defective viruses was also highly effective at generating NiV neutralizing antibody in animals. This novel approach to growing and producing a vaccine from two defective viruses could be generally applicable to vaccine production for other paramyxoviruses or for other viruses where the expression of at least two different proteins is required for viral entry. Such an approach minimizes biosafety concerns that could apply to single, replication-competent VSV recombinants expressing all proteins required for infection.     

Virulence of La Crosse virus is under polygenic control.

To identify which RNA segments of the California serogroup bunyaviruses determine virulence, we prepared reassortant viruses by coinfecting BHK-21 cells with two wild-type parents, La Crosse/original and Tahyna/181-57 viruses, which differed about 30,000-fold in virulence. The progeny clones were screened by polyacrylamide gel electrophoresis to ascertain the phenotype of the M and S RNA segments, and RNA-RNA hybridization was used to determine the genotype of selected clones. Two or three clones of each of the six possible reassortant genotypes were characterized quantitatively for neuroinvasiveness by determining the PFU/50% lethal dose (LD50) ratio after subcutaneous injection into suckling mice. The reassortants fell into two groups. (i) Six of seven reassortants with a La Crosse M RNA segment were as virulent as the parent La Crosse virus (about 1 PFU/LD50); the one exception was strikingly different (about 1,000 PFU/LD50) and probably represents a spontaneous mutant. (ii) The seven reassortants with a Tahyna M RNA segment were about 10-fold more virulent than the parent Tahyna virus (median 1,600 PFU/LD50 for reassortants and 16,000 PFU/LD50 for Tahyna virus). A comparative pathogenesis study in suckling mice of one reassortant virus and the parent Tahyna virus confirmed the greater neuroinvasiveness of the reassortant virus. From these data it was concluded that the M RNA segment was the major determinant of virulence, but that the other two gene segments could modulate the virulence of a nonneuroinvasive California serogroup virus.     

Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

Herpes simplex virus type 1 ANG (HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, mice died of encephalitis within 4 to 5 days after intracerebral inoculation with this strain. HSV-1 ANG was serially passaged in mouse brains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed infections both nonpathogenic and pathogenic viruses replicated at the primary site of infection and spread to various organs. However, only the pathogenic phenotype could be recovered from the spinal cord and the brain. Comparison of the restriction enzyme cleavage patterns of pathogenic ANG and nonpathogenic ANG virus DNAs revealed distinct alterations in the S-segment (US) sequences bounded by coordinates 0.953 and 0.958 in the prototype orientation and by coordinates 0.862 to 0.867 in the IS orientation of the viral genome. However, it is not known whether these alterations are physiologically relevant to the observed changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold excess, the nonpathogenic HSV-1 ANG protected mice against its own pathogenic variant as well as against other pathogenic HSV-1 strains. Pathogenic HSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passages in tissue culture at either high or low multiplicity of infection.     

Polygenic control of neuroinvasiveness in California serogroup bunyaviruses.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.     

我国新分离乙型脑炎病毒株毒力特征研究

将分离自不同年代的17株乙脑毒株在小鼠脑内传代,然后将病毒在BHK21细胞单层上观察不同毒株的空斑形成大小形态,小鼠脑内和皮下途径接种观察病毒的毒力,结果显示不同毒株在BHK21细胞上形成的噬斑大小不尽相同,减毒株形成的噬斑最小。所有毒株对小鼠的脑内毒力都很强,病毒滴度高达lg8.0/mL以上,毒株间无明显差异。毒株对9～11g小日龄小鼠的皮下毒力有一定差异但不明显,但对14～16g较大日龄小鼠则差别明显。PFU/LD50的对数值差异除一株(M47株)为8.44外,其余各株差别在3.94～0.45间。本研究结果证明自然界乙脑毒株存在明显的神经外毒力差异,毒力差异与分离年代和病毒基因型无关,但从人体分离到的毒株毒力表现较强。     

The Neuroinvasiveness of a Murine Retrovirus Is Influenced by a Dileucine-Containing Sequence in the Cytoplasmic Tail of Glycosylated Gag

The tempo and intensity of retroviral neuropathogenesis are dependent on the capacity of the virus to invade the central nervous system. For murine leukemia viruses, an important determinant of neuroinvasiveness is the virus-encoded protein glycosylated Gag, the function of which in the virus life cycle is not known. While this protein is dispensable for virus replication, mutations which prevent its expression slow the spread of virus in vivo and restrict virus dissemination to the brain. To further explore the function of this protein, we compared two viruses, CasFr^KP (KP) and CasFr^KP41 (KP41), which differ dramatically in neurovirulence. KP expresses high early viremia titers, is neuroinvasive, and induces clinical neurologic disease in 100% of neonatally inoculated mice, with an incubation period of 18 to 23 days. In contrast, KP41 expresses early viremia titers 100-fold lower than those of KP, exhibits attenuated neuroinvasiveness, and induces clinical neurologic disease infrequently, with a relatively long incubation period. The genomes of these two viruses differ by only 10 nucleotides, resulting in differences at five residues, all located within the N-terminal cytoplasmic tail of glycosylated Gag. In this study, using KP as the parental virus, we systematically mutated each of the five amino acid residues to those of KP41 and found that substitution mutation of two membrane-proximal residues, E⁵³ and L⁵⁶, to K and P, respectively produced the greatest effect on early viremia kinetics and neurovirulence. These mutations disrupted the KP sequence E⁵³FLL⁵⁶, the leucine dipeptide of which suggests the possibility that it may represent a sorting signal for glycosylated Gag. Supporting this idea was the finding that alteration of this sequence motif increased the level of cell surface expression of the protein, which suggests that analysis of the intracellular trafficking of glycosylated Gag may provide further clues to its function.     

Exchanging the yellow fever virus envelope proteins with Modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice

A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the "not-known vector" flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [(3)H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 10(3)-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys(46)-->Glu substitution in NS3 as well as a single Lys(315)-->Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.     

Differences in neuroinvasion and protective innate immune pathways between encephalitic California Serogroup orthobunyaviruses

The California serogroup (CSG) of Orthobunyaviruses comprises several members capable of causing neuroinvasive disease in humans, including La Crosse orthobunyavirus (LACV), Jamestown Canyon orthobunyavirus (JCV), and Inkoo orthobunyavirus (INKV). Despite being genetically and serologically closely related, their disease incidences and pathogenesis in humans and mice differ. We have previously shown that following intraperitoneal inoculation of weanling mice, LACV was highly pathogenic while JCV and INKV were not. To determine why there were differences, we examined the ability of these viruses to invade the CNS and compared the host innate immune responses that regulated viral pathogenesis. We found that LACV was always neuroinvasive, which correlated with its high level of neuroinvasive disease. Interestingly, JCV was not neuroinvasive in any mice, while INKV was neuroinvasive in most mice. The type I interferon (IFN) response was critical for protecting mice from both JCV and INKV disease, although in the periphery JCV induced little IFN expression, while INKV induced high IFN expression. Despite their differing neuroinvasive abilities, JCV and INKV shared innate signaling components required for protection. The presence of either cytoplasmic Rig-I-Like Receptor signaling or endosomal Toll-Like Receptor signaling was sufficient to protect mice from JCV or INKV, however, inhibition of both pathways rendered mice highly susceptible to neurological disease. Comparison of IFN and IFN-stimulated gene (ISG) responses to INKV in the brains of resistant wild type (WT) mice and susceptible immune knockout mice showed similar IFN responses in the brain, but WT mice had higher ISG responses, suggesting induction of key ISGs in the brain is critical for protection of mice from INKV. Overall, these results show that the CSG viruses differ in neuroinvasiveness, which can be independent from their neuropathogenicity. The type I IFN response was crucial for protecting mice from CSG virus-induced neurological disease, however, the exact correlates of protection appear to vary between CSG viruses.     

Strain-dependent structural variants of herpes simplex virus type 1 ICP34.5 determine viral plaque size,efficiency of glycoprotein processing,and viral release and neuroinvasive disease potential

The ability of certain strains of herpes simplex virus type 1 (HSV-1) to cause encephalitis or neuroinvasive disease in the mouse upon peripheral infection is dependent on a combination of activities of specific forms of viral proteins. The importance of specific variants of ICP34.5 to neuroinvasive disease potential and its correlation with small-plaque production, inefficient glycoprotein processing, and virus release were suggested by comparison of ICP34.5 from the SP7 virus, originally obtained from the brain of a neonate with disseminated disease, and the tissue culture-passaged progeny of SP7 (SLP5 and SLP10) and the KOS321 virus. SLP5, SLP10, and KOS321 are attenuated and exhibit a large-plaque phenotype, including efficient glycoprotein processing and viral release. We show that expression of the KOS321 ICP34.5 protein in cells infected with SP7 or ICP34.5 deletion mutants promotes large plaque formation and efficient viral glycoprotein processing, while expression of the SP7 ICP34.5 protein decreases efficiency of viral glycoprotein processing. In addition, a recombinant virus, 4hS1, with the SP7 ICP34.5 gene replacing the KOS321-like ICP34.5 gene in the SLP10a background, rescues the small-plaque phenotype and neuroinvasive disease. The major difference in the ICP34.5 gene product is the number of Pro-Ala-Thr repeats in the middle region of the protein, with 18 for SP7 and 3 for KOS321. Strain-dependent differences in the ICP34.5 protein can therefore alter the tissue culture behavior and the virulence of HSV-1.     

Isolation and Molecular Characterization of a Poliovirus Type 1 Mutant That Replicates in the Spinal Cords of Mice

The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.     

Mechanism of selection of class II recombinant murine leukemia viruses in the highly leukemic strain CWD

The development of spontaneous lymphomas in CWD mice is associated with the expression of endogenous ecotropic murine leukemia viruses (MuLV) and the formation of recombinant viruses. However, the pattern of substitution of nonecotropic sequences within the envelope genes of the CWD class II recombinant viruses differs from that seen in class I recombinant MuLVs of AKR, C58, and HRS mice. To determine how CWD host genes might influence the envelope gene structure of the recombinant viruses, we characterized the responses of these mice to two different types of exogenous MuLVs. Neonatal mice injected the HRS class I recombinant PTV-1 became infected and developed T-cell lymphomas more rapidly than controls did. The inoculation of CWD mice with the leukemogenic AKR ecotropic virus SL3-3 led to the formation of recombinant MuLVs with a novel genetic structure and class II-like envelope genes, although SL3-3 generates class I recombinants in other strains. These results suggest that the absence of class I recombinant MuLVs in CWD mice is not related to the restriction of the replication or oncogenicity of class I viruses or to the absence of an appropriate ecotropic virus that can generate class I recombinants. More likely, the genes of CWD mice that direct the formation or selection of class II recombinant viruses affect the process of recombination between the ecotropic and nonecotropic envelope gene sequences.