Attenuation Of Neurovirulence Research Articles

Wide circulation of type 1 vaccine-derived poliovirus strains in clinical specimens from suspected cases of poliomyelitis, their contacts and in wastewater in Madagascar since late 2020

Madagascar has faced three major outbreaks of vaccine-derived polioviruses (VDPVs) in recent decades, with VDPV type 1 reemerging in late 2020. Here, we report the molecular characterization of these cVDPV1 strains. WHO protocols were used for poliovirus detection in stool and wastewater samples. Molecular genotyping was based on the 5′ non-coding (5′NC), VP1, and 3Dpol regions. From 2020 to 2022, 92 of 5690 stool samples and 129 of 1046 wastewater samples tested positive for cVDPV1. Genetic analysis of the VP1 gene revealed 1.3%–6.1% variability compared to the Sabin strain. Most sequences showed mutations at neurovirulence attenuation sites. Phylogenetic analysis distributed strains into four genogroups originating from Southern Madagascar. All analyzed cVDPV1 strains were recombinant, containing mutated oral polio vaccine sequences in VP1 and type C enterovirus sequences in other regions. This study demonstrated that all strains were closely related during this epidemic.

Attenuation of neurovirulence of chikungunya virus by a single amino acid mutation in viral E2 envelope protein

BackgroundChikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications.MethodsIn the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors.ResultsCHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood–brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedesalbopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice.ConclusionsThese findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.

Single dose of chimeric dengue-2/Zika vaccine candidate protects mice and non-human primates against Zika virus

The development of a safe and effective Zika virus (ZIKV) vaccine has become a global health priority since the widespread epidemic in 2015-2016. Based on previous experience in using the well-characterized and clinically proven dengue virus serotype-2 (DENV-2) PDK-53 vaccine backbone for live-attenuated chimeric flavivirus vaccine development, we developed chimeric DENV-2/ZIKV vaccine candidates optimized for growth and genetic stability in Vero cells. These vaccine candidates retain all previously characterized attenuation phenotypes of the PDK-53 vaccine virus, including attenuation of neurovirulence for 1-day-old CD-1 mice, absence of virulence in interferon receptor-deficient mice, and lack of transmissibility in the main mosquito vectors. A single DENV-2/ZIKV dose provides protection against ZIKV challenge in mice and rhesus macaques. Overall, these data indicate that the ZIKV live-attenuated vaccine candidates are safe, immunogenic and effective at preventing ZIKV infection in multiple animal models, warranting continued development.

A single-dose live attenuated chimeric vaccine candidate against Zika virus

The mosquito-borne Zika virus is an emerging pathogen from the Flavivirus genus for which there are no approved antivirals or vaccines. Using the clinically validated PDK-53 dengue virus vaccine strain as a backbone, we created a chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus. VacDZ demonstrates key markers of attenuation: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in interferon deficient adult AG129 mice. VacDZ may be administered as a traditional live virus vaccine, or as a DNA-launched vaccine that produces live VacDZ in vivo after delivery. Both vaccine formulations induce a protective immune response against Zika virus in AG129 mice, which includes neutralising antibodies and a strong Th1 response. This study demonstrates that VacDZ is a safe and effective vaccine candidate against Zika virus.

Chimeric Japanese Encephalitis Virus SA14/SA14-14-2 Was Virulence Attenuated and Protected the Challenge of Wild-Type Strain SA14.

The attenuated Japanese encephalitis virus (JEV) live vaccine SA14-14-2 prepared from wild-type (WT) strain SA14 was licensed to prevent Japanese encephalitis (JE) in 1989 in China. Many studies showed that the premembrane (prM) and envelope (E) protein were the crucial determinant of virulence and immunogenicity of JEV. So we are interested in whether the substitution of prM/E of JEV WT SA14 with those of vaccine strain SA14-14-2 could decrease neurovirulence and prevent the challenge of JEV WT SA14. Molecular clone technique was used to replace the prM/E gene of JEV WT strain SA14 with those of vaccine strain SA14-14-2 to construct the infectious clone of chimeric virus (designated JEV SA14/SA14-14-2), the chimeric virus recovered from BHK21 cells upon electrotransfection of RNA into BHK21 cells. The results showed that the recovered chimeric virus was highly attenuated in mice, and a single immunization elicited strong protective immunity in a dose-dependent manner. This study increases our understanding of the molecular mechanisms of neurovirulence attenuation and immunogenicity of JEV.

Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice.

The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5' UTR (5'-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5'-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety. The live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue cultures and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared, and mutations were identified throughout the vaccine virus genome, but their contributions to attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.

Attenuation of Semliki Forest Virus Neurovirulence by MicroRNA-Mediated Detargeting

Artificial target sequences for tissue-specific miRNAs have recently been introduced as a new means for altering the tissue tropism of viral replication. This approach can be used to improve the safety of oncolytic viruses for cancer virotherapy by restricting their replication in unwanted tissues, such as the liver. Semliki Forest virus (SFV) is a positive-strand RNA virus and, similar to the related alphaviruses, like Sindbis virus, has potential as a gene therapy vector and an oncolytic virotherapy agent, but this potential is limited by the neurovirulence of these alphaviruses. Here, we have generated a replicative SFV4 carrying six tandem targets for the neuron-specific miR124 between the viral nonstructural protein 3 and 4 (nsp3 and nsp4) genes. When administered intraperitoneally into adult BALB/c mice, SFV4-miRT124 displayed an attenuated spread into the central nervous system (CNS) and greatly increased survival. Peripheral replication was not affected, indicating neuron-specific attenuation. Moreover, a strong protective SFV immunity was elicited in these animals. Intracranial infection of adult mice with SFV4-miRT124 showed greatly reduced infection of neurons in the brain but led to the infection of oligodendrocytes in the corpus callosum. Taken together, our data show that miR124-mediated attenuation of neurovirulence is a feasible and promising strategy for generating safer oncolytic alphavirus virotherapy agents.

Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied.

Antiviral Activity of 3(2H)- and 6-Chloro-3(2H)-Isoflavenes against Highly Diverged, Neurovirulent Vaccine-Derived, Type2 Poliovirus Sewage Isolates

BackgroundSubstituted flavanoids interfere with uncoating of Enteroviruses including Sabin-2 polio vaccine strains. However flavanoid resistant and dependent, type-2 polio vaccine strains (minimally-diverged), emerged during in vitro infections. Between 1998–2009, highly-diverged (8 to >15%) type-2, aVDPV2s, from two unrelated persistent infections were periodically isolated from Israeli sewage.AimTo determine whether highly evolved aVDPV2s derived from persistent infections retained sensitivity to isoflavenes.MethodsSabin-2 and ten aVDPV2 isolates from two independent Israeli sources were titered on HEp2C cells in the presence and absence of 3(2H)- Isoflavene and 6-chloro-3(2H)-Isoflavene. Neurovirulence of nine aVDPV2s was measured in PVR-Tg-21 transgenic mice. Differences were related to unique amino acid substitutions within capsid proteins.Principal FindingsThe presence of either flavanoid inhibited viral titers of Sabin-2 and nine of ten aVDPV2s by one to two log10. The tenth aVDPV2, which had unique amino acid substitution distant from the isoflavene-binding pocket but clustered at the three- and five-fold axies of symmetry between capsomeres, was unaffected by both flavanoids. Genotypic neurovirulence attenuation sites in the 5′UTR and VP1 reverted in all aVDPV2s and all reacquired a full neurovirulent phenotype except one with amino acid substitutions flanking the VP1 site.ConclusionBoth isoflavenes worked equally well against Sabin 2 and most of the highly-diverged, Israeli, aVDPV2s isolates. Thus, functionality of the hydrophobic pocket may be unaffected by selective pressures exerted during persistent poliovirus infections. Amino acid substitutions at sites remote from the drug-binding pocket and adjacent to a neurovirulence attenuation site may influence flavanoid antiviral activity, and neurovirulence, respectively.

Resistance of Herpes Simplex Viruses to Nucleoside Analogues: Mechanisms, Prevalence, and Management

Herpes simplex viruses (HSV) type 1 and type 2 are responsible for recurrent orolabial and genital infections. The standard therapy for the management of HSV infections includes acyclovir (ACV) and penciclovir (PCV) with their respective prodrugs valacyclovir and famciclovir. These compounds are phosphorylated by the viral thymidine kinase (TK) and then by cellular kinases. The triphosphate forms selectively inhibit the viral DNA polymerase (DNA pol) activity. Drug-resistant HSV isolates are frequently recovered from immunocompromised patients but rarely found in immunocompetent subjects. The gold standard phenotypic method for evaluating the susceptibility of HSV isolates to antiviral drugs is the plaque reduction assay. Plaque autoradiography allows the associated phenotype to be distinguished (TK-wild-type, TK-negative, TK-low-producer, or TK-altered viruses or mixtures of wild-type and mutant viruses). Genotypic characterization of drug-resistant isolates can reveal mutations located in the viral TK and/or in the DNA pol genes. Recombinant HSV mutants can be generated to analyze the contribution of each specific mutation with regard to the drug resistance phenotype. Most ACV-resistant mutants exhibit some reduction in their capacity to establish latency and to reactivate, as well as in their degree of neurovirulence in animal models of HSV infection. For instance, TK-negative HSV mutants establish latency with a lower efficiency than wild-type strains and reactivate poorly. DNA pol HSV mutants exhibit different degrees of attenuation of neurovirulence. The management of ACV- or PCV-resistant HSV infections includes the use of the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir. There is a need to develop new antiherpetic compounds with different mechanisms of action.

Harnessing Endogenous miRNAs to Control Virus Tissue Tropism as a Strategy for Developing Attenuated Virus Vaccines

Live attenuated vaccines remain the safest, most cost-effective intervention against viral infections. Because live vaccine strains are generated empirically and the basis for attenuation is usually ill defined, many important viruses lack an efficient live vaccine. Here, we present a general strategy for the rational design of safe and effective live vaccines that harnesses the microRNA-based gene-silencing machinery to control viral replication. Using poliovirus as a model, we demonstrate that insertion of small miRNA homology sequences into a viral genome can restrict its tissue tropism, thereby preventing pathogenicity and yielding an attenuated viral strain. Poliovirus strains engineered to become targets of neuronal-specific miRNAs lost their ability to replicate in the central nervous system, leading to significant attenuation of neurovirulence in infected animals. Importantly, these viruses retained the ability to replicate in nonneuronal tissues. As a result, these engineered miRNA-regulated viruses elicited strong protective immunity in mice without producing disease.

Virulence attenuation of Dengue virus due to augmented glycosaminoglycan-binding affinity and restriction in extraneural dissemination

To gain insight into the role of cell surface glycosaminoglycans (GAG) in dengue virus (DEN) cell tropism and virulence, DEN-2 mouse brain-adapted vaccine candidate, neurovirulent prototype strain (NGC) and low-passage strain, PUO-218, were passaged in BHK-21 and SW13 cells to isolate variants with high affinity for GAG. Sequence comparisons of parent and passage variants revealed five GAG-binding determinants, which all cluster in a surface-exposed region in domain II of the three-dimensional structure of the DEN envelope protein. Using an infectious cDNA clone of NGC and an NGC/PUO-218 prM-E chimeric clone, it was demonstrated that the GAG-binding determinants augment the specific infectivity for BHK-21 and/or SW13 cells by 10- to 170-fold and in some cases marginally reduce that for Vero cells. This altered cell tropism was due to a greater dependence of the variants on cell surface GAG for attachment/entry, given their increased susceptibility to heparin inhibition. The effect of the GAG-binding determinants on virulence was examined in mice deficient in alpha/beta/gamma interferon responses. High GAG affinity strongly correlated with low neuroinvasiveness due to rapid virus clearance from the blood. It was speculated that this mechanism accounts for the attenuation in primates of some DEN vaccine candidates. Interestingly, the GAG-binding variants did not display marked attenuation of neurovirulence and the opposing effect of enhanced neurovirulence was associated with one determinant (Lys126) already present in mouse brain-adapted NGC. This discrepancy of attenuated neuroinvasiveness and augmented neurovirulence may be reconciled by the existence of different mechanisms of virus dissemination in the brain and in extraneural tissues.

Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is sometimes associated with serious neurological disorders. In this study, an attempt was made to identify molecular determinants of EV71 attenuation of neurovirulence in a monkey infection model. An infectious cDNA clone of the virulent strain of EV71 prototype BrCr was constructed; temperature-sensitive (ts) mutations of an attenuated strain of EV71 or of poliovirus (PV) Sabin vaccine strains were then introduced into the infectious clone. In vitro and in vivo phenotypes of the parental and mutant viruses were analysed in cultured cells and in cynomolgus monkeys, respectively. Mutations in 3D polymerase (3D(pol)) and in the 3' non-translated region (NTR), corresponding to ts determinants of Sabin 1, conferred distinct temperature sensitivity to EV71. An EV71 mutant [EV71(S1-3')] carrying mutations in the 5' NTR, 3D(pol) and in the 3' NTR showed attenuated neurovirulence, resulting in limited spread of virus in the central nervous system of monkeys. These results indicate that EV71 and PV1 share common genetic determinants of neurovirulence in monkeys, despite the distinct properties in their original pathogenesis.

Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.

Impaired binding of standard initiation factors mediates poliovirus translation attenuation.

In the oral poliovirus vaccine, three attenuated virus strains generated by Albert Sabin are used. However, insufficient genetic stability of these strains causes major problems in poliovirus eradication. In infected cells, translation of the plus-strand poliovirus RNA genome is directed by the internal ribosome entry site (IRES), a cis-acting RNA element that facilitates the cap-independent binding of ribosomes to an internal site of the viral RNA. In each Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence attenuation. Here we report how these decisive mutations in the IRES confer a reduction in poliovirus translation efficiency. These single-nucleotide exchanges impair the interaction of the standard translation initiation factor eIF4G with the IRES domain V. Moreover, binding of eIF4B and the polypyrimidine tract-binding protein and the association of ribosomes with the viral RNA are affected by these mutations. However, the negative effects of the IRES mutations are completely relieved by addition of purified eIF4F. This indicates that eIF4G is the crucial factor that initially binds to the poliovirus IRES and recruits the IRES to the other components of the translational apparatus, while impaired binding of eIF4G plays a key role in attenuation of poliovirus neurovirulence.

Low-neurovirulence Theiler's viruses use sialic acid moieties on N-linked oligosaccharide structures for attachment.

Low-neurovirulence BeAn and DA Theiler's murine encephalomyelitis viruses (TMEV) cause persistent infection in the central nervous system (CNS) of susceptible mouse strains, leading to an inflammatory demyelinating process. A role for a specific virus-cell receptor interaction has been posited to explain why only low- and not high-neurovirulence TMEV cause persistent CNS infections. Low- but not high-neurovirulence TMEV use sialic acid for attachment to mammalian cells, which may contribute to neurovirulence attenuation and viral persistence. Analysis of BeAn virus binding and infection in cells with altered (mutated) cell-surface expression of sialic acid containing glyconjugates indicated that both binding and infection are mediated entirely by N-linked glycoproteins. By contrast, GDVII virus binding and infection appears to be dependent only in part on N-linked glycoproteins and not on O-linked glycoproteins or glycolipids. These results indicate that low-neurovirulence BeAn virus uses a sialic acid moiety expressed on an N-linked carbohydrate of a glycoprotein that serves as the protein entry receptor.

Dual stem loops within the poliovirus internal ribosomal entry site control neurovirulence.

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5' nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.

A Herpes Simplex Virus DNA Polymerase Mutation That Specifically Attenuates Neurovirulence in Mice

Herpes simplex virus can infect the mammalian brain causing lethal encephalitis (neurovirulence). Previously, herpes simplex virus mutants that are attenuated for neurovirulence have exhibited defects in replication in brain and/or blocks to replication in neuronal cells. We investigated the attenuation of neurovirulence of mutant PAAr5, which exhibits resistance to antiviral drugs due to altered viral DNA polymerase. Following intracerebral inoculation of 7-week-old CD1 mice, PAAr5 was 30-fold attenuated for neurovirulence compared to its wild-type parent. A drug-sensitive virus derived by marker rescue with DNA polymerase gene sequences exhibited neurovirulence that was essentially indistinguishable from that of wild-type virus, demonstrating that attenuation was due to a polymerase mutation. PAAr5 replicated in brain similarly to wild-type virus unlike another polymerase mutant, 615.8, that exhibited a similar degree of attenuation. The attenuation of PAAr5 was not associated with altered particle to PFU ratios nor with any obvious reductions in viral antigen expression in neurons, spread, histopathology, or TUNEL staining suggestive of apoptotic cells. Thus PAAr5 differs from other mutants that are attenuated for neurovirulence. Understanding how a polymerase mutation specifically attenuates neurovirulence may shed light on how herpes simplex virus can cause lethal encephalitis.

Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins.

The three flavivirus glycoproteins prM, E and NS1 are formed by post-translational cleavage and are glycosylated by the addition of N-linked glycans. NS1 may form homodimers, whereas E may form homodimers, homotrimers or heterodimers (prM-E). Modification of these processes by mutagenesis of the proteins has the potential to generate viruses that are restricted in growth and are possible vaccine candidates. Using an SV40-based expression system, we previously analysed dimerization and secretion of the NS1 protein of dengue virus type 2 (DEN-2) with mutations in the conserved Cys residues, or within hydrophilic or hydrophobic regions, or at glycosylation sites. In this study, mutations which reduce cleavage at the DEN-2 prM/E signalase cleavage site are described. On the basis of earlier and current results with transient expression, six mutations which reduced NS1 dimerization and two mutations which inhibited prM/E cleavage were analysed individually for their effects on virus growth using a genomic length cDNA clone. Two viruses were obtained that showed reduced growth in cell culture and attenuation of neurovirulence when inoculated into 3-day-old mice. One of these viruses encoded NS1 that lacked the second glycosylation site, the other encoded a Ser --> Ile change at the -3 position of the prM/E cleavage site. A third virus encoding a mutation in NS1 within a hydrophilic region grew as well as the parental virus. No virus was detected for the remaining five mutations.

Attenuation of neurovirulence of Theiler's murine encephalomyelitis virus strain GDVII is not sufficient to establish persistence in the central nervous system.

Virus recombinants constructed from Theiler's murine encephalomyelitis virus (TMEV) strain GDVII, which causes a rapidly fatal encephalitis in mice, and the less virulent BeAn, which persists in the murine central nervous system (CNS) and causes inflammatory demyelination, and a GDVII mutant deleted of 46 of 76 leader protein amino acids were analysed for virus persistence in the CNS. The two recombinant and mutant viruses principally contain GDVII sequences including the nucleotides encoding the polyprotein and 3' untranslated region. These viruses were found to replicate in the CNS of mice but they did not produce acute encephalitis or paralysis, i.e. they were attenuated in neurovirulence compared to the GDVII parent. More important, none of the viruses persisted in the mouse CNS nor caused chronic demyelination. Thus, attenuation of GDVII neurovirulence alone is not sufficient to establish TMEV persistence. This result is discussed in the context of a genomic determinant for persistence.