Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus.

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Historical Perspective: What Constitutes Discovery (of a New Virus)?

A historic review of the discovery of new viruses leads to reminders of traditions that have evolved over 118 years. One such tradition gives credit for the discovery of a virus to the investigator(s) who not only carried out the seminal experiments but also correctly interpreted the findings (within the technological context of the day). Early on, ultrafiltration played a unique role in "proving" that an infectious agent was a virus, as did a failure to find any microscopically visible agent, failure to show replication of the agent in the absence of viable cells, thermolability of the agent, and demonstration of a specific immune response to the agent so as to rule out duplicates and close variants. More difficult was "proving" that the new virus was the etiologic agent of the disease ("proof of causation")-for good reasons this matter has been revisited several times over the years as technologies and perspectives have changed. One tradition is that the discoverers get to name their discovery, their new virus (unless some grievous convention has been broken)-the stability of these virus names has been a way to honor the discoverer(s) over the long term. Several vignettes have been chosen to illustrate several difficulties in holding to the traditions (vignettes chosen include vaccinia and variola viruses, yellow fever virus, and influenza viruses. Crimean-Congo hemorrhagic fever virus, Murray Valley encephalitis virus, human immunodeficiency virus 1, Sin Nombre virus, and Ebola virus). Each suggests lessons for the future. One way to assure that discoveries are forever linked with discoverers would be a permanent archive in one of the universal virus databases that have been constructed for other purposes. However, no current database seems ideal-perhaps members of the global community of virologists will have an ideal solution.

The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production.

Flaviviruses are emerging human pathogens and worldwide health threats. During infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5'→3' host cell exonuclease Xrn1 through an unknown RNA structure-based mechanism. Here, we present the crystal structure of a complete Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly conserved nucleotides. The RNA's three-dimensional topology creates a ringlike conformation, with the 5' end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity.

Internal ribosome entry site-based attenuation of a flavivirus candidate vaccine and evaluation of the effect of beta interferon coexpression on vaccine properties.

Infectious clone technologies allow the rational design of live attenuated viral vaccines with the possibility of vaccine-driven coexpression of immunomodulatory molecules for additional vaccine safety and efficacy. The latter could lead to novel strategies for vaccine protection against infectious diseases where traditional approaches have failed. Here we show for the flavivirus Murray Valley encephalitis virus (MVEV) that incorporation of the internal ribosome entry site (IRES) of Encephalomyocarditis virus between the capsid and prM genes strongly attenuated virulence and that the resulting bicistronic virus was both genetically stable and potently immunogenic. Furthermore, the novel bicistronic genome organization facilitated the generation of a recombinant virus carrying an beta interferon (IFN-ß) gene. Given the importance of IFNs in limiting virus dissemination and in efficient induction of memory B and T cell antiviral immunity, we hypothesized that coexpression of the cytokine with the live vaccine might further increase virulence attenuation without loss of immunogenicity. We found that bicistronic mouse IFN-ß coexpressing MVEV yielded high virus and IFN titers in cultured cells that do not respond to the coexpressed IFN. However, in IFN response-sufficient cell cultures and mice, the virus produced a self-limiting infection. Nevertheless, the attenuated virus triggered robust innate and adaptive immune responses evidenced by the induced expression of Mx proteins (used as a sensitive biomarker for measuring the type I IFN response) and the generation of neutralizing antibodies, respectively. IMPORTANCE The family Flaviviridae includes a number of important human pathogens, such as Dengue virus, Yellow fever virus, Japanese encephalitis virus, West Nile virus, and Hepatitis C virus. Flaviviruses infect large numbers of individuals on all continents. For example, as many as 100 million people are infected annually with Dengue virus, and 150 million people suffer a chronic infection with Hepatitis C virus. However, protective vaccines against dengue and hepatitis C are still missing, and improved vaccines against other flaviviral diseases are needed. The present study investigated the effects of a redesigned flaviviral genome and the coexpression of an antiviral protein (interferon) on virus replication, pathogenicity, and immunogenicity. Our findings may aid in the rational design of a new class of well-tolerated and safe vaccines.

A flavivirus signal peptide balances the catalytic activity of two proteases and thereby facilitates virus morphogenesis.

Two cleavages on either side of a signal peptide separating capsid and prM on the nascent flavivirus polyprotein are uniquely regulated, such that cytosolic capsid cleavage triggers signalase cleavage of prM. Here, we show, using two experimental approaches, that this sequential order of cleavages facilitates virus morphogenesis: (i) A Murray Valley encephalitis virus (MVEV) variant, in which both cleavages occurred efficiently and independently of each other, displayed an assembly defect. (ii) Replicon particle assembly was assayed in packaging cells encoding the MVEV structural proteins; bicistronic expression of either mature or membrane-anchored capsid in addition to that of the prM and E proteins showed enhanced particle production in the latter cell line. Taken together, this study demonstrates that efficient flavivirus assembly requires a cleavable transmembrane anchor of C protein and an obligatory order of cleavages at the C-prM junction, both controlled by sequence elements in the prM signal peptide.

In situ reactions of monoclonal antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 protein.

Studies on the NS1 protein of flaviviruses have concluded that formation of a stable homodimer is required for virus replication. However, previous work has reported that substitution of a conserved proline by leucine at residue 250 in NS1 of Kunjin virus (KUNV) eliminated dimerization, but allowed virus replication to continue. To assess whether this substitution has similar effects on other flaviviruses, it was introduced into an infectious clone of Murray Valley encephalitis virus (MVEV). Consistent with studies of KUNV, the mutant virus (MVEV(NS1-250Leu)) produced high levels of monomeric NS1 and the NS1 homodimer could not be detected. In contrast, wild-type MVEV cultures contained predominantly dimeric NS1. Retarded virus growth in Vero cells and loss of neuroinvasiveness for weanling mice revealed further similarities between MVEV(NS1-250Leu) and the corresponding KUNV mutant. To confirm that the lack of detection of dimeric NS1 in mutant virus samples was not due to denaturation of unstable dimers during Western blotting, a mAb (2E3) specific for the MVEV NS1 homodimer was produced. When NS1 protein was fixed in situ in mammalian and arthropod cells infected with wild-type or mutant virus, 2E3 reacted strongly with the former, but not the latter. These results confirmed that Pro(250) in NS1 is important for dimerization and that substitution of this residue by leucine represents a conserved marker of attenuation for viruses of the Japanese encephalitis virus serocomplex. The inability to detect dimeric NS1 in supernatant or cell monolayers of cultures productively infected with mutant virus also suggests that dimerization of the protein may not be essential for virus replication.

Virus spread, tissue inflammation and antiviral response in brains of flavivirus susceptible and resistant mice acutely infected with Murray Valley encephalitis virus.

Inborn resistance to flaviviruses, conferred by a single chromosome 5 locus Flv, is a genetic trait operative in wild mice and a few strains of laboratory mice. In this study we have used in situ hybridisation to trace the spread of flavivirus genomic RNA within the brains of flavivirus susceptible C3H/HeJARC and congenic resistant C3H.PRI- Flv(r) mice following infection with Murray Valley encephalitis virus (MVE) in parallel to studying a brain histopathology and induction of cellular genes involved in antiviral response. We find that in contrast to a high viral RNA content in brains of susceptible mice, viral RNA was markedly reduced in the cortex, olfactory bulb, thalamus and hypothalamus of resistant mice. Trace amounts of viral RNA were detected in the medulla oblongata while it was completely absent from the hippocampus, pons and cerebellum of resistant mice at different time points post infection. The low virus titres within brains of resistant mice coincided with a very mild inflammation, low counts of infiltrating inflammatory cells, and lower IFN I/II and TNFalpha gene induction than in susceptible mice. Furthermore, transcripts of several genes belonging to a 2',5'-oligoadenylate synthetase ( OAS) family, implicated in IFN I-inducible OAS/RNase L antiviral pathway, showed similar brain tissue induction in both strains of mice suggesting only minor contribution of this pathway to the resistance phenotype.

Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice.

Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody-mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.

Murray Valley encephalitis virus recombinant subviral particles protect mice from lethal challenge with virulent wild-type virus.

We report on the development and characterisation of a recombinant Murray Valley encephalitis virus (MVE) envelope glycoprotein expression system that results in the secretion of subviral particles (SVPs) upon transfection of the murine fibroblast (COS-7) cell line. Initially, aspects of the physical and antigenic structure of cell-associated and secreted forms of the MVE envelope glycoproteins (prM and E) are presented. We then show that BALB/c mice inoculated with SVPs purified from pcDNA(3)-prM/E-transfected COS-7 cell supernatants are protected from lethal challenge with the virulent prototype strain MVE-1-51 and that this protection correlates with the development of a neutralising humoral immune response by the host. By contrast, prior immunisation with cell-associated, recombinant MVE envelope glycoproteins did not protect mice from challenge with MVE-1-51 and this was associated with the development of antibody that was unable to neutralise virus infectivity in vitro. These studies demonstrate that SVPs derived from the in vitro expression of recombinant MVE prM and E genes are an effective candidate vaccine for the prevention of encephalitis in the mouse model.

Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus.

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu(306) and Asp(390) for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp(390) on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA.

An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3' UTR was found to contain elements conserved throughout the genus FLAVIVIRUS: RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD(50) values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.

A mouse-attenuated envelope protein variant of Murray Valley encephalitis virus with altered fusion activity.

A neutralization escape variant of Murray Valley encephalitis virus (MVE), of low neuroinvasiveness in mice and with low haemagglutination activity, had a reduced rate of replication in cultured cells during the early phase of infection compared to wild-type MVE. The variant was internalized by Vero cells at a similar rate to wild-type MVE at pH 7.4, but had reduced pH-dependent membrane fusion activity. In fusion-from-within experiments in infected mosquito (C6/36) cells, the variant had a lowered pH threshold for induction of fusion, which occurred at a reduced rate and to a lesser extent than for wild-type virus. Fusion was inhibited by monoclonal antibodies specific for envelope protein epitopes E-5 and E-8, which were implicated as determinants of fusion. These observations are discussed in relation to the regulation of MVE replication by fusion of the viral envelope with endosome membranes and, in turn, how rates of replication may affect neuroinvasion.

A comparison of the spread of Murray Valley encephalitis viruses of high or low neuroinvasiveness in the tissues of Swiss mice after peripheral inoculation.

A Murray Valley encephalitis virus (MVE) field isolate of high neuroinvasiveness (BH3479) and a neutralization escape variant of low neuroinvasiveness (BHv1) selected from BH3479 (which differ by a single amino acid at residue 277 in the envelope glycoprotein) were examined for their distribution in the tissues of weanling Swiss mice at various times after footpad inoculation. BH3479 was first detected in lymph nodes draining the inoculated limb at 24 hr postinoculation (pi) and was found in serum between 36 and 72 hr pi. BH3479 was first detected in the central nervous system (CNS) at 4 days pi and reached maximum CNS titers ( > 10(9) PFU/g) between 6 and 9 days pi. All BH3479-infected mice developed encephalitis and died before 10 days pi. In contrast, BHv1 was not detected in lymph nodes draining the footpad at any time after inoculation; BHv1 was first detected in the serum between 60 and 72 hr pi-24 hr later, and at a 20-fold lower titer than for BH3479. BHv1 was first detected in the CNS at 7 days pi 3 days later and at a 300-fold lower titer than for BH3479. After 10 days pi, BHv1 could not be isolated from the CNS or from other host tissues. Most BHv1-infected mice experienced a subclinical infection; the mortality rate from BHv1 infection was less than 1%. Both viruses appeared to enter the CNS via the olfactory lobes. BH3479 spread throughout the CNS in a rostral to caudal direction over 3-4 days. In contrast, BHv1 infection in the CNS was restricted to the olfactory lobes and adjacent structures of the forebrain.

Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice.

Neutralization escape variants of Murray Valley encephalitis virus were selected using a type-specific, neutralizing, and passively protective anti-envelope protein (E) monoclonal antibody (4B6C-2) which defines epitope E-1c. Nucleotide sequence analysis revealed single nucleotide changes in the E genes of 15 variants resulting in nonconservative amino acid substitutions in all cases. One variant had a three-nucleotide deletion in the E gene which resulted in loss of serine at residue 277. Changes were clustered into two separate regions of the E polypeptide (residues 126-128 and 274-277), indicating that E-1c is a discontinuous epitope. One variant (BHv1), altered at residue 277 (Ser-->Ile), failed to hemagglutinate across the pH range 5.5-7.5, in contrast to parental virus and the other escape variants which hemagglutinated at an optimal pH of 6.6. BHv1 was also of reduced neuroinvasiveness in 21-day-old mice following intraperitoneal inoculation compared to the other viruses. Parental virus and the neutralization escape variants grew equally well in both vertebrate and invertebrate cell cultures, indicating that the reduced neuroinvasiveness of BHv1 was not due to a major abnormality of replication.

Neurovirulence and neuroinvasiveness of Murray Valley encephalitis virus mutants selected by passage in a monkey kidney cell line.

Variants of the prototype Murray Valley encephalitis virus (MVE-1-51) were selected by serial plaque purification and amplification in monkey kidney (Vero) cells. Four clones (C1-C4) at passage levels two and nine (P2 and P9) were examined in 21-day-old Swiss outbred mice for neuroinvasiveness (assessed from LD50 values after intraperitoneal inoculation) and neurovirulence (LD50 values after intracranial inoculation). The growth characteristics of the clones were determined in intracranially inoculated mouse brain and in mouse neuroblastoma, Vero and mosquito (C6/36) cell lines. Genomic RNA of the cloned virus stocks was sequenced through the structural protein genes (E, prM/M and C) and the 5' untranslated region. Clone C2P2 was of high neuroinvasiveness whereas C2P9 was of low neuroinvasiveness; there were also decreased yields of C2P9 in C6/36 cells compared to C2P2 and MVE-1-51. These changes were associated with the substitution of valine for phenylalanine at amino acid position 141 of the C2P9 E protein. Clone C4P2 was of high neurovirulence and low neuroinvasiveness; C4P9 was of low neurovirulence, a change accompanied by a further reduction in neuroinvasiveness. Concomitantly, C4P9 showed a pronounced reduction in growth rates and yields in 21-day-old Swiss mouse brain, in mouse neuroblastoma cells and in C6/36 cells compared to parental virus. The phenotypic changes in clone 4 appear to be due to mutation(s) within non-structural protein genes.