Alfred Walter Campbell's return to Australia.

Alfred Walter Campbell (1868-1937) established the basic cytoarchitectonic structure of the human brain while he was working as a pathologist at the Rainhill Lunatic Asylum near Liverpool in the United Kingdom. He returned to Australia in 1905 and continued doing research while establishing a neurological practice. His research over the next 17 years focused on four topics: (a) localisation in the cerebellum, (b) the neuroses and psychoses in war, (c) localisation in the cerebral cortex of the gorilla, and (d) the causes and pathology of the mysterious Australian "X" Disease (later known as Murray Valley encephalitis). In this article, I elaborate on his research in these areas, which provided evidence (a) against Louis Bolk's thesis that variation in the size of the cerebellar cortex reflected variation in the amount of cortex controlling various groups of muscle, (b) against the view that the neuroses and psychoses in war were different from those in civilian life, (c) for a parcelation of the cortex of the gorilla brain that supported his earlier findings in the higher apes, and (d) on the cause and pathophysiology of Australian "X" disease. Much of this research was overlooked, but it remains of considerable value and historical significance.

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.

End-point disease investigation for virus strains of intermediate virulence as illustrated by flavivirus infections.

Viruses of intermediate virulence are defined as isolates causing an intermediate morbidity/mortality rate in a specific animal model system, involving specific host and inoculation parameters (e.g. dose and route). Therefore, variable disease phenotype may exist between animals that develop severe disease or die and those that are asymptomatic or survive after infection with these isolates. There may also be variability amongst animals within each of these subsets. Such potential variability may confound the use of time-point sacrifice experiments to investigate pathogenesis of this subset of virus strains, as uniformity in disease outcome is a fundamental assumption for time-course sacrifice experiments. In the current study, we examined the disease phenotype, neuropathology, neural infection and glial cell activity in moribund/dead and surviving Swiss white (CD-1) mice after intraperitoneal infection with various Australian flaviviruses, including West Nile virus (WNV) strains of intermediate virulence (WNVNSW2011 and WNVNSW2012), and highly virulent Murray Valley encephalitis virus (MVEV) isolates. We identified notable intragroup variation in the end-point disease in mice infected with either WNVNSW strain, but to a lesser extent in mice infected with MVEV strains. The variable outcomes associated with WNVNSW infection suggest that pathogenesis investigations using time-point sacrifice of WNVNSW-infected mice may not be the best approach, as the assumption of uniformity in outcomes is violated. Our study has therefore highlighted a previously unacknowledged challenge to investigating pathogenesis of virus isolates of intermediate virulence. We have also set a precedent for routine examination of the disease phenotype in moribund/dead and surviving mice during survival challenge experiments.

Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction.

BACKGROUND: Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed. METHODS AND RESULTS: Using site-directed mutagenesis at the NS1-2A cleavage site of Murray Valley encephalitis virus (MVEV), we confirmed the dominant role of conserved octapeptide residues for efficient NS1-2A cleavage, while changes at variable and the P1' residues were mostly tolerated. However, digressions from the consensus cleavage motif derived from studies on DENV were also found. Thus, comparison of the impact on cleavage of mutations at the NS1-2A junction of MVEV and DENV showed virus-specific differences at both conserved and variable residues. CONCLUSION: We show, with subgenomic expression and infectious clone-derived mutants of MVEV that conserved residues in the flavivirus octapeptide motif can be replaced with a different amino acid without markedly reducing cleavage efficiency of NS1 and NS2A.

A new insect-specific flavivirus from northern Australia suppresses replication of West Nile virus and Murray Valley encephalitis virus in co-infected mosquito cells.

Recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. To date, there is no information on the presence or prevalence of these insect-specific flaviviruses (ISFs) in Australian mosquito species. To assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (RT-PCR) and flavivirus universal primers that are specific to the NS5 gene to detect these viruses in mosquito pools collected from the Northern Territory. Of 94 pools of mosquitoes, 13 were RT-PCR positive, and of these, 6 flavivirus isolates were obtained by inoculation of mosquito cell culture. Sequence analysis of the NS5 gene revealed that these isolates are genetically and phylogenetically similar to ISFs reported from other parts of the world. The entire coding region of one isolate (designated 56) was sequenced and shown to have approximately 63.7% nucleotide identity and 66.6% amino acid identity with its closest known relative (Nakiwogo virus) indicating that the prototype Australian ISF represents a new species. All isolates were obtained from Coquillettidia xanthogaster mosquitoes. The new virus is tentatively named Palm Creek virus (PCV) after its place of isolation. We also demonstrated that prior infection of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10-43 fold (1 to 1.63 log) at 48 hr post-infection, suggesting that superinfection exclusion can occur between ISFs and vertebrate-infecting flaviviruses despite their high level of genetic diversity. We also generated several monoclonal antibodies (mAbs) that are specific to the NS1 protein of PCV, and these represent the first ISF-specific mAbs reported to date.

Influence of hosts on the ecology of arboviral transmission: potential mechanisms influencing dengue, Murray Valley encephalitis, and Ross River virus in Australia.

Ecological interactions are fundamental to the transmission of infectious disease. Arboviruses are particularly elegant examples, where rich arrays of mechanisms influence transmission between vectors and hosts. Research on host contributions to the ecology of arboviral diseases has been undertaken within multiple subdisciplines, but significant gaps in knowledge remain and multidisciplinary approaches are needed. Through our multidisciplinary review of the literature we have identified five broad areas where hosts may influence the ecology of arboviral transmission: host immunity; cross-protective immunity and antibody-dependent enhancement; host abundance; host diversity; and pathogen spillover and dispersal. Herein we discuss the known and theoretical roles of hosts within these topics and then apply this knowledge to three epidemiologically important mosquito-borne arboviruses that occur in Australia: dengue virus (DENV), Murray Valley encephalitis virus (MVEV), and Ross River virus (RRV). We argue that the underlying mechanisms by which hosts influence arboviral activity are numerous and attempts to delineate these mechanisms further are needed. Investigations that focus on hosts of vector-borne diseases are likely to be rewarding, particularly where the ecology of vectors is relatively well understood. From an applied perspective, enhanced knowledge of host influences upon vector-borne disease transmission is likely to enable better management of disease burden. Finally, we suggest a framework that may be useful to identify and determine host contributions to the ecology of arboviruses.

Genetic and phenotypic differences between isolates of Murray Valley encephalitis virus in Western Australia, 1972-2003.

Murray Valley encephalitis virus (MVEV) is a medically important mosquito-borne flavivirus found in Australia and Papua New Guinea (PNG). Partial envelope gene nucleotide sequences of 28 isolates of MVEV from Western Australia (WA) between 1972 and 2003 were aligned and compared phylogenetically with the prototype MVE-1-51 from Victoria in 1951 and isolates from northern Queensland and PNG. Monoclonal antibody-binding patterns were also investigated. Results showed that the majority of isolates of MVEV from widely disparate locations in WA were genetically and phenotypically homogeneous. Furthermore, isolates of MVEV from WA and northern Queensland were almost identical, confirming results from earlier studies. Recent isolates of MVEV from Western Province in PNG were more similar to Australian isolates of MVEV than to isolates from PNG in 1956 and 1966, providing further evidence for the movement of flaviviruses between PNG and Australia. Additional representatives of a unique variant of MVEV (OR156) from Kununurra in the northeast Kimberley region of WA were also detected. This suggests that the OR156 lineage is still intermittently active but may be restricted to a small geographic area in northern WA, possibly due to altered biological characteristics.

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.

Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes.

The mechanism for efficient nucleocapsid (NC) uptake into flavivirus particles which form by budding through the membranes of the endoplasmic reticulum (ER) was investigated by using Murray Valley encephalitis virus as a model. Budding of flavivirus membranes is driven by the viral transmembrane proteins prM and E independently of NC interaction. We show that control of signalase cleavage of the multimembrane-spanning flavivirus polyprotein by the catalytic function of the viral protease is critical for efficient virus morphogenesis. In wild-type virus, signalase cleavage of prM remains inefficient until cleavage of capsid at the cytosolic side of the signal sequence separating the two proteins has occurred. This obligatory sequence of cleavages was uncoupled in a mutant virus with the consequence of greatly reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles. Efficient signalase cleavage of prM in the mutant virus resulted in partial inhibition of cleavage of capsid by the viral NS2B-3 protease. Our results support a model for flavivirus morphogenesis involving temporal and spatial coordination of NC assembly and envelopment by regulated cleavages of an ER membrane-spanning capsid-prM intermediate.

Innate resistance to flavivirus infection in mice controlled by Flv is nitric oxide-independent.

Innate resistance to flaviviruses in mice is active in the brain where it restricts virus replication. This resistance is controlled by a single genetic locus, FLV, located on mouse chromosome 5 near the locus encoding the neuronal form of nitric oxide synthase (Nos1). Since nitric oxide (NO) has been implicated in antiviral activity, its involvement in natural resistance to flaviviruses has been hypothesized. Here we present data on NO production before and during flavivirus infection in both brain tissue and peritoneal macrophages from two flavivirus-resistant (FLV(r)) and one congenic susceptible (FLV(s)) mouse strains. This study provides evidence that NO is not involved in the expression of flavivirus resistance controlled by FLV since: (a) there is no difference in brain tissue NO levels between susceptible and resistant mice, and (b) lipopolysaccharide-induced NO does not abrogate the difference in flavivirus replication in peritoneal macrophages from susceptible and resistant mice.

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.

Stimulation of dengue virus replication in cultured Aedes albopictus (C6/36) mosquito cells by the antifungal imidazoles ketoconazole and miconazole.

Replication of dengue type 3 virus in Aedes albopictus C6/36 cells was enhanced more than 50-fold by addition of the antifungal imidazole derivative ketoconazole within the first 4 h of infection. The stimulatory effect was reflected in the yield of infectious virus and in levels of viral RNA and protein synthesis. Enhanced yields were observed also for other flaviviruses, including dengue type 2 virus and Murray Valley encephalitis virus. Increased yields of dengue type 3 virus were not observed in African green monkey kidney (Vero) cells, human monocytic (U-937) cells, or cells of the mosquito Toxorhynchites amboinensis (TRA-171).

Characterization of defective viral RNA produced during persistent infection of Vero cells with Murray Valley encephalitis virus.

Defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal RNA viruses. Due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. In many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. In this paper, we describe for the first time the presence of truncated or defective interfering viral RNAs produced in Vero cells persistently infected with the flavivirus Murray Valley encephalitis virus. While these RNAs have not been detected in acutely infected Vero cells, their appearance coincided with the establishment of persistent infection. We also show for the first time that the defective viral RNAs replicate well in both cell culture and cell-free virus replication systems, indicating that they may interfere with the replication of parental virus at the level of viral RNA synthesis. Significantly, structural analyses of these RNA species including nucleotide sequencing have revealed that they carry similar nucleotide deletions encompassing the genes coding for the prM and E proteins and various gene segments coding for the N terminus of the NS1 protein. These deletions are in frame, allowing the synthesis of truncated NS1 proteins to occur in persistently infected cells. This may have further implications for the interference with the parental virus at the level of viral RNA synthesis in addition to a major one at the level of virion assembly and release.

Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.

Natural resistance to flaviviruses in mice is controlled by a single genetic locus, FIv, on chromosome 5. Although the mechanism of this resistance is not fully understood, it is believed to operate at the level of virus replication rather than the immune response. It has been hypothesized that enhanced production of viral defective interfering (DI) particles is responsible for a substantial reduction in the titres of infectious virus in resistant mice. However, this has never been established at the molecular level since such particles have not been isolated and characterized. We have studied the products of virus replication in the brains of flavivirus-susceptible C3H/HeJ (Flv(s)) and -resistant congenic C3H/RV (Flv(r)) mice after an intracerebral challenge (i.c.) with Murray Valley encephalitis (MVE) virus and have found no evidence for the accumulation of truncated viral RNA in the brains of resistant mice. All three major viral RNA species, the replicative intermediate (RI), replicative form (RF) and virion RNA (vRNA) together with a subgenomic RNA species of 0.6 kb, which has not been previously described, were present in the brains of both mouse strains. However, the viral RF and RI RNA forms preferentially accumulated in the brains of resistant mice. Thus, we confirm that the resistance allele Flv(r) interferes with discrete steps in flavivirus replication, although the precise mechanism remains to be determined.

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.

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.

Two possible mechanisms for survival and initiation of Murray Valley encephalitis virus activity in the Kimberley region of Western Australia.

Two possible mechanisms are described for the initiation of Murray Valley encephalitis (MVE) virus activity in arid, epizootic regions of tropical Australia. Virus isolations were made from mosquitoes trapped shortly after the first heavy wet season rains and flooding in the east Kimberley, which followed approximately nine months of drought. A number of isolates of MVE virus were obtained, including isolates from pools of blood-engorged Culex annulirostris mosquitoes and from a single pool of male Aedes tremulus mosquitoes. The results strongly suggested that MVE virus activity was due both to its introduction in viremic vertebrate hosts, from which first-generation mosquitoes became infected following blood meals, and also to reactivation of vertically transmitted virus from desiccation-resistant eggs of Ae. tremulus. Both mechanisms are discussed with respect to environmental conditions.

A novel complex formed between the flavivirus E and NS1 proteins: analysis of its structure and function.

We examined the structural features and functional significance of a novel complex which forms between the envelope (E) protein and nonstructural protein NS1 of Murray Valley encephalitis (MVE) virus. Western blot analysis of virus-infected C6/36 cell lysates revealed that the undenatured form of this E-NS1 complex was a heat-sensitive E-(NS1 dimer) complex. Furthermore, the E-NS1 complex was observed in cells infected with Kunjin, Japanese encephalitis, West-Nile and Kokobera viruses which indicates the complex is a common feature of flavivirus infection. E-NS1 complex which had been immunoaffinity purified from MVE-infected cell lysates or eluted from gel slices exhibited partial breakdown into the individual monomers, demonstrating that the complex arose from the association of E and NS1 proteins and was not a single polypeptide created from incomplete gene cleavage. Radioimmunoprecipitation and western blot analysis of MVE-infected cell lysates and culture fluid preparations collected at various times after infection revealed that the E-NS1 complex has a long half life, accumulates in the virus-infected cell with time and is not secreted into the extracellular fluid. We have postulated that the E-NS1 complex, or at least a major portion of the complex, is a non-specific aggregation with no functional significance in the viral life cycle.