Osteoblasts from osteoarthritis patients show enhanced susceptibility to Ross River virus infection associated with delayed type I interferon responses.

BACKGROUND: Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) have caused widespread outbreaks of chronic polyarthritis. The inflammatory responses in alphavirus-induced arthritis and osteoarthritis (OA) share many similar features, which suggests the possibility of exacerbated alphavirus-induced bone pathology in individuals with pre-existing OA. Here, we investigated the susceptibility of osteoblasts (OBs) from OA patients to RRV infection and dissected the immune mechanisms elicited from infection. METHODS: Primary hOBs obtained from trabecular bone of healthy donors and OA patients were infected with RRV. Infectivity and viral replication were determined using flow cytometry and plaque assay, respectively. Real-time PCR was performed to determine expression kinetics of type I interferon (IFN)-related immune mediators and osteotropic factors. RESULTS: OA hOBs showed enhanced RRV infectivity and replication during infection, which was associated with delayed induction of IFN-ß and RIG-I expression. Enhanced susceptibility of OA hOBs to RRV was associated with a more pronounced increase in RANKL/OPG ratio and expression of osteotropic factors (IL-6, IL-1ß, TNF-α and CCL2) in comparison to RRV-infected healthy hOBs. CONCLUSIONS: Delayed activation of type I IFN-signalling pathway may have contributed to enhanced susceptibility to RRV infection in hOBs from OA patients. RRV-induced increases in RANKL/OPG ratio and expression of osteotropic factors that favour bone resorption, which may be exacerbated during osteoarthritis. This study provides the novel insight that osteoarthritis may be a risk factor for exacerbated arthritogenic alphaviral infection.

Differential induction of type I interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses.

Dendritic cells (DCs) are an important early target cell for many mosquito-borne viruses, and in many cases mosquito-cell-derived arboviruses more efficiently infect DCs than viruses derived from mammalian cells. However, whether mosquito-cell-derived viruses differ from mammalian-cell-derived viruses in their ability to induce antiviral responses in the infected dendritic cell has not been evaluated. In this report, alphaviruses, which are mosquito-borne viruses that cause diseases ranging from encephalitis to arthritis, were used to determine whether viruses grown in mosquito cells differed from mammalian-cell-derived viruses in their ability to induce type I interferon (IFN) responses in infected primary dendritic cells. Consistent with previous results, mosquito-cell-derived Ross River virus (mos-RRV) and Venezuelan equine encephalitis virus (mos-VEE) exhibited enhanced infection of primary myeloid dendritic cells (mDCs) compared to mammalian-cell-derived virus preparations. However, unlike the mammalian-cell-derived viruses, which induced high levels of type I IFN in the infected mDC cultures, mos-RRV and mos-VEE were poor IFN inducers. Furthermore, the poor IFN induction by mos-RRV contributed to the enhanced infection of mDCs by mos-RRV. These results suggest that the viruses initially delivered by the mosquito vector differ from those generated in subsequent rounds of replication in the host, not just with respect to their ability to infect dendritic cells but also in their ability to induce or inhibit antiviral type I IFN responses. This difference may have an important impact on the mosquito-borne virus's ability to successfully make the transition from the arthropod vector to the vertebrate host.

Ecological mechanisms that promote arbovirus survival: a mathematical model of Ross River virus transmission.

Many assessments of host and vector competence for arboviruses focus on level and length of infectivity and ignore ecological mechanisms that contribute to virus survival. In this paper, mathematical models are used to compare local survival mechanisms for a range of scenarios, using Ross River virus as a case study. Ross River virus is an Australian arbovirus with many mosquito vectors and reservoir hosts. The mechanisms for maintaining long-term transmission of the virus vary between salt and freshwater mosquito vectors, and according to the availability of susceptible hosts. The models demonstrate that overwintering of virus in adult freshwater mosquitoes requires a large host population, while overwintering of virus in infected eggs of saltwater mosquitoes is an effective survival strategy when filial infection rates are high. The virus survives longer when both salt and freshwater mosquito species are included in the model than when only one mosquito species is present. When the marsupial host is replaced by a host with higher birth rate and shorter infectious period, the virus survived longer under all models. This suggests that birth rate can be a key factor when assessing the competence of reservoir hosts to maintain virus transmission.

Modelling the transmission dynamics of Ross River virus in Southwestern Australia.

During the 1995-1996 Australian financial year, over 1300 notifications of Ross River (RR) virus disease were notified in humans from Southwestern Australia. Due to the mild symptoms of the disease, it is difficult to diagnose and subclinical infections are common. However, these subclinical infections do give rise to immunity. For planning and control, it is important for public health authorities to estimate the true number of people who have contracted the disease and to assess the impact of key epidemiological parameters. A mathematical model was developed to describe the transmission of RR virus between its hosts (humans and kangaroos) and its vectors (mosquitoes). For this model, the threshold conditions and relative removal rates were calculated and interpreted. Finally, a computer program was written to simulate the model in order to estimate the total number, both clinical and sub clinical human infections given known and hypothetical epidemiological parameter values. Within this simulation sensitivity of the results to changes in the parameters were examined. The analysis of the threshold conditions conformed well to established principles of arboviral transmission and control. It was observed that conditions which can prevent an outbreak occuring include reducing the number of susceptibles in host and vector populations, reducing the infection rates between hosts and vectors and shortening the duration of viraemia. Results on the sensitivity analysis showed that some parameters such as the extrinsic incubation period, mosquito mortality rate in winter and the proportion of Western Grey Kangaroos in the marsupial population have little effect on human incidence. However, the transmission rate between hosts and vectors, vector-mortality rate in summer and the proportion of infectious vectors among infected vectors have pronounced effects. The simulation results on the ratio of clinical to subclinical human infections predicted a minimum ratio of 1:2 and a maximum ratio of 1:65, which is consistent with data obtained during previous sero-epidemiological studies.

Persistent Ross River virus infection of murine macrophages: an in vitro model for the study of viral relapse and immune modulation during long-term infection.

A clinical feature of Ross River virus disease (RRVD) is the periodic relapse of symptoms months after the initial onset of disease. The underlying mechanisms responsible for this relapse have not been determined. In a long-term (148 days) in vitro study of persistently infected murine macrophages we established that RRV infection periodically fell to undetectable biological levels that required genetic detection. However, the virus concentration spontaneously relapsed to biologically detectable levels that corresponded with enhanced viral mRNA expression, cellular detachment, and cytopathic effect. By altering the cell culture conditions we found that relapse could also be induced. We propose that the periodic relapse of symptoms in RRVD may be associated with spontaneous or stress-induced increases in RRV within persistently infected macrophages. This study also established that RRV enhanced macrophage phagocytic activity and dysregulated the immunoregulatory molecules CD80, IFN-gamma, and TNF-alpha that may facilitate persistence of RRV and avoidance of immune responses.

Geographic variation of notified Ross River virus infections in Queensland, Australia, 1985-1996.

The spatial and temporal variations of Ross River virus infections reported in Queensland, Australia, between 1985 and 1996 were studied by using the Geographic Information System. The notified cases of Ross River virus infection came from 489 localities between 1985 and 1988, 805 between 1989 and 1992, and 1,157 between 1993 and 1996 (chi2(df = 2) = 680.9; P < 0.001). There was a marked increase in the number of localities where the cases were reported by 65 percent for the period of 1989-1992 and 137 percent for 1993-1996, compared with that for 1985-1988. The geographic distribution of the notified Ross River virus cases has expanded in Queensland over recent years. As Ross River virus disease has impacted considerably on tourism and industry, as well as on residents of affected areas, more research is required to explore the causes of the geographic expansion of the notified Ross River virus infections.

Adaptive mutations in Sindbis virus E2 and Ross River virus E1 that allow efficient budding of chimeric viruses.

Alphavirus glycoproteins E2 and E1 form a heterodimer that is required for virus assembly. We have studied adaptive mutations in E2 of Sindbis virus (SIN) and E1 of Ross River virus (RR) that allow these two glycoproteins to interact more efficiently in a chimeric virus that has SIN E2 but RR E1. These mutations include K129E, K131E, and V237F in SIN E2 and S310F and C433R in RR E1. Although RR E1 and SIN E2 will form a chimeric heterodimer, the chimeric virus is almost nonviable, producing about 10(-7) as much virus as SIN at 24 h and 10(-5) as much after 48 h. Chimeras containing one adaptive change produced 3 to 20 times more virus than did the parental chimera, whereas chimeras with two changes produced 10 to 100 times more virus and chimeras containing three mutations produced yields that were 180 to 250 times better. None of the mutations had significant effects upon the parental wild-type viruses, however. Passage of the triple variants eight or nine times resulted in variants that produced virus rapidly and were capable of producing >10(8) PFU/ml of culture fluid within 24 h. These further-adapted variants possessed one or two additional mutations, including E2-V116K, E2-S110N, or E1-T65S. The RR E1-C433R mutation was studied in more detail. This Cys is located in the putative transmembrane domain of E1 and was shown to be palmitoylated. Mutation to Arg-433 resulted in loss of palmitoylation of E1. The positively charged arginine residue within the putative transmembrane domain of E1 would be expected to alter the conformation of this domain. These results suggest that interactions within the transmembrane region are important for the assembly of the E1/E2 heterodimer, as are regions of the ectodomains possibly identified by the locations of adaptive mutations in these regions. Further, the finding that four or five changes in the chimera allow virus production that approaches the levels seen with the parental SIN and exceeds that of the parental RR illustrates that the structure and function of SIN and RR E1s have been conserved during the 50% divergence in sequence that has occurred.

Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes.

Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.

Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus.

We have constructed a full-length cDNA clone of the virulent T48 strain of Ross River virus, a member of the alphavirus genus. Infectious RNA can be transcribed from this clone using SP6 or T7 RNA polymerase. The rescued virus has properties indistinguishable from those of the T48 strain of Ross River virus. We have used this clone, together with a full-length cDNA clone of Sindbis virus, to construct chimeric plasmids in which the 5' and the 3' nontranslated regions of the Sindbis and Ross River genomes were exchanged. The nontranslated regions of the two viral genomes differ in both size and sequence although they maintain specific conserved sequence elements. Virus was recovered from all four chimeras. Chimeras containing heterologous 3' nontranslated regions had replicative efficiencies equal to those of the parents. In contrast, the chimeras containing heterologous 5' nontranslated regions were defective in RNA synthesis and virus production, and the severity of the defect was dependent upon the host. Replication of a virus containing a heterologous 5' nontranslated region may be inefficient due to the formation of defective protein-RNA complexes, whereas, the presumptive complexes formed between host or virus proteins and the 3' nontranslated region to promote RNA synthesis appear to function normally in the chimeras.

Anti-mosquito antibodies reduce the susceptibility of Aedes aegypti to arbovirus infection.

Aedes aegypti (L.) mosquitoes showed a significant reduction in susceptibility to infection with Ross River virus and Murray Valley encephalitis virus when they were fed on a blood-virus mixture containing rabbit antibodies to mosquito midgut components. Presence of the antibodies did not demonstrably affect virus titres in infected mosquitoes, nor the transmission of virus from infected mosquitoes to vertebrates.

Genetic and phenotypic studies on Ross River virus variants of enhanced virulence selected during mouse passage.

We have passaged Ross River virus (RRV) in mice to generate variants with increased mouse virulence and attempted to relate changes in virulence to genome sequence changes. RRV NBO (zero passage in mice) is a plaque-purified clone of the mouse-avirulent strain RRV NB5092, and is of low virulence for day-old mice. During RRV NBO replication in infant mice, its virulence for day-old mice increased markedly with time. By 7 days postinfection the LD50 value of harvested virus (passage level one) was congruent to 10(4)-fold less than that of the parental virus. No further decrease in LD50 followed 10 serial passages in infant mice. However, 10th passage level virus showed increased clinical effects in week-old mice by comparison with virus from passage levels one and two. The growth kinetics of RRV variants in mice suggested that the rate and extent of RRV replication in the brain tissue determined the enhanced mouse virulence of serially passaged virus. Seven out of eight independently passaged, 10th passage level variants had changes in the E2 gene leading to one or two amino acid substitutions. The changes were at residues 212, 232, 234, 251, 341, 27 and 172, and 72 and 134 in these variants; all changes except two were nonconservative. Residues 212, 234, and 251 form part of a neutralization determinant in RRV. Changes in epitope b2 (which includes amino acids 246, 248, and 251) alter the kinetics of RRV entry into cells (P. Kerr, R. C. Weir, and L. Dalgarno, unpublished data). First and second passage level virus of enhanced virulence was unchanged in E2 or E1 gene sequences from RRV NBO. However, 1st, 2nd, and 10th passage level virus induced higher levels of virus-specific RNA synthesis than did RRV NBO in cultured BHK cells. We propose a model for the mechanism of virulence enhancement on passaging RRV NBO in mice.

Prolonged infection of human synovial cells with Ross River virus.

Primary cultures of human synovial cells shed infectious virus for 14 to 35 days following infection with isolates of Ross River virus which had been passaged in the C6/36 line of Aedes albopictus mosquito cells. No frank cytopathic effect was seen in infected synovial cells and they continued to replicate for the duration of the experiments.

Ross River virus mutant with a deletion in the E2 gene: properties of the virion, virus-specific macromolecule synthesis, and attenuation of virulence for mice.

A mutant of RRV T48 the prototype strain of Ross River virus has been isolated with a 21-nucleotide deletion in the gene coding for the envelope glycoprotein E2. Direct sequencing of the 26 S subgenomic RNA, together with HaeIII and TaqI restriction digest analysis of cDNA to RNAs from cells infected with the mutant virus (RRV dE2) and with RRV T48, were consistent with the deletion being the only major alteration in the mutant genome. The E2 protein of RRV dE2 virions had a higher electrophoretic mobility than that of RRV T48 E2 protein. Neither RRV dE2 nor RRV T48 virions contained more than trace amounts of E3, the small envelope glycoprotein found in Semliki Forest virus. RRV dE2 generated small plaques on Vero cell monolayers; plaque formation was not temperature-sensitive between 32 and 41 degrees. By comparison with RRV T48 the infectivity of RRV dE2 virions was thermolabile at 50 degrees. In BHK cells RRV dE2 grew with similar kinetics to RRV T48. Rates of synthesis of 26 S RNA and 49 S RNA were higher in cells infected with RRV dE2 than in cells infected with RRV T48. Virus-specific protein synthesis and shut-down of host protein synthesis occurred 2-3 hr earlier in RRV dE2-infected cells than in cells infected with RRV T48. Minor differences between the two viruses were observed in the profiles of virus-specific proteins generated in infected cells. In day-old mice RRV dE2 induced less severe symptoms of hind leg paralysis than did RRV T48. A small increase in LD50 and average survival time was observed in RRV dE2-infected mice by comparison with RRV T48 infected mice. Peak titers reached by RRV dE2 in the hind leg muscle, brain, and blood of day-old mice were 3-4 log units less than the titers reached during infection with RRV T48. In week-old mice the differences in virulence between the two strains were magnified: RRV dE2 induced no detectable symptoms even when injected at high doses (8 X 10(6) PFU) whereas the LD50 and average survival time for RRV T48 were unchanged from those in day-old mice. Peak RRV dE2 titers in hind leg muscle, brain, and blood, respectively, were 2, 5, and 5 log units less than the corresponding titers for RRV T48. Peak muscle titers reached by RRV dE2 were similar (approximately 10(8) PFU/g tissue) in day-old mice where lethality was high and in week-old mice where the virus was avirulent.(ABSTRACT TRUNCATED AT 400 WORDS)

An investigation of the potential of Aedes camptorhynchus (Thom.) as a vector of Ross River virus.

Aedes camptorhynchus (Thom.) collected on the mid-south coast of New South Wales during the winter of 1982 were highly susceptible to infection (ID50 = 10(2.4) VERO pfu/mosquito) when fed on rat tail skins containing blood and serial dilutions of the T48 strain of Ross River (RR) virus. After 2 d, when no virus was detectable, rapid proliferation allowed transmission from 5 d post ingestion. A maximum transmission rate occurred 9 d post-feeding when 4 of 4 infected mosquitoes transmitted virus. The susceptibility of Ae camptorhynchus to RR virus infection was compared with that of a laboratory colony of Ae aegypti (L.) (ID50 = 10(3.8) VERO pfu/mosquito).

Interaction of Ross River virus with the complement system.

In the absence of virus-specific antibody, Ross River virus failed to activate either the classical or alternative complement pathways. Instead, it inhibited the cleavage of C3 via both pathways. The virus did not appear to act by disrupting C3bBb complexes or by preventing cleavage of factor B by factor D. Instead Ross River virus was found to interfere with the actual cleavage of C3 by activated factor B (C3bBb) of the alternative pathway and C4b2a of the classical pathway.

Pathogenesis of in utero infections with abortogenic and non-abortogenic alphaviruses in mice.

The initial stages of infection of pregnant mice at gestation day 11 with either the T48 strain of Ross River virus or avirulent Semliki Forest virus are similar. With both infections, a hematogenous spread of virus to the placenta occurs. The viruses subsequently replicate to high titer in all placentas and are able to persist in the presence of specific maternal antiviral antibodies. There is a delay of at least 1 to 2 days between the initial detection of virus in the placenta and the onset of fetal infection. With Semliki Forest virus, abortion occurred in all mothers and appeared to be preceded by infection of all fetuses. However, when Semliki Forest virus was given at other stages of pregnancy, abortion was less common, and in all non-aborted pregnancies at least one uninfected fetus was observed. This situation was similar to that with Ross River virus, in which abortion was not observed and fetal infection and death were only seen in a proportion of fetuses. Within each pregnancy, the outcome of the two in utero infections appeared to result from similar mechanisms, with the fate of an individual fetus depending upon the timing of the passive transfer of anti-viral immunoglobulin G from the mother relative to the timing of fetal infection by virus from the placenta. Although the passive maternal immunoglobulin G protected susceptible fetuses against infection, antibody did not mediate in utero recovery of infected fetuses or clear placental infection.

Polypeptide synthesis in alphavirus-infected Aedes albopictus cells during the establishment of persistent infection.

Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cell, RRV reached peak titres at 34--48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and less than 5 per cent of cells assayed as infected. There was no shut-down of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s). The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persitently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK celos host protein synthesis was severly inhibited and by 9--11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity.

Replication of standard and defective Ross River virus in BHK cells: patterns of viral RNA and polypeptide synthesis.

Virus-specific macromolecule synthesis has been examined in BHK cells infected with Ross River virus. Unpassaged virus (R-0) and tenth-passage virus (R-10) have been compared. In infected cells R-0 generates i) 45S, 28S, 33S and 26S viral RNAs, ii) virus-specific precursor polypeptides of mol. wt. 127,000, 95,000 and 61,000 and iii) viral envelope proteins (mol. wts. 52,000 and 49,000) and nucleocapsid protein (mol. wt. 32,000). Thus in terms of virus-specific RNA and polypeptide synthesis, the replication of standard RRV is analogous to that of Semliki Forest virus and Sindbis virus. R-10 interferes with the replication of standard Ross River virus and generates large amounts of 19S and 24S defective RNA species; 45S and 26S RNA synthesis was not markedly affected. Defective RNAs are associated with RNAse-sensitive, 50S cytoplasmic particles which contain a variety of (mainly host) proteins but no nucleocapsid protein. No evidence for translation of defective RNAs was obtained. R-10 infection is also characterized by a relatively early shut down of host protein syntehsis and by a reduction in virus-specific polypeptide synthesis and nucleocapsid formation. The data suggest that defective Ross River virus interferes primarily at the translational level.