Ross River virus

Epidemiologically, Ross River Virus (RRV) is Australia’s most important mosquito- borne disease, causing 4660 (range 1451–9551) human clinical notifications at an estimated economic cost of $15 million annually (Aaskov, Fokine, & Liu, 2012; Harley, Sleigh, & Ritchie, 2001), and this cost does not include the substantive investment in mosquito control – estimated at $9 million for the State of Queensland alone (Tomerini, 2005). RRV naturally cycles among marsupial hosts and mosquito vectors, spilling over to humans (Russell, 2002; Harley et al., 2001; Carver et al., 2009; Koolhof & Carver, 2016). The primary reservoirs for enzootic RRV transmission appear to be marsupials, particularly grey kangaroos. Annual and multi-annual seasonality is fundamentally characteristic of RRV epidemics across all areas of Australia (Harley et al., 2001; Russell, 2002). Further, the transmission ecology of RRV is broadly emblematic to that of other globally distributed alphaviruses and flaviviruses (Carver et al., 2009), which are significant sources of human morbidity and mortality, such as chikungunya, Sindbis virus, yellow fever, West Nile and Japanese encephalitis viruses.

Ross River virus (RRV) is an indigenous Australian arthropod-borne alphavirus responsible for epidemic polyarthritis (EPA), myalgia, and lethargy in humans. Macrophages and monocytes have been associated with human RRV disease, and previous studies have shown that RRV is capable of infecting macrophages via both a natural virus receptor and by Fc receptor-mediated antibody-dependent enhancement (ADE). Similar to oth- er viruses, such as human immunodeficiency virus and dengue virus, ADE infection results in dramatic RRV growth increases for in vitro macrophage cultures. This study demonstrates that RRV could resist lipopoly- saccharide (LPS)-induced antiviral activity in macrophage cultures when infection was via the ADE pathway. Investigation of this infection pathway found that RRV was able to suppress the transcription and translation of key antiviral genes (tumor necrosis factor and inducible nitric oxide synthase) in LPS-stimulated macro- phages by disrupting the transcription into mRNA of the genes coding for the associated transcription factors IRF-1 and NF- ␬ B. The transcription of non-antiviral control genes was not perturbed by RRV-ADE infection, and de novo protein synthesis also was not significantly affected in RRV-ADE infected cells. The ADE pathway of infection allowed RRV to specifically target antiviral genes in macrophages, resulting in unrestricted virus replication. As ADE has been observed for several virus families and associated with disease and adverse vaccination outcomes, these findings may have broad relevance to viral disease formation and antiviral vac- cination strategies.

Infection of humans with arthritogenic alphaviruses, such as Ross River virus (RRV), chikungunya virus, o’nyong-nyong virus, mayaro virus, and others, is a global cause of debilitating musculoskeletal disease (12, 37). These viruses are also of serious concern due to their ability to cause explosive epidem- ics that can involve thousands to millions of patients and po- tentially lead to emergence in new geographic regions. This has been highlighted by the recent epidemic reemergence of chikungunya virus in the southeastern islands of the Indian Ocean, as well as India. Since January 2005, an estimated 244,000 individuals have been infected on Re ´union island alone, as well as thousands of additional cases on other islands, including Comoros, Mayotte, Seychelles, and Mauritius (34). Since many of these islands are popular tourist destinations, chikungunya virus-infected individuals returning from these areas have been identified in France, Germany, Canada, and other regions where the virus is not endemic. In addition, past epidemics include an epidemic in 1979 and 1980 of RRV disease in the South Pacific that involved more than 60,000 patients (14) and an epidemic from 1959 to 1962 of o’nyong- nyong fever in Africa that involved at least 2 million patients (41).

Old world alphaviruses (e.g., Chikungunya, O’nyong-nyong, and Ross River) cause fever, rash, and arthritis in humans and are associated with infrequent, but major, outbreaks of disease (5). Ross River virus (RRV) infects more than 20 vertebrate hosts, causing clinical disease in humans and horses, and has been recovered from more than 20 mosquito species (18). Since the ability to generate genetic diversity (20) enables RNA viruses to rapidly exploit new ecological niches, including new host species, and may be a key determinant of virulence (9, 14), it is clearly of central importance to determine the extent and structure of genetic diversity in human pathogens such as RRV. In particular, it is important to determine what proportion of intrahost genetic variation is created de novo by mutation and what might be due to mixed infections of indi- vidual hosts with phylogenetically diverse lineages. To date, measures of genetic diversity in alphaviruses, including RRV, have relied on comparisons of population consensus se- quences, often of isolates and occasionally from pools of mos- quitoes that may contain more than one infected insect (16). There is only one report of an attempt to quantify genetic diversity in alphaviruses within individual hosts (26), and that study was constrained by the technology of the time (T1 nu- clease fingerprinting) (6) and by the use of isolates that had been passaged in vitro rather than taken directly from host tissues.

IMPORTANCE This study gives further insight into mechanisms of type I IFN modu- lation by the medically important alphaviruses Ross River virus (RRV) and Sindbis vi- rus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was high- lighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen- associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease patho- genesis. This study also highlights the importance of the host innate immune re- sponse in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new ap- proaches for rational attenuation of alphaviruses for construction of vaccine candi- dates.

Arthritis/myositis-associated alphaviruses, such as Ross River virus (RRV), chikungunya virus, o’nyong-nyong virus, mayaro virus, and others, are mosquito-transmitted viruses that cause debilitating inflammatory disease in humans (18, 37, 46). In addition to causing endemic disease, this group of viruses is capable of causing explosive epidemics that can in- volve millions of infected individuals. Past epidemics include a 1979-to-1980 epidemic of RRV disease in the South Pacific that involved more than 60,000 patients (20) and a 1959-to- 1962 epidemic of o’nyong-nyong fever in Africa that involved at least 2 million patients (57). More recently, a reemergence of chikungunya virus has resulted in an unprecedented epi- demic in multiple countries, including Indian Ocean islands such as the Comoros, Reunion, Mayotte, Seychelles, and Mau- ritius as well as India, Sri Lanka, and Indonesia (36, 37). The number of infected individuals involved thus far in this ongoing epidemic is in the millions, with an estimated 1.4 to 6.5 million cases in India alone (29). In addition, from July to September 2007, an outbreak of chikungunya virus occurred in northeast- ern Italy, resulting in 205 confirmed human cases and detection of the virus within the local mosquito population (4, 40).

A rthritogenic alphaviruses (genus Alphavirus, family Togaviridae), including Ross River virus (RRV), chikungunya virus (CHIKV), o’nyong-nyong virus, and Mayaro virus, are a group of mosquito- transmitted viruses with positive-sense, single-stranded RNA ge- nomes that cause musculoskeletal inflammatory diseases in humans (1). In addition to causing endemic disease in Australia, Africa, Asia, and South America, these viruses are capable of causing explosive epidemics. Previous outbreaks include a 1959-1962 epidemic of o’nyong-nyong fever in Africa involving at least 2 million cases (2) and a 1979-1980 epidemic of RRV disease in Australia and islands in the South Pacific, which involved more than 60,000 cases (3). Since 2004, CHIKV has caused a series of epidemics in the Indian Ocean region, resulting in millions of cases of severe, debilitating, and often persistent arthralgia (4). Furthermore, autochthonous transmission resulting in the first CHIKV disease outbreaks in Europe and the Pacific region occurred in Italy in 2009 (5), in France in 2010 (6), and in New Caledonia in 2011 (7). These examples illustrate the ability of these viruses to reemerge and to spread to new geographical regions. Clinical manifestations which follow infection with an arthri- togenic alphavirus develop after an incubation period of between 2 and 12 days. Human disease is most commonly characterized by fever, maculopapular rash, intense pain in the peripheral joints, myalgia, and difficulty ambulating (3, 8). A multitude of studies have indicated that musculoskeletal pain persists for months to years in a subset of patients infected with RRV or CHIKV; how- ever, the underlying cause of these persistent symptoms remains unclear (1, 9). No specific therapies or licensed vaccines are cur- rently available. Treatment is limited to supportive care with an- algesics and anti-inflammatory drugs (1, 9).

Table 3. Relationships and importance of individual covariates in explaining the Ross river virus infection count per area across three spatial scales. Vector abundance and (Shannon) diversity of mosquitoes were only available for the subset of locations for which mosquito data was available, thus AIC and % deviance explained can only be compared within that analysis, not between that analysis and the state-wide analysis.

We systematically identified original research papers on RRV reservoir as follows. First, we searched electronic da- tabases (Web of Science, ProQuest, Science Direct, PubMed and Google Scholar) for articles published be- tween 1950 and May 2016 using combinations (Additional file 1: Table S1) of the following keywords: ‘Ross River virus’ , ‘Ross River fever’ , ‘endemic polyarthritis’ , ‘host’ , ‘reser- voir’ , ‘wild*’ , ‘captive’ , ‘population’ , ‘serolog*’ , ‘serosurvey*’ , ‘anti- bod*’ , ‘virus’ , ‘viral’ , ‘viraemia’ , ‘viremia’ , ‘PCR’ , ‘patholog*’ , ‘serum’ , ‘RNA ’ , ‘vector*’. The asterisk (*) operator was used as a wildcard to search for all the possible variations of keywords. We then manually searched bibliographies for additional references. Review papers, studies involving only humans, and studies not reporting original data were excluded. A flow chart showing the article selection process is presented in Additional file 2: Figure S1. A list of the publications included is provided in Additional file 1: Table S2. One person (EBS) was responsible for deter- mining if a paper was included and extracting data. By fol- lowing the inclusion and exclusion criteria there were no discrepancies for selecting papers.

SIN(RRE1), in which the 6K, E1, and 3⬘-nontranslated regions were derived from Ross River virus (RR) and the rest of the genome was derived from Sindbis virus (SIN), was almost nonviable because of a defect in budding (41). Chimeric het- erodimers between SIN PE2 and RR E1 formed and were cleaved to E2/E1 heterodimers during transport to the cell plasma membrane but had an altered conformation that did not support budding (41). Nucleocapsids in the cytoplasm did not interact with chimeric E2/E1 heterodimers in the plasma membrane as determined by electron microscopy (41). When this chimera was passaged in culture, adapted variants that grew 100 times better than the original chimera arose (42). In these variants, interactions between nucleocapsids and het- erodimers were readily observed by electron microscopy. Adaptive mutations were identified in both SIN E2 and RR E1, and some of these mutations have been partially charac- terized (42; E. G. Strauss, E. M. Lenches, and J. H. Strauss, unpublished data). In this paper, we report a study of three of these adaptive mutations, the change from Lys-131 to Glu in the ectodomain of SIN E2, the change from Ser-310 to Phe in the ectodomain of RR E1, and the change from Cys-433 to Arg in the transmembrane domain of RR E1. The last change is of particular interest because it represents the introduction of a charged residue within what is believed to be a transmembrane anchor and because palmitoylation occurs on cysteine residues in the transmembrane domains of the membrane proteins of a number of enveloped viruses (1, 6, 17, 27, 30, 31, 44). Fatty acylation has been suggested to have roles in virus formation (12, 14, 16, 44), tissue invasiveness (17), or fusion activity (6, 27). Both E1 and E2 of SIN and of Semliki Forest virus, as well * Corresponding author. Mailing address: Division of Biology, Cal-

Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.

Glycoprotein PE2 of Sindbis virus will form a heterodimer with glycoprotein E1 of Ross River virus that is cleaved to an E2/E1 heterodimer and transported to the cell plasma membrane, but this chimeric heterodimer fails to interact with Sindbis virus nucleocapsids, and very little budding to produce mature virus occurs upon infection with chimeric viruses. We have isolated in both Sindbis virus E2 and in Ross River virus E1 a series of suppressing mutations that adapt these two proteins to one another and allow increased levels of chimeric virus production. Two adaptive E1 changes in an ectodomain immediately adjacent to the membrane anchor and five adaptive E2 changes in a 12-residue ectodomain centered on Asp-242 have been identified. One change in Ross River virus E1 (Gln-4113Leu) and one change in Sindbis virus E2 (Asp-2483Tyr) were investigated in detail. Each change individually leads to about a 10-fold increase in virus production, and combined the two changes lead to a 100-fold increase in virus. During passage of a chimeric virus containing Ross River virus E1 and Sindbis virus E2, the E2 change was first selected, followed by the E1 change. Heterodimers containing these two adaptive mutations have a demonstrably increased degree of interaction with Sindbis virus nucleo- capsids. In the parental chimera, no interaction between heterodimers and capsids was visible at the plasma membrane in electron microscopic studies, whereas alignment of nucleocapsids along the plasma membrane, indicating interaction of heterodimers with nucleocapsids, was readily seen in the adapted chimera. The significance of these findings in light of our current understanding of alphavirus budding is discussed.

The detection of RRV in multiple bone and joint-associated tissues, such as synovial tissue, periosteum, tendons, and liga- ments, has not previously been described. These findings are consistent with observations of RRV-infected humans in whom both viral antigen and viral RNA have been detected from synovial effusions and synovial biopsy samples (6). Addi- tionally, infectious virus was detectable in the ankle joints of RRV-infected mice by plaque assay by 12 hpi; however, infec- tious RRV has not yet been recovered from the joints of RRV-infected patients (13). Similar to previous reports, high titers of RRV were also detected within skeletal muscle tissues of infected mice (22, 26, 31). However, our targeting studies demonstrated that there were very few RRV-infected muscle fibers in the hind limbs at early times postinfection. By 48 to 72 hpi, large areas of RRV-infected muscle fibers were observed in hind limb skeletal muscle tissue. These findings raise the possibility that RRV may initially infect joint- or skeletal mus- cle-associated connective tissues and subsequently spread into skeletal muscle myofibers. Direct infection of skeletal muscle tissue by RRV in humans has not been demonstrated, although 60% of patients diagnosed with Ross River virus disease ex- perience myalgia (12). The route and spread of RRV from the initial site of infection to joint and skeletal muscle tissue have not been characterized fully. Studies performed with other alphaviruses, such as Venezuelan equine encephalitis virus, have suggested that this group of viruses may initially infect skin dendritic cells (23). The infected dendritic cells migrate to the draining lymph node, where the virus undergoes additional rounds of replication and seeds a high-titer serum viremia, resulting in viral spread to target tissues.

The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the trans- frame protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(⌬6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-( ⌬ 6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(⌬6K) resulted in a reduced viral load and acceler- ated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development.

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 differences, 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 (LD 50 ), although no change in the average survival time of infected mice was observed. These variants grew to

CHIKV-induced disease that recapitulate many aspects of the hu- man diseases (13–17). Studies with the RRV mouse model dem- onstrated that following a high-titer serum viremia, bone/joint- associated tissues and skeletal muscle tissue are the primary targets of RRV replication (15, 18). RRV replication at these sites results in a severe inflammatory response with abundant tissue damage, leading to deficits in grip strength and an altered gait. Human disease following infection by an arthritogenic alphavirus shows a similar progression, with (i) high-titer serum viremia (19, 20), (ii) the detection of virus RNA and/or antigen in musculoskeletal tis- sues (21, 22), (iii) mononuclear inflammatory infiltrates in joints and muscle tissue (19, 22–24), and (iv) disease signs, such as mus- culoskeletal pain and difficulty ambulating. Thus, understanding the host and viral factors that contribute to pathogenesis in this mouse model may aid in the development of therapies and vac- cines to treat or prevent human disease.

The aim of this study was to use RNase T1 mapping to examine the genetic relatedness of multiple isolates of RR virus from different geographical regions and to determine whether i the v[r]

viral titer resulted from the digestion of the recently acquired infectious blood meal. It is unlikely that mosquito excreta has a role as an alternative route of transmission under field conditions. Firstly, arboviruses are labile in the environment; in fact, viability of arboviruses in infected mosquitoes decreases rapidly after their death in hot and humid conditions [48]. Mos- quito excreta also contains digestive enzymes [49] which could continue to inactivate remain- ing virions once they have been excreted. Secondly, arbovirus infection via aerosol has only been observed under circumstances of high virus concentration [50]. Studies to test Japanese encephalitis virus (JEV) vaccines using Rhesus macaques exposed intranasally to JEV required at least 6.6 x 10 6 infectious units per animal to achieve infection [51, 52]. Our results obtained from batches of 5 mosquitoes with a high infection rate showed only low or trace amounts of viable virus. In the field, where only 1–2 mosquitoes out of thousands in a trap might be infected, the amount of viable virus in excreta would be even lower. Finally, it is well docu- mented that mosquito saliva plays an important role in facilitating arbovirus transmission [53] and excreta lacks salivary proteins responsible for generating favourable replication conditions in the vertebrate host.

RRV envelope glycoproteins are currently unknown. RRV, as well as its alphavirus relatives Semliki Forest and Sindbis vi- ruses, have extremely broad host ranges. They infect both invertebrate and vertebrate animals and a wide variety of cell types (41, 46). This feature suggests that alphaviruses might utilize multiple receptors to gain access to the cells or tissues or, alternatively, that these viruses use ubiquitous proteins as cellular receptors that are well conserved among diverse spe- cies (22, 45). Moreover, even though RRV, Semliki Forest virus, and Sindbis virus all belong to the alphavirus family, they appear to utilize different cellular molecules as receptors for cell binding and/or entry. For example, the glycosaminoglycan heparan sulfate is shown to participate in the binding of Sind- bis virus to cells (8). Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells leads to a large re- duction in Sindbis virus binding. However, these treatments have no effects on RRV binding, suggesting that, unlike Sind- bis virus, RRV virus does not utilize heparan sulfate as a cellular receptor (8). This difference in the use of cellular receptors may explain in part the discrepancy in the cellular tropism in the central nervous system between RRV- pseudotyped FIV vector and Sindbis virus/Semliki Forest vi- FIG. 5. RRV-pseudotyped FIV vector directs transgene expression predominantly in neuroglia. (A) At 3 weeks postinjection of 5 ⫻ 10 5 TU

Type I IFN (IFN- ␣ / ␤ ) bioassay. IFN-␣/␤ levels in cell culture supernatants were measured by an IFN bioassay as described previously (29, 35). L929 mouse fibroblasts (ATCC CCL-1) were seeded into 96-well plates and grown in the same medium as BHK-21 cells. Samples were acidified to a pH of 2.0 for 24 h and then neutralized to pH 7.4. Additional virus inactivation by UV light (as de- scribed above) was performed prior to titration by twofold serial dilutions across the plate. Twenty-four hours later, encephalomyocarditis virus was added to each well at an MOI of 5. At 18 to 24 hpi, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT; Sigma) was used to assess the viability in each well. The MTT product produced by viable cells was dissolved in isopropanol– 0.4% HCl and quantified by absorbance readings on a microplate reader at 570 nm. Each plate contained an IFN- ␤ standard (Chemicon or R&D Systems) which was used to determine the number of international units of IFN- ␣ / ␤ per milliliter of the unknown samples. Alternatively, IFN- ␤ levels were quantified using a mouse IFN-␤ enzyme-linked immunosorbent assay (ELISA) kit (PBL Biomedical Laboratories, Piscataway, NJ). ELISAs were performed per the manufacturer’s instructions except that the IFN-␤ standard was diluted in RPMI 1640 supplemented with 10% FBS, L -glutamine, penicillin and streptomycin, and 2-mercaptoethanol instead of the sample diluent provided in the kit.