Macrophage-induced muscle pathology results in morbidity and mortality for Ross River virus-infected mice.

Ross River virus (RRV) is an Australian alphavirus that is often responsible for chronic epidemic polyarthritis and myalgia in humans. Past studies have shown severe disruption of striated muscle fibers to be prominent in RRV pathology in mice; in the present study, macrophages were directly implicated as the primary mediators of muscle damage. General immunosuppressive therapies had only minor effects on mortality and morbidity in RRV-infected mice, with no inhibition of muscle damage. Treatment of mice with macrophage-toxic agents (e.g., silica) prior to RRV infection completely abrogated disease symptoms without significantly affecting titers of virus in organs. Further studies found that clinical signs of infection and muscle damage correlated with a massive influx of macrophages into hind leg muscle, whereas no such infiltrate or damage was observed for silica-treated mice. These observations are significant for the human disease context, as monocytic cells have been detected in the synovial effusions of persons with epidemic polyarthritis.

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.

Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and mouse brain.

A method is described for direct sequence analysis of selected regions of dengue virus genomic RNA in infected tissues. Using specific primers, total high-molecular-weight infected-cell RNA is reverse transcribed to single-stranded (ss) complementary DNA, amplified using the polymerase chain reaction (PCR) and sequenced using ssDNA obtained after lambda exonuclease digestion of one strand of the PCR product (R.G. Higuchi and H. Ochman, Nucleic Acids Research, 17, 5865, 1989). Sequence data for the envelope protein gene of two dengue-3 virus isolates were obtained using RNA from small numbers (10(5)) of cultured mosquito or monkey kidney cells, from one mg of infected mouse brain and from 1/300th of an infected Toxorhynchites amboinensis mosquito. Independent determinations showed that errors occurring during reverse transcription or PCR were not represented to a significant degree in the sequence of the amplified DNA. The method does not depend on extensive passaging of virus or large-scale growth to generate material for sequencing and therefore provides a means of obtaining sequence data for unadapted dengue virus isolates.

Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence.

We have passaged the prototype strain of Murray Valley encephalitis virus in SW13 (human) cells, sequenced the E and M genes, and examined the virulence of the passaged virus for 21-day-old mice following intracranial and intraperitoneal inoculation. Six independent passage series were carried out: four in the presence of mouse hyperimmune ascitic fluid and two without antibody. Changes were observed in the E protein deduced amino acid sequence for each of the six 10th passage stocks sequenced. Eleven changes were observed in total for the six stocks sequenced; these were at residues 117, 118, 390, 423, and 460. Nine of the changes were nonconservative. Five of the six passaged variants were altered at Asp 390 which is part of an Arg-Gly-Asp (RGD) sequence. This change resulted from adaptation to SW13 cells rather than from antibody selection. The RGD sequence (and residue 423) falls within a region which is highly conserved between flaviviruses and is strongly hydrophilic. All five variants which were altered at Asp 390 were attenuated in 21-day-old mice following i.p. inoculation. We propose that the domain of E encompassing the RGD sequence is an important determinant of flavivirus pathogenicity.

Murray Valley encephalitis virus field strains from Australia and Papua New Guinea: studies on the sequence of the major envelope protein gene and virulence for mice.

We have compared the nucleotide sequence of the gene encoding the major envelope (E) protein of a number of Murray Valley encephalitis virus (MVE) isolates from Australia and Papua New Guinea (PNG). The isolates, from widely separated geographic regions, were from four fatal human cases, a heron, and six mosquito pools and covered a period of 25 years. The sequences of the Australian strains were notable for their similarity, showing not more than 1.7% nucleotide sequence divergence in pairwise comparisons. There was 6.8% divergence in the E gene between the two available strains from PNG, and 9-10% divergence between each of the PNG strains and the Australian prototype. These data are consistent with previous conclusions based on HaeIII restriction digest analysis of cDNA to virion RNA (M. Lobigs, I. D. Marshall, R. C. Weir, and L. Dalgarno, 1986, Aust. J. Exp. Biol. Med. Sci. 64, 571-585). We conclude that a single MVE genetic type exists in Australia. Separate foci of MVE evolution appear to exist in PNG, generating greater strain variation. For all MVE isolates the deduced length of the E protein was 501 amino acids. The E protein differed at no more than three positions between any two Australian strains. The PNG strains differed from the Australian strains at 6-11 residues depending on the virus pair. Differences in amino acid sequence did not occur at a position corresponding to a previously demonstrated neutralization determinant in yellow fever virus (M. Lobigs, L. Dalgarno, J. J. Schlesinger, and R. C. Weir, 1987, Virology 161, 474-478). Thus selection for neutralization resistance may not be a major evolutionary pressure in the field situation. In comparisons between the E protein amino acid sequence of the prototype strain and those of a number of other MVE strains, 7 out of 14 differences were at residues seen at the corresponding position for Japanese encephalitis virus (JE), consistent with the close serological relationship of MVE and JE. Five Australian MVE strains and two from PNG were tested for virulence by comparing LD50 values after intraperitoneal and intracranial inoculation of 21-day-old mice; all strains were virulent by this test.

Genetic differentiation of Murray Valley encephalitis virus in Australia and Papua New Guinea.

The genetic relatedness of ten Murray Valley encephalitis virus (MVE) isolates from Australia has been examined by comparing HaeIII and TaqI restriction digest profiles of cDNA to virion RNA. The isolates were from the Murray Valley region of south-eastern Australia and from the Ord River region of Western Australia and spanned a period of 23 years (1951-1974). The isolates generated closely similar restriction digest profiles. The extent of similarity suggested that the level of nucleotide sequence divergence between any pair of Australian MVE isolates is probably around 1%. The genetic homogeneity of the MVE isolates contrasts with results obtained for Ross River virus, an alphavirus, using an identical methodological approach; we propose that this difference results from the important role of birds in the life cycle of MVE. Four MVE isolates from three fatal human cases showed small genetic differences one from the other. These isolates did not have a common restriction digest profile which distinguished them from strains obtained from other sources (e.g., from mosquitoes or a heron). The data do not support the view that clinical cases of MVE infection in humans are due to a particular strain of virus although this has not been rigorously excluded. The two available MVE isolates from Papua New Guinea (PNG) were from the Sepik and Port Moresby regions. They generated HaeIII and TaqI restriction digest profiles which were different both from each other and from those of the Australian type. Genetic divergence between the two PNG isolates was estimated to be approximately 6%; divergence between either of the PNG isolates and the Australian type was greater than 6%. Our data suggest that the evolution of MVE in Australia and PNG has proceeded independently and that circulating Australian MVE strains are not systematically re-seeded from regions of endemicity in PNG. Studies on the relatedness of MVE and two close antigenic relatives, Japanese encephalitis virus (JE) and Alfuy virus (ALF), showed that the genetic relatedness between any MVE isolate and JE or ALF is less than that between the most divergent of the MVE isolates, including those from Papua New Guinea.

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).

Classification of Barmah Forest virus as an alphavirus using cytotoxic T cell assays.

Barmah Forest virus, an arbovirus, does not cross-react convincingly with alpha-, flavi- or bunyavirus immune sera. Secondary cytotoxic T cells generated in vitro immune to a number of alphaviruses cross-lyse Barmah Forest virus-infected target cells. Flavivirus (West Nile and Kunjin)- and Bunyamwera virus-immune Tc cells lyse homologous virus-infected target cells, but not alphavirus-infected targets. Using cytotoxic T cell assays Barmah Forest virus can be classified as an alphavirus.

Ross River virus genetic variants in Australia and the Pacific Islands.

HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA or virion RNA were used as a guide to genetic relationships between fourteen isolates of Ross River virus (RRV) obtained from mosquitoes collected in various localities in eastern Australia where the virus is endemic. RRV isolates from Fiji, American Samoa, the Cook Islands and the Wallis Islands where major outbreaks of epidemic polyarthritis took place in 1979-1980 were also examined. Among these RRV isolates we have identified three genetic types (I-III) on the basis of differences between their restriction digest profiles. We estimate that 1.5-5% nucleotide sequence diversity exists between genetic types. Within each genetic type strain differentiation gave rise to small but significant differences in restriction digest profiles. No clear pattern of geographic distribution of RRV genetic types could be established from the limited number of RRV isolates examined. Genetic types I, II and III, respectively, were isolated from three, three and one different mosquito species, indicating there is no strong association between genetic type and the species of mosquito vector. HaeIII restriction digest analysis did not detect any genetic difference between the four Pacific Island isolates, suggesting that a single RRV variant was involved in the epidemics. Genetically, this variant was closely related to isolates of genetic type II. Virtually identical HaeIII restriction digest profiles were observed for isolates obtained at various stages of the Pacific Island epidemics, suggesting that extensive sequence evolution did not accompany Ross River virus spread.

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.

Effect of pregnancy on stimulation of alphavirus immunity in mice.

The state of pregnancy was associated with a marked enhancement of both proliferative and cytotoxic T cell antiviral immune responses after infection of mice with avirulent Semliki Forest virus. Strong responses were obtained from the spleen and the para-aortic lymph nodes that drain the uterus. This immune enhancement seemed to be in response to the increased antigenic challenge that originated from infected placental and fetal tissue and was released during the process of abortion. Immune enhancement was not observed in pregnant mice similarly infected with Ross River virus, which also established an intrauterine infection but does not cause abortion.

Characterization of Barmah forest virus: an alphavirus with some unusual properties.

Barmah Forest virus has been characterized in a number of ways including electron microscopy of infected cells; physical studies of the virion, its RNA, and associated proteins; N-terminal sequence analysis of the two envelope glycoproteins; studies of macromolecular species present in infected cells; and serological cross-reactions with alphaviruses and bunyaviruses. From these results Barmah Forest virus is clearly an alphavirus since the structure of the virion, the mode of replication, and the macromolecular species present in infected cells are typical of alphaviruses. The N-terminal regions of the two glycoproteins E1 and E2 show extensive sequence homology (approximately 50%) with those of other alphaviruses. Barmah Forest virus cross-reacts in hemagglutination inhibition tests, although not in complement fixation tests or infectivity neutralization tests, with other alphaviruses. In some of its properties Barmah Forest virus is unusual, however. It cross-reacts in complement fixation and hemagglutination inhibition tests with Umbre virus, a bunyavirus, which originally led it to be classified as a bunyavirus; the glycosylation pattern of E2 of Barmah Forest virus appears to differ from that of other alphaviruses; and the sedimentation coefficient of the virion appears to be slightly less than that of other alphaviruses.

The role of colostrum and milk in protection of the neonatal mouse against peripheral infection with Ross River virus. Brief report.

Neonatal mice can be protected against peripheral infection with Ross River virus by colostral and milk IgG after suckling on an immune mother. This protection supplements the transplacentally acquired protection against in utero infection.

Primary viraemia responses of herons to experimental infection with Murray Valley encephalitis, Kunjin and Japanese encephalitis viruses.

Rufous night herons, Pacific herons, little egrets and intermediate egrets were experimentally infected with Murray Valley encephalitis, Kunjin or Japanese encephalitis viruses. Viraemias of at least one day's duration were detected in all birds except two intermediate egrets inoculated with a very low dose of Kunjin virus and one rufous night heron inoculated with Japanese encephalitis virus. there was usually a viraemia of 3 to 5 days' duration commencing on the first or second day and continuing until day 5 or 6 and rarely until day 7. Maximum titres tended to be higher in young birds, up to 2-5 months of age (10(4)-10(5) mouse LD50/ml), than in older birds more than 8 months of age (10(3)-10(4) mouse LD50/ml). Significant differences in maximum viraemia titres were not observed in the different species or between Murray Valley encephalitis and Kunjin viruses. Japanese encephalitis viraemias were significantly lower, but this was probably due to the high mouse brain passage level of the strain used. The onset of viraemia was earlier in intermediate egrets than in rufous night herons inoculated with similar doses of Murray Valley encephalitis virus, but no difference in the susceptibility to infection was observed. With Kunjin virus there was a significant difference in the susceptibility of intermediate egrets and rufous night herons, with rufous night herons being more susceptible to infection with low doses of virus. This difference in threshold of infection, if it extends to other species with both Kunjin and Murray Valley encephalitis viruses, may, in part, be an explanation for the greater incidence of natural infections observed in rufous night herons compared with other species and orders of water birds.

Primary antibody responses of herons to experimental infection with Murray Valley encephalitis and Kunjin viruses.

Antibody responses of rufous night herons (Nycticorax caledonicus) and little egrets (Egretta garzetta) following infection with Murray Valley encephalitis and Kunjin viruses were determined. Haemagglutinin-inhibiting antibodies were first detected on day 5 or 6 after inoculation and increased rapidly, reaching maximum titres of 320 to 2560 between 10 and 20 days after inoculation. Titres declined 20-320 between 60 and 120 days after inoculation, then tended to remain stationary. Titres were 2- to 8-fold higher to infecting virus than heterologous virus. Neutralizing antibody development paralleled that of HI antibodies with titres maintained at a higher level for longer periods; however, they did eventually decline to low levels. Following MVE virus infection IgM (19S), HI antibodies were 80-100% of HI antibodies detectable on day 6 or 7 after inoculation and declined rapidly, becoming undetectable by 20 days after inoculation. With Kunjin virus infections, IgM HI antibodies represented 90-100% of HI antibodies detectable on day 6 or 7 after inoculation. Significant levels of IgM HI antibodies were still detectable 20 days after inoculation (5-30% of total HI antibodies) and, in some birds, even at 27 days after inoculation (up to 10%), IgG (7S) HI antibodies were low or undetectable on day 6 or 7 after inoculation, then increased rapidly with rapidly rising HI antibody titres. The specificity of IgM and IgG antibodies and unfractionated sera was determined by testing against Murray Valley encephalitis, Kunjin, Japanese encephalitis and West Nile virus haemagglutinating antigens. It was possible to determine with which virus a bird had been infected from the pattern of cross-reaction with these antigens. These results should provide a rational basis for the interpretation of serological results from naturally infected birds.

Viruses recovered from mosquitoes and wildlife serum collected in the Murray Valley of South-eastern Australia, February 1974, during an epidemic of encephalitis.

Pools of mosquitoes collected in the Murray Valley in February, 1974, during an encephalitis epidemic yielded 239 isolates of 11 distinct viruses. These included 39 isolates of MVE virus, an incriminated causative agent of encephalitis in man, and 111 isolates of Kunjin virus, a probable causative agent. An additional isolate of MVE virus was recovered from the serum of a white-faced heron, Ardea novaehollandiae. The other 9 viruses comprised the Alpha-viruses Ross River and Sindbis, the Flavivirus, Edge Hill, the Bunyaviruses, Koongol and Wongal, the undefined Kowanyama virus, and 3 previously undescribed viruses. One isolate of MVE virus was recovered from a total of 180 Culex australicus, and all other isolates of all viruses were from Culex annulirostris. The combined apparent infection rate of MVE and Kunjin viruses in Cx annulirostris was 1/913. The concurrent activity of so many seemingly transient arboviruses, their wide geographical distribution and temporal considerations all weaken the widely held hypothesis that during the pre-epidemic period MVE virus is introduced into the Murray Valley by viraemic waterbirds from distant areas of tropical enzooticity.

Variation in arbovirus infection rates in species of birds sampled in a serological survey during an encephalitis epidemic in the Murray Valley of South-eastern Australia, February 1974.

There was extensive and exuberant breeding of waterbirds before and during an epidemic of arboviral encephalitis in the Murray Valley of south eastern Australia in 1974. As estimated by haemagglutination inhibition tests on 432 bird sera collected between 4th and 13th February, 1974, infection with Murray Valley encephalitis virus, Kunjin virus and possibly other flaviviruses was concentrated in species of the Order Ciconiiformes (55% positive) and Pelecaniformes (41%), compared with only 5% in Anseriformes. Although Sindbis virus infections were also highest in these 2 Orders (56% and 46%, respectively), the incidence of antibodies was spread more uniformly through other Orders than with the flaviviruses: e.g. Anseriformes, 33%, Podicipitiformes, 27%. As viruses were recovered virtually only from Culex annulirostris mosquitoes, the different patterns of infection seem unrelated to host preference by mosquito species or behavioural response to mosquito attack, and suggest a specific relationship between MVE/KUN and Ciconiiformes and Pelecaniformes. The highest infection rate was 22/25 in mature rufous night herons (formerly nankeen night herons) Nycticorax calendonicus, and here tolerance to mosquito attack was probably a contributing factor. Assays of sera from 13 horses indicated high infection rates both alphaviruses and flaviviruses.

Species composition of mosquitoes collected in the Murray Valley of South-eastern Australia during an epidemic of arboviral encephalitis.

Mosquito activity at the height of an epidemic of arboviral encephalitis in the Murray Valley during the summer of 1974 was almost entirely crepuscular and nocturnal, but anecdotal evidence, indicated that day-biting Aedes species were also unusually prevalent during the first 4-5 weeks of the epidemic. Ninety-eight percent of the 138,359 mosquitoes captured in miniature light traps and by aspiration between 4th and 13th February, 1974, were Culex annulirostris, and 10 other species of 5 genera comprised the remaining 2%. In the one 'man-biting' collection 96% of 1,364 mosquitoes were Cx annulirostris. The overall capture rate for 51 light trap nights at 7 widely dispersed collecting sites was 2,675. At Barmah Forest 10 light trap nights yielded an average of 9,103 mosquitoes, with a maximum of 26,293 in one trap night.

Arboviruses of coastal south-eastern Australia.

During investigations of epidemic polyarthritis at Nelson Bay, New South Wales, 12 strains of Ross River virus, the causative agent, were recovered from pools of mosquitoes. In addition, the mosquito pools yielded 9 strains of the flavivirus Edge Hill, 4 strains of a bunyavirus, Gan Gan, 1 strain of an orbivirus Tilligerry, and 1 strain of an ungrouped probable arbovirus, Yacaaba. The latter 3 viruses were found to be antigenically distinct from previously recorded arboviruses. A case of epidemic polyarthritis probably contracted at Bawley Point, approximately 400 kilometres south of Nelson Bay on the south coast of New South Wales, is recorded. Mosquitoes collected in the nearby Termeil State Forest one year later yielded a strain of Ross River virus and 5 strains of a previously undescribed ungrouped togavirus, Termeil. The extent of arboviral activity in temperate coastal areas of south-eastern Australia was unexpected.

Bunyaviridae.

The family Bunyaviridae comprises over 200 viruses (serotypes, subtypes, and varieties) that infect vertebrates and/or invertebrates. Four genera of viruses have been defined (Bunyavirus, Nairovirus, Phlebovirus, and Uukuvirus). The main characteristics of the member viruses are: (i) the virus particles are for the most part uniformly spherical, 80-110 nm in diameter, and possess a unit membrane envelope from which protrude polypeptide spikes 5-10nm long; (ii) the viruses have three helical nucleocapsids, often in the form of supercoiled circles, each consisting of a single species of single-stranded RNA, major nucleocapsid polypeptide, N, and at least in some cases minor amounts of a large polypeptide which may be a transcriptase component; (iii) the genome is composed of three species of RNA (L, large; M, medium; and S, small), organized in end-hydrogen bonded circular structures; (iv) most viruses have three major virion polypeptides (N, and two surface polypeptides, designated G1 and G2); (v) for at least some member viruses, the virions have been shown to contain an RNA-directed RNA polymerase, believed to be responsible for the synthesis of viral complementary mRNA, so that bunyaviruses are considered to be negative-stranded viruses; (vi) at least some bunyaviruses are capable of heterologous virus genome segment reassortment and can form recombinant viruses at high or low frequency; (vii) viruses appear to mature primarily at smooth membrane surfaces and accumulate in Golgi vesicles and saccules, or nearby; (viii) transovarial, venereal and/or transstadial transmission in arthropods has been shown to occur for some members of the family.