Ockelbo Virus Research Articles

Diversity of Culex torrentium Martini, 1925 — a potential vector of arboviruses and filaria in Europe

Culex torrentium is one of the most common mosquito species in Germany. Due to its sympatric occurrence as well as its similar morphological and ecological characteristics, it has often been confused with another common species, Culex pipiens. Both species are known to be potential vectors for different arboviruses (not only in Germany) with C. torrentium being a possible vector for Sindbis or Ockelbo virus. In our study, we analyzed the genetic variability in a 658 bp fragment of the cytochrome c oxidase subunit I gene (coxI) of C. torrentium, from nine localities in the Frankfurt/Rhine-Main Metropolitan Region. The results of our genetic survey indicate a higher genetic diversity in this gene region for C. torrentium than for the morphologically similar C. pipiens. Our findings may explain the difficulties in the past to find morphological characteristics that apply to all populations of C. torrentium, when attempting to separate them clearly from C. pipiens, by any other criteria than male genitalia. Being ornithophilic, possible hybrids between C. torrentium and the humanophilic C. pipiens biotype molestus, could potentially serve as important vectors for zoonotic diseases. Therefore, we recommend that greater emphasis is placed on the ecological characteristics, population structure, and the taxonomy of this often neglected species, in the future.

The Molecular and Cellular Aspects of Arthritis Due to Alphavirus Infections

Alphaviruses such as the Sindbis-group viruses, Scandinavian Ockelbo virus, the African Asian chikungunya virus, the African O'nyong-nyong virus, the South American Mayaro virus, and the Australasian Barmah Forest and Ross River viruses, are commonly associated with outbreaks of acute and persistent arthritis and arthralgia in humans. The mechanisms by which these viruses cause arthritis/arthralgia are poorly understood. This chapter summarizes our current understanding of viral arthritides using our newly developed mouse model of Ross River virus-induced joint and muscle inflammation.

Sindbis viruses and other alphaviruses as cause of human arthritic disease

Amongst the arthritis-causing arboviruses, i.e. those spread by insects, the alphavirus group is of special interest. These viruses occasionally cause vast outbreaks, such as O'nyong-nyong in Africa in 1959. In Fennoscandia, Sindbis-related Ockelbo, Pogosta, or Karelian fever viruses have been found to cause significant morbidity. The major symptoms in addition to joint inflammation are fever, fatigue, headache and rash. The joint symptoms may persist for weeks, even months. The diagnosis is based on the clinical picture and serology. The causative viruses are closely related but not identical. It appears that at least in Finland the Pogosta disease is more common than thought, and the symptoms may often be overlooked. Several factors related to the viruses, their hosts, and global environmental changes may affect the spread of these viruses. All over the world arbovirus-caused diseases have increased, because of global changes.

Genetic Relatedness of Sindbis Virus Strains from Europe, Middle East, and Africa

The relatedness of 40 strains of Sindbis virus (SIN) from Europe, the Middle East, and Africa was investigated by limited sequencing within the gene encoding the E2 glycoprotein corresponding to amino acid residues 117 to 229 and encompassing one of the major neutralization epitopes. Phylogenetic analyses using distance matrix and parsimonious methods identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for Venezuelan equine encephalomyelitis virus with a maximum divergence of 12.4% versus 28.5%, respectively. One cluster comprising 19 strains included the HR derivate of the Egypt SIN prototype, AR339, and strains from Israel, Saudi-Arabia, Italy, Slovak Republic, Azerbaijan, as well as three Swedish strains. Another cluster of 17 strains included the Ockelbo virus (OCK) prototype, Edsbyn 5/82, and the majority of SIN strains from northern Europe including strains from Sweden, Norway, and Karelia, as well as two strains from South Africa. A third cluster, supported by the Neighbor Joining method, was made up of four strains from South Africa, Uganda, and Cameroon. Residue 212, either Ser or Thr, previously appointed important for the differences in neutralization assays between SIN and Edsbyn 5/82, respectively, correlated with the two major genetic clusters, but was a Thr for two of the three Swedish strains in the SIN prototype cluster, and a Ser in one Swedish and one Karelian strain in the OCK cluster. The finding of strains similar to prototype SIN in Middle Sweden and of strains in South Africa relating to the northern cluster of SIN strains supports the notion of the dispersal of SIN by migrating birds as previously suggested for New World alphaviruses.

Ockelbo virus (Togaviridae: Alphavirus) neutralizing antibodies in experimentally infected Swedish birds.

The ability of native Swedish birds to produce and maintain production of Ockelbo virus neutralizing (Nt) antibodies were evaluated experimentally between 6 June 1990 and 27 July 1991. After experimental infection of 57 birds of the orders Anseriformes and Passeriformes with Ockelbo virus, these birds were examined for Ockelbo virus Nt antibodies at 5 days, and at 1, 3, 6, 9, and 12 mo after inoculation. One month after inoculation, Nt antibodies were more prevalent in birds with a detectable viremia (100%, n = 22) than in non-viremic birds (65%, n = 26). The Nt antibody prevalence varied over time among taxa; detectable antibodies occurred earlier after inoculation and for a longer time in Anseriformes than in Passeriformes. By 5 days after inoculation, antibodies could be detected in 22 (71%) of 31 Anseriformes but in none of 16 Passeriformes. However, at 1 mo the antibody prevalences at 1 mo were similar: 84% among the Anseriformes and 73% among the Passeriformes; at 3 mo the prevalences were 50% in Anseriformes and 15% in Passeriformes. Forty-two percent of the Anseriformes had detectable antibodies even 12 mo after inoculation.

Development of specific antibody patterns and clinical symptoms following Ockelbo virus infection.

Sixteen patients with symptoms typical for Ockelbo disease (rash, arthralgia, fever) were enrolled in a 2 1/2 year study, during which clinical symptoms were recorded and ELISA was employed to study specific IgM, IgG and IgG subclass development. Initially, all patients presented with rash and arthralgia, and five patients still suffered from joint symptoms at the end of the study period. Ockelbo virus specific IgM was detected during the first week post onset in 6 patients and in 15 patients by day 14. One patient failed to develop specific IgM and was later diagnosed with a human parvovirus B19 infection. All patients were IgM-negative 2 1/2 years post onset. Seroconversions or significant titer rises for specific total IgG were seen in 15 patients. IgG titers generally peaked within one year but in two patients maximum titers were seen 2 1/2 years post onset. Development of IgG1 followed that of total IgG, while IgG3, after an initial increase in all Ockelbo disease patients, remained at peak levels for one year in four patients, three of whom still had detectable IgG3 at the end of the study period. Ockelbo virus specific IgG2 or IgG4 was not detected in any of the patients.

Antigenic Comparison of Ockelbo Virus Isolates from Sweden and Russia with Sindbis Virus Isolates from Europe, Africa, and Australia: Further Evidence for Variation Among Alphaviruses

The plaque-reduction neutralization test (PRNT) was used to compare 15 isolates of Ockelbo virus from Sweden, one isolate of Ockelbo virus from Russia, the Egyptian prototype Sindbis virus, and Sindbis-like viruses from Slovakia, South Africa, Cameroon, and Australia. Strains from northern Europe (Sweden and Russia) were indistinguishable by PRNT. We observed some antigenic variation between isolates of Sindbis virus from Europe, Africa, and Australia. An Australian strain (C-377) was shown to be distinct from prototype Sindbis virus, and the Acrocephalus strain from Slovakia was shown to be identical to prototype Sindbis virus. All other strains, including Ockelbo virus isolates, were shown to be subtypes of prototype Sindbis virus.

Detection of Ockelbo virus RNA in skin biopsies by polymerase chain reaction.

A sensitive assay based on the polymerase chain reaction for the detection of Ockelbo virus RNA was developed. Two primer pairs from the gene coding for the E2 glycoprotein were chosen. By use of a nested strategy for the primers, as few as 1 to 10 PFU could be detected. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The primer pairs allowed amplification of several Ockelbo and Sindbis virus isolates but discriminated between these and other alphaviruses. Ockelbo virus RNA was detected in 4 of 10 skin biopsy specimens collected during the acute stage of the disease. The identities of the amplified products were confirmed by restriction endonuclease cleavage. Acute- and convalescent-phase sera as well as lymphocytes collected during the convalescent phase were negative by the polymerase chain reaction. No infectious virus could be recovered from any of the specimens.

Viremia in three orders of birds (Anseriformes, Galliformes and Passeriformes) inoculated with Ockelbo virus.

One-hundred six birds of 14 species were inoculated with approximately 10(2.7) plaque-forming units of Ockelbo virus and bled daily for 5 days to determine viremia levels. Virus was detected in birds of all 14 species tested (four Anseriformes, one Galliformes and nine Passeriformes). The onset of viremia occurred earlier and viral titers were higher in very young anseriforms and galliforms than in older birds. Adult passeriforms had Ockelbo viremias of higher titer and longer duration than did adult anseriforms. Viremia titers in adult birds of all three orders tested were sufficient to induce high transmission rates in enzootic mosquito vectors, and viremias in passeriforms could induce high transmission rates in bridging vectors as well. Passeriforms of the genera Turdus and Fringilla could serve as amplification hosts for Ockelbo virus based on the presently demonstrated viremia of high titer and long duration in these birds, and the previously demonstrated high prevalence of Ockelbo virus neutralizing antibodies in free-ranging individuals and great population size compared to birds of other taxa. Bird species of all three orders tested, however, could function as incidental hosts of the virus.

Antibodies to Ockelbo virus in three orders of birds (Anseriformes, Galliformes and Passeriformes) in Sweden.

Sera from 324 birds collected in an Ockelbo virus disease endemic area in central Sweden were examined for the presence of specific antibodies to Ockelbo virus by a plaque reduction neutralization test. Birds examined belonged to the orders Anseriformes (n = 207), Galliformes (n = 66) and Passeriformes (n = 51). Ockelbo virus neutralizing antibodies were detected in 26 (8%) of the specimens, including species from each of the three orders tested. Specific antibodies found in caged birds and in 6- to 10-week-old birds suggested local transmission. The highest antibody prevalence (27%, 14/51) was observed in the Passeriformes in which 5 of 9 species tested contained antibodies. The high antibody prevalence in passeriforms and the very large population of this group in relation to other avian groups in Sweden gives them a high potential as amplification hosts for Ockelbo virus.

Structure of the Ockelbo virus genome and its relationship to other sindbis viruses

Ockelbo virus was first isolated in 1982 in Sweden. It is the causal agent of disease in humans characterized by arthritis, rash, and fever and is antigenically very closely related to Sindbis virus. We have determined the nucleotide and translated amino acid sequences of the prototype Ockelbo virus isolate (82-5) to determine the relatedness of Ockelbo virus to Sindbis virus at the genomic level and clarify the taxonomic position of Ockelbo virus within the alphavirus genus. The numbers of nucleotides and of translated amino acids in each region of the Ockelbo virus genome were exactly the same as those for the prototype AR339 strain of Sindbis virus except for three small deletions and insertions in the C-terminal half of nsP3 and for three single nucleotide insertions and deletions in the 3′ untranslated region. Overall there were 672 nucleotide differences (5.7% divergence), resulting in 97 amino acid changes (2.6% divergence), between the two viruses: more than 85% of the nucleotide changes were silent. Only the C-terminal domain of nsP3 and the E2 glycoprotein showed a higher degree of amino acid substitution than the overall average. The former domain is not conserved among alphaviruses, and the latter is primarily responsible for antigenic variation. Sequence analysis of 420 nucleotides at the 3′ end of a number of other Sindbis-like alphaviruses, including Karelian fever virus and South African, Indian, and Australian isolates of Sindbis virus, demonstrated that Ockelbo virus is more closely related to South African strains of Sindbis virus than it is to the prototypic Egyptian AR339 strain. Thus the South African strains, which have caused epidemic disease in humans, may have been introduced into Northern Europe by man or by migratory birds to establish Ockelbo disease there. The Indian and Australian strains form a distinct branch of the evolutionary tree and differ from prototypic AR339 Sindbis virus in 17% of the nucleotides sequenced.

Geographical and temporal distribution of Ockelbo disease in Sweden.

The incidence of Ockelbo disease and the prevalence of Ockelbo virus neutralizing antibodies were investigated in a sample of the Swedish population. The disease occurs throughout most of Sweden but with higher incidence and antibody prevalence rates in the central part of the country. It generally affects middle-aged men and women, with equal incidence between sexes, and is uncommon in people younger than 20 years of age. The disease occurs during a short period each year between the third week of July and the first week of October, with a peak during the second half of August. During the 8 years studied (1981-8), an average of 31 Ockelbo patients/year were diagnosed. The antibody prevalence rates in the oldest age groups were 20-40 times higher than the accumulated life-risk of being diagnosed and reported as an Ockelbo disease patient, which suggests that many cases are asymptomatic and/or unreported.

Effect of Environmental Temperature on the Vector Competence of Aedes Aegypti and Ae. Taeniorhynchus for Ockelbo Virus

Studies were conducted to determine the effect of environmental temperature on the ability of Aedes aegypti and Ae. taeniorhynchus to transmit Ockelbo (OCK) virus. Temperatures tested were 10 degrees C, 17 degrees C, 24 degrees C, and a cyclic (10-24 degrees C, mean = 17 degrees C) regimen designed to mimic continuously temperatures to which a mosquito might be exposed in July and August in central Sweden. Both species were highly susceptible to oral infection, with no consistent association between incubation temperature and infection rates. However, dissemination of OCK virus to the hemocoel in infected mosquitoes was directly related both to the dose of virus ingested and to the extrinsic incubation temperature. After greater than or equal to 14 days extrinsic incubation, once a disseminated infection was achieved, transmission by bite to a susceptible chick did not appear to be affected by either the initial dose ingested, the holding temperatures of 17 degrees C, 24 degrees C, nor the cyclic regimen. When re-fed, 93% (68/73) and 82% (67/82) of the disseminated Ae. taeniorhynchus and Ae. aegypti, respectively, transmitted virus. In contrast, when mosquitoes were held at 10 degrees C, fewer disseminated Ae. taeniorhynchus (0/5) and Ae. aegypti (2/6) transmitted virus. There were no significant differences in infection, dissemination, or transmission rates for Ae. taeniorhynchus held at the cyclic temperature regimen vs. those held at a constant 17 degrees C. Environmental temperature affected the vectorial capacity of Ae. aegypti and Ae. taeniorhynchus for OCK virus, with transmission occurring earlier and at a higher rate in mosquitoes held at higher temperatures.

Effect of Environmental Temperature on the Vector Competence of Culex Pipiens and Cx. Torrentium for Ockelbo Virus

The effects of environmental temperature on Ockelbo virus infection, dissemination, and transmission were studied in Culex torrentium and in the Uppsala and the El Gabal strains of Cx. pipiens. Temperatures tested included 10 degrees C, 17 degrees C, 24 degrees C, and a cyclic (10-24 degrees C, mean = 17 degrees C) regimen designed to mimic typical hourly temperatures in an area endemic for Ockelbo disease in Sweden during the transmission season. The vector competence of both the Uppsala and the El Gabal strains of Cx. pipiens was directly related to environmental temperature. Mosquitoes held at 10 degrees C had a reduced ability to transmit virus as compared to those held at 17 degrees C, 24 degrees C, or in the cyclic temperature regimen. A rapid increase in dissemination rates was observed in mosquitoes exposed to a shift in temperature from 10 degrees C to 24 degrees C. After 4 days at 24 degrees C, these mosquitoes had vector competence similar to those held at 24 degrees C for the entire incubation time. In contrast, virus dissemination in Cx. torrentium was rapid at all temperatures tested and appeared unaffected by environmental temperature; infection and dissemination rates were consistently higher in Cx. torrentium than in either the Uppsala or the El Gabal strains of Cx. pipiens. It seems the transmission of Ockelbo virus by Cx. pipiens might be interrupted by a prolonged period of cold weather, while transmission by Cx. torrentium would continue.

Swedish Culex torrentium and Cx. pipiens (Diptera: Culicidae) as Experimental Vectors of Ockelbo Virus

The ability of the sibling species Culex pipiens (L.) and Culex torrentium (Martini) from central Sweden to transmit Ockelbo (OCK) virus was determined. Both species became infected after ingesting OCK virus from a viremic chicken; they transmitted this virus to chickens after 21-28 d of extrinsic incubation. In Cx. torrentium, infection rates were 90% or higher, and all 10 refeeding mosquitoes transmitted virus after feeding on chickens with a viremia of at least 10(3.0) plaque-forming units (PFU)/ml. In contrast, only 1 of 28 (4%) of the Cx. pipiens that ingested blood containing 10(3.0-3.9) PFU/ml became infected, and none of 16 refeeding mosquitoes transmitted virus. However, 98 of 184 (53%) of the Cx. pipiens that ingested a blood meal containing at least 10(6.0) PFU/ml became infected. Transmission rates in Cx. pipiens increased with increasing virus concentration in the blood meal to a maximum of 37% in mosquitoes that ingested greater than 10(8.0) PFU/ml. Based on these data, both Cx. pipiens and Cx. torrentium are capable of transmitting OCK virus in an enzootic cycle involving birds as hosts. However, Cx. torrentium appears to be physiologically a more efficient vector than Cx. pipiens.

Transmission of Ockelbo Virus by Aedes cinereus, Ae, communis, and Ae. excrucians (Diptera: Culicidae) Collected in an Enzootic Area in Central Sweden

Studies were conducted to determine the ability of Aedes excrucians (Walker), Ae. cinereus Meigen, and Ae. communis (De Geer) group mosquitoes, collected in an Ockelbo (OCK) virus enzootic area in central Sweden, to transmit this virus. All three species were highly susceptible to infection; at least 96% of the specimens of each species became infected after ingesting blood from a viremic chicken. Recovery of virus from the legs of all 61 of the Ae. excrucians and from 51% (24 of 47) and 75% (6 of 8) of the Ae. cinereus and Ae. communis tested, respectively, indicated that OCK virus readily disseminated from the midgut to the hemocoel in these species. Although none of the Ae. communis refed, Ae. cinereus and Ae. excrucians successfully transmitted OCK virus by bite. Because these Aedes species are attracted to avian and mammalian hosts and because OCK virus has been isolated from field-collected specimens in Sweden and the USSR, Aedes mosquitoes should be considered a possible link between human infections and the enzootic cycle involving birds and Culex and Culiseta mosquitoes.

Ecologic Studies of Mosquitoes and Birds as Hosts of Ockelbo Virus in Sweden and Isolation of Inkoo and Batai Viruses from Mosquitoes

Field studies were conducted in central Sweden from 1983 through 1985 to obtain information on the etiologic agent of Ockelbo disease, described in Sweden in the 1960s and probably identical to Pogosta disease in Finland and to Karelian fever in the western USSR. Mosquitoes (63,644) collected during this 3 year period yielded 21 virus strains. Ockelbo virus isolations were from Culiseta morsitans (5 strains), Culex pipiens and/or Cx. torrentium (6 strains), and Aedes cinereus (3 strains). Inkoo (6 strains) and Batai (1 strain) viruses were recovered from Ae. communis. Blood samples collected March-May from migrating birds on the southeast and est coast of Sweden and in July and August from resident birds in east-central Sweden were tested for neutralizing antibody to Ockelbo virus. Antibody was not detected in 328 birds sampled during spring migrations. Two of 58 (3.4%) birds bled in July and 8 of 78 birds (10%) bled in August had antibody to Ockelbo virus. Ockelbo virus circulates in a mosquito-bird-mosquito cycle, with Cs. morsitans and Cx. pipiens and/or Cx. torrentium as enzootic vectors. Antibody was detected in passerine birds. Other classes of birds or other vertebrates were not sampled. Aedes cinereus may serve primarily to transmit virus to people. The role of other mosquito species as vectors for people is unknown.