Thogoto Research Articles

Rhipicephalus appendiculatus (Acari: Ixodidae): dynamics of Thogoto virus infection in female ticks during feeding on guinea pigs

Engorged nymphs ( Rhipicephalus appendiculatus) were inoculated parenterally with Thogoto (THO) virus (∼1 μl per nymph; 10 6–10 7 PFU/ml). The adult females which resulted were used as the source of infected ticks for this study. Hemolymph, salivary glands, synganglion, gut, ovary, and Malpighian tubules were collected on each day of the blood meal and titrated for THO virus by plaque assay. The percent of tissues infected with virus was 16% or less on the day of attachment. Percent infection rose for all tissues throughout 6–7 days of feeding, reaching 40–100% infection during the rapid phase of engorgement. For the first 4 days of feeding, virus titer in the synganglion was higher than in salivary glands (means of 6.4–34.7 PFU/synganglion and 1.6–8.8 PFU/salivary gland pair). From days 5–7, virus titer was generally higher in the salivary gland than the synganglion (means of 422, 408, and 817 PFU/gland pair and means of 62, 811, and 9 PFU/synganglion). However, because a salivary gland pair is much heavier than a synganglion, the virus concentration in the synganglion was much higher than in the salivary gland during the slow phase of feeding. During the rapid phase of feeding, the difference in virus titer between the synganglion and salivary gland reduced. This difference between the early and late stages of feeding may explain why a previous study [J. Gen. Virol. 70 (1989) 1093], using immunofluorescence and immuno-gold labelling, failed to detect virus in the salivary gland early in feeding. These data provide evidence to explain that R. appendiculatus can transmit THO virus within 24 h of attachment, an important epidemiological finding. Index Descriptors and Abbreviations: Arboviruses, arthropod-borne viruses; PFU, plaque-forming units; Rhipicephalus appendiculatus (Acari: Ixodidae); THO virus, Thogoto virus; ticks

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection.

Adult Rhipicephatus appendiculatus ticks, infected with Thogoto (THO) virus or control, were fed on guinea pigs and removed at intervals throughout the feeding cycle. Salivary fluid secretion was measured by an in vitro technique. The salivary glands of infected, partially-fed ticks secreted fluid in vitro at about 75% the rate of controls, but the difference between infected and controls among engorged ticks was not statistically significant. Basal and DA-stimulated levels of cyclic AMP (cAMP) were determined in isolated glands and were significantly affected by THO virus infection. The differences in secretory rate among control and infected ticks could not be explained in terms of altered cAMP levels. Haemolymph volume was measured by a tracer-dilution technique using 3H-inulin. The mean haemolymph volume for both THO-infected and control groups was between 23-24% body weight throughout the feeding cycle, indicating that infection by this arbovirus did not influence salivary fluid secretion via altered haemolymph volume. The mechanism by which THO virus affects secretory activity of its tick vector remains unknown.

The 5' ends of Thogoto virus (Orthomyxoviridae) mRNAs are homogeneous in both length and sequence.

Thogoto (THO) virus is a tick-borne member of the Orthomyxoviridae whose genome consists of six segments of linear, negative sense, single-stranded RNA. To gain insight into the mechanism by which viral mRNA transcripts are initiated, poly(A)+ RNA isolated from THO virus-infected cells was characterized by (i) primer extension experiments, (ii) immunoprecipitation studies with an anticap monoclonal antibody, (iii) direct sequencing analysis of the isolated RNA, and (iv) cloning and sequencing of individual mRNA molecules. The results indicated that THO virus mRNAs are capped and homogeneous in both length and sequence at their 5' end. These findings contrast with the situation found in all other segmented, negative sense or ambisense, single-stranded RNA viruses so far analyzed in which the 5' ends of viral mRNAs are heterogeneous in length and sequence. These results are discussed in terms of the mechanism used by THO virus to initiate mRNA synthesis.

Amblyomma variegatum(Acari: Ixodidae): Mechanism and Control of Arbovirus Secretion in Tick Saliva

Saliva is considered to be the conduit by which pathogens are transmitted from blood-sucking arthropod vectors to their vertebrate hosts, but supporting evidence for this is fragmentary. To determine if Thogoto (THO) virus, a tick-borne member of the influenza virus family, is transmitted via tick saliva, and whether virus replication is a prerequisite for such transmission, two experimental conditions were compared: (1) “biological transmission” and (2) “mechanical transmission.” In (1), THO virus was allowed to infect and replicate in a natural vector,Amblyomma variegatum: virus was detected in saliva collected from 3/22 (14%) ticks. In (2), virus was inoculated directly into the hemocoel with the drug used to induce salivation and saliva was collected immediately to preclude the possibility of virus replication: virus was detected in saliva collected from 31/170 (18%) ticks. The results demonstrate that THO virus is secreted in tick saliva and that virus can pass from the hemolymph to the salivary glands independently of viral replication within the tick. The comparatively low numbers of ticks that yielded virus-positive saliva samples together with the results from assays of serial saliva samples suggested that virus secretion may not be a continuous process during salivation. Ticks in which THO virus had established an infection showed an impaired secretory response compared with uninfected ticks and ticks used for mechanical transmission.

Immune Protection Conferred by the Baculovirus-Related Glycoprotein of Thogoto Virus (Orthomyxoviridae)

The coding region of segment 4 of Thogoto (THO) virus, a tick-borne member of the Orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. This construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (MAbs). Immunization of hamsters with Spodoptera frugiperda (Sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against virus challenge; control hamsters developed clinical disease with high-titer viremia 3 days postchallenge. In contrast to hamsters, guinea pigs are comparatively resistant to THO virus infection but support nonviremic transmission between cofeeding infected and uninfected ticks. However, when uninfected ticks fed on guinea pigs immunized with the baculovirus recombinant, only 2% became infected following virus challenge of the animals compared to 26% of ticks on control nonimmune guinea pigs. Furthermore, neutralizing MAbs specific for THO viral glycoprotein protected mice against lethal challenge with THO virus; nonneutralizing MAbs specific for the glycoprotein, which inhibit THO viral agglutinating activity, did not induce a protective response. Thus at least in the murine model, protective immunity is conferred by antibodies directed against the neutralizing epitope(s) of the baculovirus-related glycoprotein of THO virus.

Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein.

Thogoto (THO) virus is a tick-borne virus which shares morphological and genetic features with members of the Orthomyxoviridae family although the viral glycoprotein appears to be related to gp64 of baculoviruses. Characterization of THO virus was undertaken to clarify its taxonomic position. Purified virus preparations contained at least six virus-encoded polypeptides with apparent M(r) values ranging from 29K to 92K. A 75K polypeptide was identified as an envelope-associated glycoprotein by Triton X-100 and salt dissociation studies, and by proteolytic degradation of the exposed proteins of the virion. By the same criteria, the nucleoprotein and the matrix protein were identified as the 52K and 29K polypeptides, respectively. Immunofluorescence studies using monoclonal antibodies (MAbs) located the glycoprotein on the external cell membrane and the nucleoprotein in the nucleus of infected cells indicating that virus replication involved a nuclear phase. In addition, the virus displayed haemagglutination and haemolytic activities with an optimum at pH 6. These activities are functions of the viral glycoprotein since they were inhibited by anti-glycoprotein MAbs. The data reported here support the notion that THO virus is a member of the Orthomyxoviridae family but that it should be classified in a group distinct from the other influenza viruses.

Modification of the skin feeding site by tick saliva mediates virus transmission.

A tick vector of Thogoto (THO) virus was shown to secrete a factor in saliva which potentiates the transmission of THO virus to uninfected ticks feeding on an apparently non-viraemic host. The effect of the saliva activated transmission (SAT) factor on the virus occurred at the site of inoculation in the skin and was apparent even when the virus was introduced 3 days after the SAT factor. The results suggest that tick saliva can play an important role in disease transmission by virtue of host modification at the site of feeding.

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands of Rhipicephalus appendiculatus, Amblyomma variegatum, and Boophilus microplus ticks.

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor(s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guinea-pigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species, Rhipicephalus appendiculatus and Amblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected female Boophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.

The glycoprotein of Thogoto virus (a tick-borne orthomyxo-like virus) is related to the baculovirus glycoprotein GP64

Thogoto (THO) virus is a tick-transmitted virus which shares morphological and biochemical characteristics with members of the Orthomyxoviridae. The genome of Thogoto virus comprises six segments of single-stranded, negative sense RNA. The complete nucleotide sequence of the fourth largest RNA segment of THO virus has been determined from cDNA analyses. This RNA segment is 1574 nt long and has a coding capacity for a glycoprotein of 512 amino acids with a predicted molecular weight of 57,550 Da. The sequence of this protein has extensive homology with the putative envelope protein of Dhori (DHO) virus, the only other recorded tick-borne orthomyxo-like virus, but not with the envelope glycoproteins of the influenza viruses. A search of the translation of the available nucleotide database has produced evidence for a relationship between the glycoproteins of THO and DHO viruses and the gp64 glycoprotein of two DNA-containing insect baculoviruses, Autographa californica nuclear polyhedrosis virus and Orgyia pseudotsugata nuclear polyhedrosis virus. The baculovirus gp64 protein is a membrane protein implicated in endocytotic fusion events during infection. A multiple alignment between these four glycoproteins gave significance scores of >28 standard deviations, indicating that the homologies between them are highly significant. The distribution of cysteine residues is conserved between all four proteins which also have similar hydropathy profiles, suggestive of a type I membrane protein topology.

The Effect of Host Resistance to Tick Infestation on the Transmission of Thogoto Virus by Ticks

Tick-borne virus transmission was examined using guinea-pigs and hamsters previously infested with ticks. Guinea-pigs developed immunity to Rhipicephalus appendiculatus after a single exposure to the ticks. Nymphal and adult stages that fed on resistant guinea-pigs had increased mortality during feeding, and reduced engorged weights. Egg production from female ticks fed on resistant hosts fell by at least 50%. Guinea-pigs maintained high levels of immunity to tick infestation for at least 210 days after the initial exposure. In contrast, hamsters did not develop resistance to ticks even after three or four infestations. R. appendiculatus adults infected with Thogoto (THO) virus (donors) were allowed to co-feed with uninfected nymphs (recipients) on either resistant or naive guinea-pigs. The number of recipient ticks that acquired virus was significantly reduced on resistant guinea-pigs. In contrast, feeding on pre-infested hamsters did not affect tick-borne transmission of THO virus. Host resistance to tick infestation, if prevalent in nature, may severely limit the spread of tick-borne viruses. Such an effect could result directly from a reduction in the number of ticks that acquire virus, or indirectly from poor egg production (in the case of viruses maintained in ticks by vertical transmission) and reduced survival of ticks fed on resistant hosts.

Sequence analyses of Thogoto viral RNA segment 3: evidence for a distant relationship between an arbovirus and members of the Orthomyxoviridae.

The genome of Thogoto (THO) virus, an unclassified tick-borne virus, comprises six segments of single-stranded RNA. The complete sequence of the third largest RNA segment has been determined from overlapping cDNA clones and by primer extension studies. Segment 3 RNA consists of 1865 nucleotides (approx. 6.2 x 10(5) Mr). It has a large open reading frame (ORF1;597 amino acids, 68.6K) in its virus-complementary sequence, confirming that the RNA has a negative-sense coding strategy. A transcription termination (polyadenylation) site located after the end of ORF1 has been identified. A second ORF (ORF2;98 amino acids in length), overlapping ORF1, is also present in the virus-complementary sequence although whether it is translated is not known. The 3' and 5' sequences of the segment 3 RNA are complementary and similar to those of the tick-borne Dhori (DHO) and the mammalian and avian influenza viruses. Protein database searches have identified regions of homology between the sequence of the THO ORF1 gene product and regions of the PA protein of influenza virus strain A/NT/60/68 (approx. 20% aligned homology) and the corresponding protein of influenza B/Sing/222/79 virus (approx. 15% aligned homology). Although the THO protein sequence is not as closely related to those of the influenza viruses as they are to each other (40% aligned homology), the indicated sequence data provide further evidence of relationships between the tick-borne THO and DHO viruses and the vertebrate orthomyxoviruses.

The effect of virus-immune hosts on Thogoto virus infection of the tick, Rhipicephalus appendiculatus

Thogoto (THO) virus infections of Rhipicephalus appendiculatus ticks were examined using tick hosts immune to the virus. In the first set of experiments, ticks were infected by feeding on viraemic hamsters. Inter-stadial infection of THO virus was not affected when ticks ingested a virus-immune bloodmeal but there was an effect on persistence of the virus. The incidence of intra-stadial infection was reduced by at least 40% when nymphs partially fed on viraemic hamsters and completed their bloodmeal on a virus-immune guinea pig. When the reverse situation was examined — feeding on a virus-immune host and then a viraemic host- no difference was observed in the number of ticks infected. In the second set of experiments, uninfected ticks acquired virus by co-feeding with infected ticks on apparently non-viraemic guinea pigs. Non-viraemic transmission of the viruses was inhibited when the guinea pigs were immune to either the Sicilian (SiAr 126) or prototype (IIA) isolates of THO virus. The laboratory data indicate that virus-immune hosts may have a significant effect on the role played by ticks in the epidemiology of tick-borne viruses.

Viral interference in the tick, Rhipicephalus appendiculatus. II. Absence of interference with Thogoto virus when the tick gut is by-passed by parenteral inoculation.

Genetic reassortment of Thogoto (THO) virus has been demonstrated in dually infected Rhipicephalus appendiculatus ticks. However previous results showed that oral superinfection is inhibited by interference. To ascertain the site of THO viral interference, ticks were infected parenterally or orally with a temperature-sensitive (ts) mutant of THO virus. Infected ticks were then challenged with wild-type (wt) THO virus via parenteral inoculation. Intra-stadial superinfection was carried out by parenteral inoculation of newly infected engorged ticks whereas inter-stadial superinfection involved inoculation of engorged ticks infected at the previous stage. In both instances viral interference was not observed, i.e. the challenge virus replicated and was delivered by bite to susceptible hosts. Therefore when the gut is bypassed, R. appendiculatus ticks are apparently permissive to dual infection even when there is a delay in the presentation of the superinfecting virus. These results demonstrate that interference following superinfection per os does not occur in the salivary glands, but may occur in the gut and possibly in a secondary site of viral replication such as the synganglion.

Viral interference in the tick, Rhipicephalus appendiculatus. I. Interference to oral superinfection by Thogoto virus.

Interference between arboviruses in a naturally infected tick vector is reported for the first time. Rhipicephalus appendiculatus nymphs were dually infected with Thogoto (THO) virus, a tick-borne virus, similar to members of the family Orthomyxoviridae. In the first series of experiments examining 'inter-stadial' interference, larvae were orally infected with a temperature-sensitive (ts) mutant, and after moulting the nymphs were superinfected with the wild-type (wt) virus. In the second series of experiments examining 'intra-stadial' interference, nymphs were dually infected by interrupted feeding; the time interval between infective feeds was either shorter than 24 h or lasted for 10 days. Interference was demonstrated by the inability of wt virus to replicate in ticks previously infected with ts virus. Both inter- and intra-stadial interference were observed and complete interference was detected in 78% of dually infected nymphs. A pool of dually infected ticks, in which intra-stadial interference had been detected, failed to transmit the superinfecting virus after moulting.

Enhancement of Virus Transmission by Tick Salivary Glands

Previous studies have demonstrated that Thogoto (THO) virus is transmitted from infected to uninfected ticks cofeeding on an uninfected guinea-pig, although the guinea-pig does not develop a detectable viraemia. To investigate this mode of transmission, guinea-pigs were infected with uninfected Rhipicephalus appendiculatus nymphs prior to inoculation with either a mixture of THO virus and tick salivary gland extract, or with THO virus alone. The number of ticks that acquired the virus from feeding on animals inoculated with a mixture of virus and salivary gland extract was 10-fold greater than the number that became infected by feeding on animals inoculated with virus alone. The increase in the number of ticks that became infected was greatest when the salivary glands used in the inoculum were derived from uninfected ticks, which had partially fed for a period of 6 days. Viraemia was not detected in any of the guinea-pigs tested during the experiments. These results indicate that THO virus transmission is enhanced by factor(s) associated with the salivary glands of ticks, and that these factor(s) may facilitate 'non-viraemic' transmission between infected and uninfected ticks.

Anatomical Basis of Thogoto Virus Infection in BHK Cell Culture and in the Ixodid Tick Vector, Rhipicephalus appendiculatus

Infection by Thogoto (THO) virus, a tick-borne virus related to the orthomyxoviruses, has been compared in vertebrate cell culture and in Rhipicephalus appendiculatus ticks using infectivity titrations, immunofluorescence, and immune electron microscopy with colloidal gold markers to detect cell surface and intracellular antigens. Morphogenesis of THO virus in cell culture was similar to that of influenza virus, with polymorphic virus particles budding at the plasma membrane. In the tick, THO viral infection caused no obvious pathology; virions or budding profiles were not observed in electron micrographs, although replication, trans-stadial persistence and transmission to a susceptible host occur. THO virus was not detected in the salivary glands of trans-stadially infected ticks until about 7 days after the commencement of feeding on a host. The synganglion (brain) appears to be the major organ involved in trans-stadial persistence of the virus; viral antigens were detected in the neural cortex (cell bodies) but not in nerve fibres and axons. The detection of THO viral antigen in basement membranes and connective tissue, but its absence from nerve fibres, suggests that dissemination occurs via the haemolymph rather than a neural route.

Vector capacity of Rhipicephalus appendiculatus and Amblyomma variegatum for Thogoto and Dhori viruses

Experimental studies were undertaken to ascertain the vector potential of Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius for Thogoto (THO) and Dhori (DHO) viruses, candidate members of the Orthomyxoviridae. In the first set of experiments, ticks were infected orally by feeding on viraemic hamsters. THO virus replicated in R. appendiculatus and A. variegatum, persisted trans-stadially in both tick species, and was transmitted to susceptible hosts during feeding. In contrast, both R. appendiculatus and A. variegatum were refractory to per os infection by DHO virus. In the second set of experiments, engorged R. appendiculatus and A. variegatum nymphs were parenterally inoculated with DHO virus. The virus persisted trans-stadially in both tick species and was transmitted by bite to susceptible hosts. These results indicate that the midgut acts as a barrier to per os infection of R. appendiculatus and A. variegatum by DHO virus. However, when this barrier is bypassed, i.e. by parenteral inoculation, both R. appendiculatus and A. variegatum can serve as efficient vectors of DHO virus.

Experimental studies on the transmission cycle of Thogoto virus, a candidate orthomyxovirus, in Rhipicephalus appendiculatus.

Thogoto (THO) virus, a candidate orthomyxovirus, replicated in and was transmitted by larvae, nymphs, and adults of the brown ear tick, Rhipicephalus appendiculatus. Larvae fed on viremic hamsters (10(7-8) PFU/ml blood) acquired an average of 10(2.5) PFU per tick. Following engorgement the titer dropped to 10(1.9) PFU on day 2 but increased by day 6 to 10(3.3) PFU. Virus survived transstadially in these ticks as demonstrated by the fact that, on day 10, newly moulted nymphs contained, on average, 10(3.5) PFU/tick. When 10 such infected nymphs were placed on a hamster a fatal infection of the animal developed involving a viremia of 10(6.7) PFU/ml blood. Another group of 6 infected nymphs did not elicit a detectable viremia in a hamster, or cause death. However the animal seroconverted to THO, virus indicating that virus transmission had occurred. Following acquisition of THO virus at the larval stage, virus was detected in adult ticks 138 days later. Uninfected nymphs fed on viremic hamsters acquired an average of 10(4) PFU/nymph. No virus was detected in the nymphs 4 days post-engorgement. Virus was, however, recovered by 6 days post-engorgement (10(4.7) PFU/nymph). Virus persisted transstadially as shown by the presence of an average of 10(3.4) PFU in newly moulted adults. Three groups of these infected adults (5-6 ticks/group) induced viremia in hamsters with blood titers of the order 10(2.8-3.5) PFU/ml. Virus persisted in engorged adults for up to 66 days following nymphal engorgement.(ABSTRACT TRUNCATED AT 250 WORDS)

Arbovirus isolations from ixodid ticks infesting livestock, Kano Plain, Kenya

In a study conducted on the Kano Plain, Kenya, virus isolation attempts were made on ixodid ticks collected, over a 14-month period, from livestock held in family enclosures (bornas) before releasing the animals for daily foraging. 8735 Amblyomma variegatum (Fabricius) were tested, 98·6% of which were taken from cattle, yielding 36 strains of Dugbe (DUG), four strains of Nairobi sheep disease (NSD), three strains of Bhanja (BHA), one strain of Thogoto (THO) and five strains of virus which could not be characterized. 6549 Rhipicephalus spp. ticks were collected (60·3% from cattle). NSD, DUG and BHA viruses were each isolated twice from ticks taken from cattle. One BHA virus strain was recovered from ticks from a sheep. One strain recovered from ticks on cattle could not be characterized.