Linda D. Jones

Transmission of louping ill virus between infected and uninfected ticks co‐feeding on mountain hares

Abstract. Most of the data on oral infection of ticks by louping ill virus have been obtained from experiments in which animals were infected by syringe inoculation with infectious material. Using infected ticks to mimic the natural situation, we have demonstrated that louping ill (LI) virus transmission can occur from infected to uninfected Ixodes acinus feeding in close proximity on mountain hares (Lepus timidus). Under these conditions the hares developed either low or undetectable viraemias. Highest prevalence of LI virus infection was observed in recipient nymphs which had fed to repletion between days 3 and 7 post‐attachment of virus‐infected adults; following engorgement, 56% of nymphs acquired virus. These results demonstrate the efficient transmission of LI virus between co‐feeding ticks on naive mountain hares. However, when ticks were allowed to co‐feed on virus‐immune hares a significant reduction in the frequency of infection was observed. Neither red deer (Cervus elaphus) nor New Zealand White rabbits supported transmission of LI virus. The significance of virus transmission between cofeeding ticks on LI virus epidemiology is discussed.

Persistence and transmission of tick-borne viruses: Ixodes ricinus and louping-ill virus in red grouse populations

The population dynamics of tick-borne disease agents and in particular the mechanisms which influence their persistence are examined with reference to the flavivirus that causes louping-ill in red grouse and sheep. Pockets of infection cause heavy mortality and the infection probably persists as a consequence of immigration of susceptible hosts. Seroprevalence is positively associated with temporal variations in vectors per host, although variation between areas is associated with the abundance of mountain hares. The presence of alternative tick hosts, particularly large mammals, provides additional hosts for increasing tick abundance. Grouse alone can not support the vectors and the pathogen but both can persist when a non-viraemic mammalian host supports the tick population and a sufficiently high number of nymphs bite grouse. These alternative hosts may also amplify virus through non-viraemic transmission by the process of co-feeding, although the relative significance of this has yet to be determined. Another possible route of infection is through the ingestion of vectors when feeding or preening. Trans-ovarial transmission is a potentially important mechanism for virus persistence but has not been recorded with louping-ill and Ixodes ricinus. The influence of non-viraemic hosts, both in the multiplication of vectors and the amplification of virus through non-viraemic transmission are considered significant for virus persistence.

Tick-borne encephalitis virus in northern Italy: molecular analysis, relationships with density and seasonal dynamics of Ixodes ricinus

Abstract. Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12 390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick‐borne encephalitis (TBE)‐specific antiserum. The identity of the virus isolates was determined by RT‐PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of I. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.

Amplification of tick-borne encephalitis virus infection during co-feeding of ticks.

Abstract. Following engorgement of Rhipicephalus appendiculatus larvae on guinea‐pigs infected with tick‐borne encephalitis (TBE) virus, none of the engorged larvae or emergent nymphs contained detectable infectious virus. However, one of twelve pools, each containing three of the unfed nymphs, was positive when screened by polymerase chain reaction (PCR), indicating a low prevalence of TBE virus infection in the unfed nymphs. After engorgement of the nymphs on four uninfected guinea‐pigs, 19/24 (79%) fed nymphs from one guinea‐pig and 4/25 (16%) fed nymphs from a second guinea‐pig were infected; all the ticks examined from the other two guinea‐pigs were uninfected. The results suggest that TBE virus was transmitted from a low proportion of infected nymphs (infected as larvae) to uninfected nymphs as they fed together on an uninfected guinea‐pig. Such amplification of the initial infection, at the population level, could play an important role in maintaining TBE virus infections in nature, particularly if there is a low level of vertical transmission from one tick generation to the next.

Saliva activated transmission (SAT) of Thogoto virus: relationship with vector potential of different haematophagous arthropods

Abstract. Tick saliva (or salivary gland extract) potentiates the transmission of Thogoto (THO) virus to uninfected ticks feeding on a non‐viraemic guinea‐pig. This phenomenom has been named saliva activated transmission (SAT). To investigate the potential of different haematophagous arthropods to mediate SAT, guinea‐pigs were infested with uninfected R.appendiculatus Neumann nymphs and inoculated with THO virus and salivary gland extract (SGE) derived from a range of ixodid (metastriate and prostriate) or argasid ticks, or mosquitoes; control guinea‐pigs were inoculated with virus alone. Enhancement of THO virus transmission was observed only when SGE was derived from metastriate ticks. Comparison with the vector potential of these various arthropod species revealed that enhancement of THO virus transmission was specific for ticks which were competent vectors of the virus. The data indicate a correlation between vector competence and the ability of haematophagous arthropods to mediate SAT of THO virus.

THE NON-STRUCTURAL PROTEINS OF BLUETONGUE VIRUS ARE A DOMINANT SOURCE OF CYTOTOXIC T CELL PEPTIDE DETERMINANTS

Virus-specific, CD8+ cytotoxic T lymphocytes (CTLs) were generated in two strains of mice (BALB/c, CBA/Ca) against bluetongue virus serotype 10 (BTV-10). Recombinant vaccinia viruses (VV) expressing the individual structural and non-structural proteins of BTV were used to infect syngeneic target cells. We found that in both BALB/c (H-2d) and CBA/Ca (H-2k) mice, polyclonal CTL populations recognized target cells expressing the non-structural proteins better than those expressing the structural proteins. CTLs generated against other BTV serotypes also predominantly recognized the non-structural proteins. However, the extent of cross-reactivity was dependent on the H-2 background of the animals immunized. No CTLs cross-reactive to the BTV-10 heterotype were demonstrated with the panel of molecularly cloned recombinants in the H-2d haplotype. The outer capsid proteins VP2 and VP5 which vary considerably between serotypes were not recognized by heterotypic CTLs. Using this murine model we have determined which BTV proteins are the major targets of the CTL response. The implications for the design and development of subunit vaccines are discussed.

Comparison of the major structural core proteins of tick-borne and Culicoides-borne orbiviruses

Comparison of sequence data for Broadhaven (BRD) virus, a tick-borne orbivirus, and bluetongue virus (BTV), the type species of the genus, indicated that RNA segments 2 and 7 of BRD virus encode the two structural core proteins, VP2 and VP7, respectively. Segment 2 is 2792 nucleotides in length with a coding capacity for a protein (VP2) of 908 amino acids and a net charge of +8.5 at neutral pH. Segment 7 is 1174 nucleotides in length with a coding capacity for a protein (VP7) of 356 amino acids and a net charge of +11.5 at neutral pH. Comparison of the two sequences with BTV serotype 10 revealed amino acid identity of 35% between the product of segment 2 and BTV VP3, and 21% between the product of segment 7 and BTV VP7. The core proteins therefore show evidence of significant evolutionary divergence compared with that shown between different insect-borne orbiviruses. In particular, the amino terminus of BRD virus VP7 differed markedly from the equivalent region in VP7 of BTV and African horse sickness virus. This region is thought to interact with the outer capsid layer of insect-borne orbiviruses.

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.

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.

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.