Figure 2

Figure 2. Nucleotide alignments of novel AHFV sequences obtained from Hyalomma marginatum sensu lato ticks (likely H. rufipes) with reference AHFV and KFDV sequences. A) Partial alignment of 5′ untranslated region of AHFV...

We collected ticks from birds migrating northward initially at 2 bird observatories on the Mediterranean islands Capri (Italy; 40°33′N, 14°15′E) and Andikíthira (Greece; 35°51′N, 23°18′E) during spring of 2009 and 2010. We captured 14,824 birds (78 species) during their yearly migration, presumably leaving Africa for breeding grounds in Europe or Asia. We collected 747 ticks, 88% of which were identified morphologically as members of the complex H. marginatum sensu lato (s.l.), most likely H. rufipes and H. marginatum. We screened cDNA for AHFV by real-time PCR with primers targeting the 5′ untranslated region of AHFV (12). Five fully engorged ticks, morphologically and molecularly determined to be H. marginatum s.l., likely H. rufipes (GenBank accession nos. MH061004–MH061008, MH061010–MH061014), tested positive for AHFV RNA in 2 separate analyses. These 5 ticks (4 nymphs, 1 larva) were collected at Andikíthira in 2010 (Table) from 3 bird species that winter in sub-Saharan Africa and breed in Europe (Figure 1, panels A–C). One sedge warbler (Acrocephalus schoenobaenus) carried 2 AHFV-positive nymphs. Sequencing of the 5′ untranslated region amplicons revealed 2 identical 72-bp sequences. BLAST (http://www.ncbi.nlm.nih.gov/blast/) and comparative analyses revealed high identity to AHFV and KFDV reference sequences, despite a region with 7 consecutive mismatching nucleotides and a 14-nt deletion (Figure 2, panel A). Apart from this difference, 100% identity was seen with all other AHFV and KFDV sequences available in GenBank.

Because of these results, we included additional bird observatories and collection sites along with Capri and Andikíthira in spring of 2014 and 2015: the Anapodaris River, Crete, Greece (34°59′N, 25°17′E); Huelva Province, Spain (37°30′N, 5°30′W); Sevilla Province, Spain (37°33′N, 6°55′W); Canary Islands, Spain (28°9′N, 15°25′W); Jerusalem, Israel (31°47′N, 35°13′E); and the Kizilirmak Delta, Turkey (41°38′N, 36°05′E). We ringed 22,069 birds (137 species) and collected 1,024 ticks. RNA was extracted at Uppsala University (Uppsala, Sweden) and sent to Agence Nationale de Sécurité Sanitaire de l’Alimentation (Paris, France) for high-throughput screening by microfluidic real-time PCR (Biomark Dynamic Arrays, Fluidigm, South San Francisco, CA, USA) targeting multiple tickborne viruses (S. Moutailler, unpub. data). One fully engorged adult tick, molecularly determined to be H. marginatum s.l. (GenBank accession nos. MH061009, MH061015), likely H. rufipes, collected from a common redstart (Phoenicurus phoenicurus) in Turkey tested positive for AHFV RNA (Table) when using a set of primers and probe that amplifies part of the KFDV/AHFV premembrane region (Kyasanur_poly_F, 5′-ACACGATGCACACACCTGC-3′; Kyasanur_poly_R, 5′- CACCAATGAAACTCTAGTCGTC-3′; Kyasanur_poly_P, 5′-AGAACCGGGACTTTGTCTCAGGGAC-3′). To confirm this finding, a subsequent real-time PCR was performed with the same primers and probe. Alignment of the 31-bp fragment revealed a single nucleotide change compared with available AHFV sequences and 3 or 4 differences compared with available KFDV sequences (Figure 2, panel B). The methods used did not enable virus isolation and propagation; therefore, acquisition of further sequence information proved difficult. The common redstart also breeds in Europe and winters in sub-Saharan Africa, similarly to the 3 previously mentioned bird species (Figure 1, panel D).

We detected AHFV/KFDV RNA in 6 of 1,771 investigated ticks. Although we could not differentiate between KFDV and AHFV on the basis of the short sequences analyzed, we suggest these sequences represent AHFV because this supposition is geographically and ecologically more plausible. Our findings are insufficient to establish the role of the H. rufipes tick as an AHFV vector. However, detection of AHFV RNA in Europe and Asia Minor raises concerns for conceivable dissemination of the virus to these areas, facilitated by climate change resulting in increased distribution of Hyalomma spp. ticks.

H. rufipes ticks are widely distributed in Africa and are also present on the Arabian Peninsula, along the Red Sea coast (13). The H. rufipes species is a 2-host tick; that is, the nymph remains attached to ingest blood on the same host it did for its first blood meal as a larva. Immature ticks usually infest birds or small mammals (e.g., hares), whereas adult ticks feed on larger mammals (e.g., camels, buffaloes, cattle, and large birds) (13). Consequently, the AHFV-positive ticks (except the 1 adult) most likely had fed only on the bird to which they were attached. This assumption implies that an immature tick could have acquired the virus vertically from its mother, from its putatively viremic avian host, or by co-feeding transmission, which can occur when ticks feed close to >1 infective tick (14,15). However, in this case, co-feeding transmission is less plausible because the 2 ticks attached to the same bird were not located close to each other (on the beak and nape). For several tickborne viruses, including KFDV, vertical transmission from the adult female tick to the eggs occurs. However, the prevalence of transovarial infection in nature is considered low (14). The 1 positive adult tick could have acquired virus in any of these ways or from a previous mammalian blood meal.

To our knowledge, birds have not been shown to play a role in the ecology or epidemiology of AHFV, and this tick species has not been identified previously as a possible vector. Our finding of this virus in ticks infesting birds en route from Africa to Europe and Asia, together with clinical cases in southern Egypt, the detection of AHFV RNA in ticks, and cases of seropositivity in Djibouti, could indicate a wider geographic distribution of the virus throughout eastern Africa and in novel regions. Further investigations of AHFV ecology and modes of transmission, including the potential role of Hyalomma ticks as vectors and passerine birds as reservoirs or distributors of potentially infected ticks, as well as increased surveillance, are needed.