by Carola Maffioli, Denis Grandgirard, Stephen L. Leib, Olivier Engler

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.

by Yann Bertrand, Mats Töpel, Annelie Elväng, Wessam Melik, Magnus Johansson

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

Phlebotomine sandflies are known to transmit leishmaniases, bacteria and viruses that affect humans and animals in many countries worldwide. These sandfly-borne viruses are mainly the Phlebovirus, the Vesiculovirus and the Orbivirus. Some of these viruses are associated with outbreaks or human cases in the Mediterranean Europe. In this paper, the viruses transmitted by Phlebotomine sandflies in Europe (Toscana virus, Sicilian virus, sandfly fever Naples virus) are reviewed and their medical importance, geographical distribution, epidemiology and potential spreading discussed. Data on vertebrate reservoirs is sparse for sandfly fever viruses. The factor currently known to limit the spread of diseases is mainly the distribution areas of potential vectors. The distribution areas of the disease may not be restricted to the areas where they have been recorded but could be as wide as those of their vectors, that is to say Larroussius and P. papatasi mainly but not exclusively. Consequently, field work in form of viral isolation from sandflies and possible reservoirs as well as laboratory work to establish vectorial competence of colonised sandflies need to be encouraged in a near future, and epidemiological surveillance should be undertaken throughout the European Union.

We report the first worldwide case of Usutu virus (USUV) neuroinvasive infection in a patient with diffuse large B cell lymphoma who presented with fever and neurological symptoms and was diagnosed with meningoencephalitits. The cerebrospinal fluid was positive for USUV, and USUV was also demonstrated in serum and plasma samples by RT-PCR and sequencing. Partial sequences of the premembrane and NS5 regions of the viral genome were similar to the USUV Vienna and Budapest isolates.

We report a case of Usutu virus (USUV)-related illness in a patient that underwent an orthotropic liver transplant (OLT). Post transplant, the patient developed clinical signs of a possible neuroinvasive disease with a significant loss of cerebral functions. USUV was isolated in Vero E6 cells from a plasma sample obtained immediately before the surgery, and USUV RNA was demonstrated by RT-PCR and sequencing. This report enlarges the panel of emerging mosquito-borne flavivirus-related disease in humans.

Arboviruses are arthropod-borne viruses, which include West Nile fever virus (WNFV), a mosquito-borne virus, Rift Valley fever virus (RVFV), a mosquito-borne virus, and Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus. These arthropod-borne viruses can cause disease in different domestic and wild animals and in humans, posing a threat to public health because of their epidemic and zoonotic potential. In recent decades, the geographical distribution of these diseases has expanded. Outbreaks of WNF have already occurred in Europe, especially in the Mediterranean basin. Moreover, CCHF is endemic in many European countries and serious outbreaks have occurred, particularly in the Balkans, Turkey and Southern Federal Districts of Russia. In 2000, RVF was reported for the first time outside the African continent, with cases being confirmed in Saudi Arabia and Yemen. This spread was probably caused by ruminant trade and highlights that there is a threat of expansion of the virus into other parts of Asia and Europe. In the light of global warming and globalisation of trade and travel, public interest in emerging zoonotic diseases has increased. This is especially evident regarding the geographical spread of vector-borne diseases. A multi-disciplinary approach is now imperative, and groups need to collaborate in an integrated manner that includes vector control, vaccination programmes, improved therapy strategies, diagnostic tools and surveillance, public awareness, capacity building and improvement of infrastructure in endemic regions.

West Nile disease in humans has been detected for the first time in Italy in two regions, Emilia-Romagna and Veneto. We conclude that also West Nile fever cases should be specifically targeted by surveillance.

by Milan Labuda, Adama R Trimnell, Martina Ličková, Mária Kazimírová, Gillian M Davies, Olga Lissina, Rosie S Hails, Patricia A Nuttall

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a “dual action” in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: “transmission,” number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; “support,” number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and “survival,” number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.