Of phlebotomines (sandflies) and viruses: a comprehensive perspective on a complex situation.

Old World sandfly-borne phleboviruses are classified into three serological complexes: Sandfly fever Sicilian, Sandfly fever Naples and Salehabad. Human pathogens (febrile illness ['sandfly fever'], neuroinvasive infections) belong to the two first complexes. The increasing number of newly discovered sandfly-borne phleboviruses raises concerns about their medical and veterinary importance. They occupy a wide geographic area from Mediterranean basin to North Africa and the Middle East to the central Asia. At least nine species of sandflies can transmit these viruses. Recent results suggest that sandfly vectors are not as specific for viruses as initially believed. Recent seroprevalence studies demonstrate that humans and domestic animals are heavily exposed. Specific molecular diagnostic methods must be developed and implemented in clinical microbiology laboratories.

High Rates of Neutralizing Antibodies to Toscana and Sandfly Fever Sicilian Viruses in Livestock, Kosovo.

Toscana and sandfly fever Sicilian viruses (TOSV and SFSV, respectively), both transmitted by sand flies, are prominent human pathogens in the Old World. Of 1,086 serum samples collected from cattle and sheep during 2013 in various regions of Kosovo (Balkan Peninsula), 4.7% and 53.4% had neutralizing antibodies against TOSV and SFSV, respectively.

A serological study of severe fever with thrombocytopenia syndrome using a virus neutralization test and competitive enzyme-linked immunosorbent assay.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by the SFTS virus (SFTSV). The SFTSV appears to have a wide host range, as SFTSV-positive ticks have been isolated from both farm animals and wild rodents. Therefore, it is important to monitor SFTSV-positive animals to prevent the transmission of SFTSV from animals to humans. Previously, we developed a competitive enzyme-linked immunosorbent assay (cELISA) to detect SFTSV-specific antibodies from field animals and compared the cELISA results to those from an indirect immunofluorescence assay (IFA). In this study, cELISA results were compared to and evaluated against the results from both an IFA and a virus neutralization (VN) test of 193 bovine serum samples (including two bovine positive control sera) and 70 horse serum samples. The consistency (98.9%) between cELISA and VN results was higher than that (97.4%) between cELISA and IFA for the bovine serum samples. Similarly, for the horse serum samples, the consistency (88.6%) between cELISA and VN results was higher than that (84.3%) between the cELISA and IFA. These findings indicate that our newly developed cELISA can be used for surveillance or epidemiological studies of SFTSV in animals.

Novel and emergent sandfly-borne phleboviruses in Asia Minor: a systematic review.

Sandfly-transmitted phleboviruses are globally spread agents causing febrile diseases and central nervous system infections. The activity of pathogenic phleboviruses, as well as several novel strains, has been reported from Turkey, a transboundary country connecting Asia, Europe, and Africa with suitable habitats for sandflies. This study overviews all published data on phleboviruses from Turkey and evaluates the impact from the virological, epidemiological, and public health perspectives. A systematic review of Web-based global and local resources was performed. Comparison and phylogenetic analyses of particular phlebovirus sequences were also undertaken. Through the evaluation of 1693 international and regional entries, 31 manuscripts providing data on case reports or outbreaks, serological surveillance, animal infections and exposure, virus characterization, vector surveillance, and/or diagnostics were accessed. Detailed information on 5 novel phleboviruses completely or partially characterized during 2008-2015 as well as on clinical and epidemiological features of major phleboviruses established as human pathogens such as Toscana virus and sandfly fever Sicilian virus has been compiled. The ongoing activity of these agents, as indicated by consistently reported symptomatic cases and confirmed exposure in vertebrates including humans, was noted. The circulation in the Anatolian peninsula of phleboviruses with surprising diversity as well as distinct virus species is documented. Specific phlebovirus strains constitute a public health threat for local populations and travelers and must be considered in the diagnostic workup of clinically compatible cases. Human health impact and epidemiological aspects of certain viruses require further investigation via intensive surveillance.

Seroprevalence of Sandfly-Borne Phleboviruses Belonging to Three Serocomplexes (Sandfly fever Naples, Sandfly fever Sicilian and Salehabad) in Dogs from Greece and Cyprus Using Neutralization Test.

Phleboviruses transmitted by sandflies are endemic in the Mediterranean area. The last decade has witnessed the description of an accumulating number of novel viruses. Although, the risk of exposure of vertebrates is globally assessed, detailed geographic knowledge is poor even in Greece and Cyprus where sandfly fever has been recognized for a long time and repeatedly. A total of 1,250 dogs from mainland Greece and Greek archipelago on one hand and 422 dogs from Cyprus on the other hand have been sampled and tested for neutralising antibodies against Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV), Arbia virus, and Adana virus i.e. four viruses belonging to the 3 sandfly-borne serocomplexes known to circulate actively in the Mediterranean area. Our results showed that (i) SFSV is highly prevalent with 71.9% (50.7-84.9% depending on the region) in Greece and 60.2% (40.0-72.6%) in Cyprus; (ii) TOSV ranked second with 4.4% (0-15.4%) in Greece and 8.4% (0-11.4%) in Cyprus; (iii) Salehabad viruses (Arbia and Adana) displayed also substantial prevalence rates in both countries with values ranging from 0-22.6% depending on the region and on the virus strain used in the test. These results demonstrate that circulation of viruses transmitted by sand flies can be estimated qualitatively using dog sera. As reported in other regions of the Mediterranean, these results indicate that it is time to shift these viruses from the "neglected" status to the "priority" status in order to stimulate studies aiming at defining and quantifying their medical and veterinary importance and possible public health impact. Specifically, viruses belonging to the Sandfly fever Sicilian complex should be given careful consideration. This calls for implementation of direct and indirect diagnosis in National reference centers and in hospital microbiology laboratories and systematic testing of unelucidated febrile illness and central and peripheral nervous system febrile manifestations.

Neutralization-based seroprevalence of Toscana virus and sandfly fever Sicilian virus in dogs and cats from Portugal.

Sandfly-borne phleboviruses are endemic in the Mediterranean basin. However, levels of exposure of human and animal populations are inadequately researched. Toscana virus (TOSV) is present in Portugal where it causes human infection and disease; in contrast there are few data for sandfly fever Sicilian virus (SFSV) which has neither been isolated nor detected by molecular tests and for which there are only limited serological data. The sera collected from 1160 dogs and 189 cats in southern Portugal were tested for the presence of neutralizing antibodies against TOSV and SFSV, two viruses recognized as distinct serocomplexes in the Mediterranean region. Our data showed (i) seropositivity to TOSV and SFSV in dogs at a rate of 6.8 and 50.8 %, respectively, and (ii) that 3.7 % of cats were seropositive for TOSV. TOSV findings are in line with previous results obtained with less stringent serological assays. Our results for SFSV in dogs clearly indicate that the virus is circulating widely and that humans may be exposed to infection via the dogs. Although the presence of SFSV was suggested by haemagglutination inhibition in 4/1690 human sera in 1974, this is the first time, as far as we know, that SFSV has been shown to circulate so widely in dogs in Portugal. Future studies should be directed at isolating strains of SFSV in Portugal from dogs, humans and sandflies collected in high prevalence regions. As dogs appear to be good sentinels for SFSV, their role as a possible reservoir in the natural cycle should also be considered.

Novel phlebovirus with zoonotic potential isolated from ticks, Australia.

Recently discovered tick-borne phleboviruses have been associated with severe disease and death among persons in Asia and the United States. We report the discovery of a novel tick phlebovirus in Tasmania State, Australia, that is closely related to those zoonotic viruses found in Asia and North America.

Efficacy of dinotefuran, permethrin and pyriproxyfen combination spot-on on dogs against Phlebotomus perniciosus and Ctenocephalides canis.

This study was conducted to evaluate the efficacy of a new topical ectoparasiticidal spot-on containing 4.95% dinotefuran (w/w), 36.08% permethrin (w/w) and 0.44% pyriproxyfen (w/w) (Vectra 3D, Ceva, Libourne, France) against Portuguese strain of Phlebotomus perniciosus and a French strain of Ctenocephalides canis in dogs. Twelve beagle dogs were exposed for 1 h to 100 P. perniciosus on day 6 for allocation in two groups. One group was treated on day 0, and the other group was the control group. The dogs were exposed for 1 h to 100 P. perniciosus on days 1, 7, 14, 21 and 28. After each sandfly challenge, the same dogs were infested with 100 C. canis. Counts of living fleas were determined 48 h after infestation on days 4, 3, 9, 16, 23 and 30. For sandflies, the anti-feeding effect was 96.9, 99.7, 98.7, 83.5 and 87.0 % on days 1, 7, 14, 21 and 28, respectively. The mortality effect was 97.8, 99.8, 73.7, 27.5 and 39.6% on days 1, 7, 14, 21 and 28, respectively. At each challenge point, the mortality and anti-feeding effects on sandflies were significantly different between the control and treatment groups (p < 0.05). The adulticidal effect on C. canis remained above 99% throughout the study period. The results indicate that a combination with dinotefuran, permethrin and pyriproxyfen may be used as an effective part of an overall flea and sandfly control strategy in dogs for monthly use.

Severe fever with thrombocytopenia syndrome virus, Shandong Province, China.

Severe fever with thrombocytopenia syndrome, which results in severe illness and has a high case-fatality rate, is caused by a novel bunyavirus, severe fever with thrombocytopenia syndrome virus. We found that samples from 2/237 (0.8%) healthy persons and 111/134 (83%) goats in Yiyuan County, Shandong Province, China, were seropositive for this virus.

Potential role of immunomodulators for treatment of phlebovirus infections of animals.

Rift Valley fever (RVFV) is a major phlebovirus-induced epizootic disease of domestic animals (primarily cattle and sheep) in Africa. No therapies for the disease are known. A related phlebovirus, Punta Toro virus (PTV), has been adapted to induce an RVFV-like disease in C57BL/6 mice. This PTV infection has been used as a model for RVFV because it is reasonably safe and does not require high-level biologic containment. The infection model has been used to study the potential role of immunomodulating substances as therapies. A spectrum of immunomodulators has been studied; those immunomodulators most capable of preventing death and other disease manifestations are ampligen, bropirimine, poly (ICLC), AM-3, P-136, and 7-thia-8-oxoguanosine. An immunologic parameter common to all these substances has been their ability to induce interferon. Timing studies have indicated that these active substances may be administered therapeutically as well as prophylactically to inhibit markedly the progress of the disease. Further work is needed in the development of these materials for use in treating viral infections in domestic animals. As a next step, studies need to be run to compare the immunologic profiles induced by each substance in domestic animals and in mice.

Experimental studies of Rio Grande virus in rodent hosts.

Rio Grande (RG) virus, a new member of the Phlebotomus fever serogroup, was inoculated into wild wood rats (Neotoma micropus) and laboratory-reared cotton rats (Sigmodon hispidus) to determine if these potential hosts could be experimentally infected. Nine of 14 (64%) wood rats became viremic, with titers of circulating virus ranging from 10(2.3) to 10(5.3) plaque-forming units (PFU)/ml and a geometric mean titer of 10(3.7) PFU/ml. Virus was not detected in urine specimens from inoculated wood rats but was found in a single saliva specimen. RG virus was detected in the blood of 1 of 12 (8%) cotton rats. Neutralizing (N) antibody developed in 8 of 9 inoculated wood rats which survived for 30 days postinoculation and in 11 of 12 cotton rats. N antibody was still detectable in 4 of 7 wood rats which survived for 1 year, and all 7 were resistant to rechallenge with the virus, as were 3 wood rats with naturally-acquired antibody. High mortality (36%) occurred in inoculated wood rats; whereas low mortality (8%) occurred in cotton rats. The specific cause of death of the rats was not determined. Modes of transmission of the virus in nature are discussed.

Studies on the epidemiology of sandfly fever in Iran. II. The prevalence of human and animal infection with five phlebotomus fever virus serotypes in Isfahan province.

Human and animal sera from an endemic area of sandfly fever in Iran were tested by plaque reduction neutralization method against five different Phlebotomus fever virus serotypes (Naples, Sicilian, Karimabad, Salehabad, and I-47). The overall prevalence of Naples, Sicilian, and Karimabad virus antibodies among the human population was 17%, 25%, and 66%, respectively. All sera were negative against Salehabad and I-47 viruses. Age-specific antibody rates suggested that Sicilian and Karimabad viruses were endemic in the study area but that Naples virus activity was sporadic. These observations were confirmed by isolations of Sicilian and Karimabad viruses from sandflies collected in the study area. Among the animal sera tested, evidence of Phlebotomus fever virus infection was detected only in gerbils. Of 38 Rhombomys opimus tested, 34% had neutralizing antibodies against Sicilian virus and 32% against Karimabad. These results indicate that gerbils are infected with these two viruses and possibly might serve as reservoirs or amplifying hosts. The serologic studies also suggest that the ecology of Sicilian and Karimabad viruses involves chiefly sandflies, gerbils, and man, an epidemiologic pattern previously demonstrated for cutaneous leishmaniasis in the same region of Iran.