Screening for tick-borne and tick-associated viruses in ticks collected in Ghana.

Ticks are blood-sucking arthropods that transmit many pathogens, including arboviruses. Arboviruses transmitted by ticks are generally referred to as tick-borne viruses (TBVs). TBVs are known to cause diseases in humans, pets, and livestock. There is, however, very limited information on the occurrence and distribution of TBVs in sub-Saharan Africa. This study was designed to determine the presence and distribution of ticks infesting dogs and cattle in Ghana, as well as to identify the tick-borne or tick-associated viruses they harbour. A more diverse population of ticks was found to infest cattle (three genera) relative to those infesting dogs (one genus). Six phleboviruses and an orthonairovirus were detected in tick pools screened by RT-PCR. Subsequent sequence analysis revealed two distinct phleboviruses and the previously reported Odaw virus in ticks collected from dogs and a virus (16GH-T27) most closely related to four unclassified phleboviruses in ticks collected from cattle. The virus 16GH-T27 was considered a strain of Balambala tick virus (BTV) and named BTV strain 16GH-T27. Next-generation sequencing analysis of the BTV-positive tick pool detected only the L and S segments. Phylogenetic analysis revealed that BTV clustered with viruses previously defined as M-segment-deficient phleboviruses. The orthonairovirus detected in ticks collected from cattle was confirmed to be the medically important Dugbe virus. Furthermore, we discuss the importance of understanding the presence and distribution of ticks and TBVs in disease prevention and mitigation and the implications for public health. Our findings contribute to the knowledge pool on TBVs and tick-associated viruses.

RNA virome analysis of questing ticks from Hokuriku District, Japan, and the evolutionary dynamics of tick-borne phleboviruses.

Tick-borne viruses have emerged recently in many parts of the world, and the discoveries of novel tick-borne viruses have been accelerated by the development of high-throughput sequencing technology. In this study, a cost-efficient small benchtop next-generation sequencer, the Illumina MiniSeq, was used for the RNA virome analysis of questing ticks collected from Hokuriku District, Japan, and assessed for their potential utility in a tick-borne virus surveillance system. We detected two phleboviruses [Kabuto Mountain virus (KAMV) and Okutama tick virus (OKTV)], a coltivirus [Tarumizu tick virus (TarTV)], and a novel iflavirus [Hamaphysalis flava iflavirus (HfIFV)] from tick homogenates and/or cell culture supernatants after virus isolation processes. The number of sequence reads from KAMV and TarTV markedly increased when cell culture supernatants were used, indicating a successful isolation of these viruses. In contrast, OKTV and HfIFV were detected only in tick homogenates but not from cell culture supernatants, suggesting a failure to isolate these viruses. Furthermore, we performed genomic and phylogenetic analyzes of these detected viruses. OKTV and some phleboviruses discovered recently by NGS-based methods were probably deficient in the M genome segment, which are herein proposed as M segment-deficient phlebovirus (MdPV). A phylogenetic analysis of phleboviruses, including MdPV, suggested that Uukuniemi and Kaisodi group viruses and kabutoviruses evolved from an ancestral MdPV, which provides insights into the evolutionary dynamics of phleboviruses as emerging pathogens.

Dengue Virus Infection in Aedes albopictus during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan.

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed.

Characterization of a novel thogotovirus isolated from Amblyomma testudinarium ticks in Ehime, Japan: A significant phylogenetic relationship to Bourbon virus.

The genus Thogotovirus, as represented by Thogoto virus and Dhori virus, comprises a group of arthropod-borne viruses, most members of which are transmitted by ticks. Here we report the genetic and biological characterization of a new thogotovirus, designated Oz virus (OZV), isolated from the hard tick Amblyomma testudinarium in Ehime, Japan. OZV efficiently replicated and induced a cytopathic effect in Vero cells, from which enveloped pleomorphic virus particles were formed by budding. OZV could also replicate in BHK-21 and DH82 cells and caused high mortality in suckling mice after intracerebral inoculation. Phylogenetic analyses of six viral proteins indicated that OZV is clustered with Dhori and related viruses, and is most closely related in glycoprotein (GP) and matrix protein (M) sequences to Bourbon virus, a human-pathogenic thogotovirus discovered recently in the United States. Our findings emphasize the need for understanding the geographic distribution and ecology of OZV and related viruses and for reevaluation of the medical and public health importance of thogotoviruses.

Isolation and characterization of Kabuto Mountain virus, a new tick-borne phlebovirus from Haemaphysalis flava ticks in Japan.

In Japan, indigenous tick-borne phleboviruses (TBPVs) and their associated diseases first became evident in 2013 by reported human cases of severe fever with thrombocytopenia syndrome (SFTS). In this study, we report a novel member of the genus Phlebovirus designated as Kabuto Mountain virus (KAMV), which was isolated from the ixodid tick Haemaphysalis flava in Hyogo, Japan. A complete viral genome sequencing and phylogenetic analyses showed that KAMV is a novel member of TBPVs, which is closely related to the Uukuniemi and Kaisodi group viruses. However, unlike the Uukuniemi group viruses, the 165-nt intergenic region (IGR) in the KAMV S segment was highly C-rich in the genomic sense and not predicted to form a secondary structure, which are rather similar to those of the Kaisodi group viruses and most mosquito/sandfly-borne phleboviruses. Furthermore, the NSs protein of KAMV was highly divergent from those of other TBPVs. These results provided further insights into the genetic diversity and evolutionary relationships of TBPVs. KAMV could infect and replicate in some rodent and primate cell lines. We evaluated the infectivity and pathogenicity of KAMV in suckling mice, where we obtained a virulent strain after two passages via intracerebral inoculation. This is the first report showing the existence of a previously unrecognized TBPV in Japan, other than the SFTS virus.

Isolation and characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks in Japan.

During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava. Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae. This was supported by the facts that the 5'- and 3'-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus.

Complete Genome Sequencing and Phylogenetic Analysis of a Getah Virus Strain (Genus Alphavirus, Family Togaviridae) Isolated from Culex tritaeniorhynchus Mosquitoes in Nagasaki, Japan in 2012.

Getah virus (GETV; genus Alphavirus, family Togaviridae) is a mosquito-borne virus known to cause disease in horses and pigs. In 2014, for the first time in ∼30 years, a sudden GETV outbreak occurred among racehorses in Ibaraki, Japan. Two years before this outbreak, we obtained multiple GETV isolates from Culex tritaeniorhynchus mosquitoes collected in Nagasaki, Japan and determined the whole genome sequence of GETV isolate 12IH26. Our phylogenetic analysis of GETV strains revealed that the isolate 12IH26 forms a robust clade with the epidemic strains 14-I-605-C1 and 14-I-605-C2 isolated from horses in the 2014 outbreak in Ibaraki. Furthermore, the complete genomic sequence of the isolate 12IH26 was 99.9% identical to those of the 2014 epidemic strains in Ibaraki. Phylogenetic analysis also showed that the recent Japanese GETV strains, including the isolate 12IH26, are closely related to the Chinese and South Korean strains rather than the previous Japanese strains, suggesting that GETV strains may be transported from overseas into Japan through long-distance migration of the infected mosquitoes or migratory birds.

Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan.

Among the tick-borne orbiviruses (genus Orbivirus, family Reoviridae), 36 serotypes are currently classified within a single virus species, Great Island virus. In this study, we report the first characterization of a tick-borne orbivirus isolated from the tick Ixodes turdus in Japan, which we identified as a new member of the species Great Island virus. The virus isolate, designated Muko virus (MUV), replicated and induced cytopathic effects in BHK-21, Vero E6, and CCL-141 cells and caused high mortality in suckling mice after intracerebral inoculation. Full genome sequence analysis showed that MUV shared the greatest phylogenetic similarity with Tribec virus in terms of the amino acid sequences of all viral proteins except for outer capsid protein 1 (OC1; VP4 of MUV). Analysis of genome segment 9 in MUV detected an uninterrupted open reading frame that overlaps with VP6 (Hel), which putatively encodes a molecular and functional equivalent of NS4 from Great Island virus. Our study provides new insights into the geographic distribution, genetic diversity, and evolutionary history of the members of the species Great Island virus.

First isolation and characterization of a mosquito-borne orbivirus belonging to the species Umatilla virus in East Asia.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

Molecular identification of avian haemosporidia in wild birds and mosquitoes on Tsushima Island, Japan.

We investigated for the first time the prevalence of avian haemosporidia of genera Plasmodium, Haemoproteus and Leucocytozoon among birds and mosquitoes on Tsushima Island of Japan, which is located between Japan and the Korean Peninsula. Of 55 wild birds belonging to 33 species, 16 (29.1%) tested positive for haemosporidia as follows: Plasmodium spp. (11/55; 20.0%); Haemoproteus spp. (2/55; 3.6%); and Leucocytozoon spp. (3/55; 5.5%). A genetic lineage isolated from the Eurasian Sparrowhawk (Accipiter nisus) was identical to that of the known avian malaria parasite P. circumflexum. Several genetic lineages were identical or closely related to the parasite lineages that were previously detected in birds and mosquitoes in Japan and Korea. Another single identical genetic lineage was also detected in both migratory and resident birds. A total of 753 mosquitoes from 12 species were collected; and one fully fed Aedes albopictus was positive for avian Plasmodium(1/753; 0.13%) which is identical to a genetic lineage detected in both mosquitoes in Japan and birds in Korea. Blood-meal identifications of blood-fed mosquitoes showed direct contact between the mosquitoes and 4 species of mammals including humans, cattle, rodents and the endangered Tsushima leopard cat (Prionailurus bengalensis euptilura). Migratory birds use Tsushima Island as a site for wintering, breeding and resting, and our results suggest the transmission of avian haematozoa between resident and migratory birds during their stay on Tsushima Island.

Prevalence of avian haematozoa in wild birds in a high-altitude forest in Japan.

The infection dynamics of avian haematozoa, which includes the genera Plasmodium, Haemoproteus, and Leucocytozoon, are complicated by a variety of environmental factors and host-parasite interactions. In Japan, the prevalence of haematozoa in wild birds has recently been determined in several local areas. However, no information on the annual prevalence of avian haematozoa in a single study site has been reported. Here, we investigated the long-term infection dynamics of haematozoa in wild birds inhabiting a mountain forest of Japan. Blood samples were collected from 415 wild birds captured in the Chichibu mountains in Saitama Prefecture at an altitude of 1650 m between 2007 and 2010. All obtained samples were examined for haematozoan infection using nested polymerase chain reaction (PCR) of the cytochrome b (cytb) genes of haematozoa. A total of 62 out of 415 (14.9%) forest birds were PCR positive for haematozoa. Relatively high infection rates of Leucocytozoon were found among several bird species (Parus ater, 64.3%; Parus montanus, 81.8%) and may be due to the host preference of vector black flies and host nestling pattern in this forest. Phylogenetic analysis of amplified cytb sequences revealed for the first time that a variety of lineages of avian haematozoa are distributed among wild bird hosts in a high-altitude forest stand in Japan. Notably, significant seasonal changes of the prevalence of avian haematozoa were not observed; however, continuous investigation will likely provide detailed information on host-parasite interactions, including local environmental factors, that influence the dynamics of avian haematozoan infections.

Blood meal identification and prevalence of avian malaria parasite in mosquitoes collected at Kushiro wetland, a subarctic zone of Japan.

In Japan, the prevalence of avian Plasmodium in birds and mosquitoes has been partially examined in the temperate and subtropical zones; however, mosquitoes in the Japanese subarctic zone have not been adequately investigated. In this study, mosquito collections and avian Plasmodium detections from the mosquito samples were carried out to demonstrate the avian Plasmodium transmission between vector mosquitoes and birds inhabiting in Kushiro Wetland, subarctic zone of Japan. A total of 5657 unfed mosquitoes from 18 species and 320 blood-fed mosquitoes from eight species was collected in summer 2008, 2009, and 2010. Three Aedes esoensis that fed on Hokkaido Sika Deer and one unfed Culex pipiens group were found to be positive for avian Plasmodium by polymerase chain reaction. This is the first report of the detection of avian Plasmodium DNA from mosquitoes distributing in the subarctic zone of Japan. The blood meals were successfully identified to captive or wild animals, including seven mammalian species, four bird species, and one amphibian species. These results indicated that infected birds with avian Plasmodium inhabited and direct contacts occurred between the infected birds and mosquitoes in Kushiro Wetland, Hokkaido, Japan.

First record of avian Plasmodium DNA from mosquitoes collected in the Yaeyama Archipelago, southwestern border of Japan.

We studied the prevalence of avian Plasmodium in 509 mosquitoes of 9 species collected from the Ishigaki and Iriomote islands in the Yaeyama Archipelago, located southwest from the mainland of Japan. Two identical avian Plasmodium lineages were detected from Culex (Culiciomyia) nigropunctatus. Detected lineages were phylogenetically classified into different clade to avian Plasmodium lineages from birds and mosquitoes in the mainland of Japan but identical to a lineage detected from a resident bird, White-breasted Waterken (Amaurornis phoenicurus). This is the first detection of avian Plasmodium DNA from mosquitoes in the Yaeyama Archipelago and suggested that resident birds might have been infected with an avian Plasmodium lineage specific to the studied area and C. nigropunctatus could be the candidate vector mosquito species.

Entomological study on transmission of avian malaria parasites in a zoological garden in Japan: bloodmeal identification and detection of avian malaria parasite DNA from blood-fed mosquitoes.

Several species of captive and wild birds have been found to be infected with various avian blood protozoa in Japan. We investigated the prevalence and transmission of avian malaria parasite and determined the bloodmeal hosts of mosquitoes collected in a zoological garden in Tokyo, Japan, by using the polymerase chain reaction. In total, 310 unfed and 140 blood-fed mosquitoes of seven species were collected by using sweep nets and CDC traps. Bloodmeal identification indicated that mosquitoes had fed on 17 avian and five mammalian species, including captive animals. The results of avian malaria parasite detection from mosquitoes with avian bloodmeals indicated that Culex pipiens pallens Coquillet is a main vector of avian Plasmodium in the current study site and that some captive and wild birds could be infected with avian malaria parasites. Furthermore, the distances between the collection site of blood-fed mosquitoes and the locations of their blood-source captive animals were estimated. Most females with fresh bloodmeals were found within 40 m of caged animals, whereas half-gravid and gravid females were found between 10 and 350 m from caged host animals. We demonstrated that blood-fed mosquitoes can provide useful information regarding the mosquito vector species of avian malaria parasites and allows for noninvasive detection of the presence of avian malaria parasites in bird populations.

Molecular identification of blood source animals from black flies (Diptera: Simuliidae) collected in the alpine regions of Japan.

One of vector-borne avian protozoa, Leucocytozoon lovati, has been found in the Japanese rock ptarmigans (Lagopus mutus japonicus), the endangered bird species distributed in the alpine regions in Japan. Vector arthropod species of L. lovati has also been estimated as Simuliidae black flies distributed in the same habitat of the host bird, however, possible blood meals of the black flies were not identified yet. To reveal host animals of black flies, we estimated the blood resources by using molecular techniques. Black flies were collected at Mt. Chogatake, one of the alpine regions of Japan in which Japanese rock ptarmigans live in June 2005. The analyzed 144 specimens were morphologically identified into five species including Simulium japonicum (n = 87), Prosimulium hirtipes (n = 48), Prosimulium yezoense (n = 3), Twinnia japonensis (n = 3), and Cnephia mutata (n = 3). Individually extracted DNA from the black flies was subjected to polymerase chain reaction (PCR) amplification targeting the partial mitochondrial cytochrome b gene of birds or mammals to identify the blood meals. Of 144 black flies examined, 34 specimens were PCR positive for avian hosts (23.6%). No mammalian-derived bloods were detected from the samples studied through. Sequences amplified from 11 black flies consist of S. japonicum, P. hirtipes, and C. mutata showed high similarity to that of the Japanese rock ptarmigan. Therefore, present results conclusively suggest that these three species of black flies might suck the bloods of Japanese rock ptarmigans and could be the vector for L. lovati infection among this endangered bird species of Japan.

Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan.

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.

Molecular detection of Leucocytozoon lovati from probable vectors, black flies (Simuliudae) collected in the alpine regions of Japan.

Probable arthropod vectors of avian blood protozoa, Leucocytozoon lovati, were collected in the alpine regions of Japan, the habitats of the host birds of Japanese rock ptarmigan (Lagopus mutus japonicus). Seven alpine regions of Japan, Asahidake, Chogatake, Tateyama, Jiigatake, Norikura, Kitadake, and Senjyogatake were investigated for black fly collection during 2004 to 2007. The collected 490 insects were morphologically identified as six species of female black flies, including Prosimulium hirtipes group (n = 59), Prosimulium mutata (n = 13), Prosimulium yezoense (n = 10), Similium japonicum (n = 359), Similium uchidai (n = 39), and Twinnia japonensis (n = 10). Extracted DNAs from individual black fly species were utilized for the amplification of the partial mitochondrial cytochrome b gene sequences of Leucocytozoon lovati previously reported. Four S. japonicum, two S. uchidai, and two P. hirtipes group studied were positive for the nested PCR among 490 black flies collected (1.6%; 8/490). All amplified sequences from the black flies were completely identical to those of L. lovati previously detected from Japanese rock ptarmigan. Our results suggest that at least three species of black flies, S. japonicum, S. uchidai, and P. hirtipes group, studied in this area could be regarded as potential vectors for L. lovati in the rock ptarmigan. This is the first detection case of Leucocytozoon from black flies of Japan.

Detection of avian Plasmodium spp. DNA sequences from mosquitoes captured in Minami Daito Island of Japan.

Several species of birds in Minami Daito Island, an oceanic island located in the far south from the main islands of Japan, were found to be infected with avian Plasmodium. However, no vector species of the avian malaria in this island have been revealed yet. To speculate potential vectors, we collected mosquitoes there and investigated using a PCR procedure whether the mosquitoes harbor avian malaria or not. Totally 1,264 mosquitoes including 9 species were collected during March 2006 to February 2007. The mosquitoes collected were stored every species, sampled date and location for DNA extraction. Fifteen out of 399 DNA samples showed positive for the partial mtDNA cytb gene of avian Plasmodium. Estimated minimum infection rate among collected mosquitoes was 1.2% in this study. Four species of mosquitoes; Aedes albopictus, Culex quinquefasciatus, Lutzia fuscanus and Mansonia sp. had avian Plasmodium gene sequences. Detected DNA sequences from A. albopictus and L. fuscanus were identical to an avian Plasmodium lineage detected in bull-headed shrike (Lanius bucephalus) captured in the island. Different sequences were detected from C. quinquefasciatus, which were corresponding to an avian Plasmodium from a sparrow (Passer montanus) and Plasmodium gallinaceum. Our results suggest that A. albopictus, Lutzia fuscanus, C. quinquefasciatus, and Mansonia sp. could be potential vectors of avian malaria in Minami Daito Island. This study was the first report of molecular detection of avian Plasmodium from mosquitoes in Japan.