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Kasokero virus (KASV; genus Orthonairovirus) was first isolated in 1977 at Uganda Virus Research Institute from serum collected from Rousettus aegyptiacus bats captured at Kasokero Cave, Uganda. During virus characterization studies at the institute, 4 laboratory-associated infections resulted in mild to severe disease. Although orthonairoviruses are typically associated with vertebrate and tick hosts, a tick vector of KASV never has been reported. We tested 786 Ornithodoros (Reticulinasus) faini tick pools (3,930 ticks) for KASV. The ticks were collected from a large R. aegyptiacus bat roosting site in western Uganda. We detected KASV RNA in 43 tick pools and recovered 2 infectious isolates, 1 of which was derived from host blood-depleted ticks. Our findings suggest that KASV is maintained in an enzootic transmission cycle involving O. (R.) faini ticks and R. aegyptiacus bats and has the potential for incidental virus spillover to humans.

Genetic relationships between bunyaviruses from Australia and pathogenic bunyaviruses from elsewhere indicate emergence potential., To better understand the diversity of bunyaviruses and their circulation in Australia, we sequenced 5 viruses (Gan Gan, Trubanaman, Kowanyama, Yacaaba, and Taggert) isolated and serologically identified 4 decades ago as members of the family Bunyaviridae. Gan Gan and Trubanaman viruses almost perfectly matched 2 recently isolated, purportedly novel viruses, Salt Ash and Murrumbidgee viruses, respectively. Kowanyama and Yacaaba viruses were identified as being related to members of a large clade containing pathogenic viruses. Taggert virus was confirmed as being a nairovirus; several viruses of this genus are pathogenic to humans. The genetic relationships and historical experimental infections in mice reveal the potential for these viruses to lead to disease emergence.

To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV) (Nairovirus, Bunyaviridae), the causative agent of Crimean-Congo hemorrhagic fever, has been detected in sub-Saharan Africa, southeastern Europe, the Middle East, and central Asia. The virus has been detected in >31 species of ticks and is transmitted to humans by bite of infected ticks (mainly of the genus Hyalomma) or by contact with body fluids or tissue of viremic patients or livestock. The disease is characterized by fever, myalgia, headache, vomiting, and sometimes hemorrhage; reported mortality rate is 10%–50% (1). CCHFV strains currently constitute 7 evolutionary lineages, 1 of which (Europe 2) contains the prototype strain AP92, which was isolated in 1975 from Rhipicephalus bursa ticks collected from goats in Greece (2). This strain seems to have low or no pathogenicity for humans; only a few mild cases have been reported (3). This observation is supported by the relatively high (14.4%) seroprevalence but no clinical cases in humans in northwestern Greece (4). The documented tick carriers of this strain are R. bursa and Hyalomma marginatum (5). Hyalomma aegyptium ticks are highly host specific; adults feed almost entirely on tortoises of the genus Testudo (6) and occassionally on hedgehogs and hares. Unlike adult ticks, the larvae and nymphs are less host specific and feed on a wide spectrum of hosts (e.g., other reptiles, birds, and mammals [including humans]) (7). This trait elevates the epidemiologic role of the tick as a possible bridge vector connecting wildlife, domestic animals, and humans. To determine the biological and epidemiological role of H. aegyptium ticks, during 2009–2010, we collected 56 adult ticks from 12 Testudo graeca tortoises at a locality near the city of Aflou in Laghouat Province, Algeria. We tested the ticks for probable CCHFV infection by using nested reverse transcription PCR (8), which amplifies a partial fragment of the CCHFV small RNA segment. We slightly modified the assay: reverse transcription time was 60 minutes and annealing temperature was 52°C (9). In total, 16 (28.6%) ticks were positive for CCHFV. The PCR products of 15 (26.8%) positive samples were sequenced. BLAST (http://blast.ncbi.nlm.nih.gov//Blast.cgi) analysis identified all 15 sequences as CCHFV with 98%–100% identity to the AP92 strain (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ211638","term_id":"78191750","term_text":"DQ211638"}}DQ211638). Two variants of AP92 were detected and differed by 0.6%. A phylogenetic tree was constructed by Bayesian inference, using MrBayes version 3.1.2 (http://mrbayes.sourceforge.net/index/php) under a general time-reversible plus gamma distribution plus invariable site model with 107 generations setup (Figure). Sequences are available in GenBank (accession nos. KT99097 and KT99098). Figure Phylogenetic analysis of Crimean-Congo hemorrhagic fever virus small RNA segment sequences, performed by using Bayesian inference in MrBayes version 3.1.2. (http://mrbayes.csit.fsu.edu/) under a general time-reversible plus gamma distribution plus invariable ... Our findings demonstrate the presence of CCHFV in Algeria, either recently introduced or overlooked. The nearest location where CCHFV has been reported is the Zouala region in Morocco, where the virus was detected in H. marginatum tick larvae and nymphs collected from migratory birds (10). It also confirms association of AP92-like sequences with H. aegyptium ticks. This study shows that the Europe 2 lineage is not restricted to the Balkan region and Turkey. The role of H. aegyptium ticks as CCHFV vectors should be further tested. Further investigation of the distribution of CCHFV in ticks in Algeria is also needed. To date, CCHFV strains of lineage Europe 2 have not been associated with severe disease in humans. However, physicians in Algeria should be aware of potential Crimean-Congo hemorrhagic fever cases.

To the Editor: Nairobi sheep disease virus (NSDV; genus Nairovirus, family Bunyaviridae) causes acute hemorrhagic gastroenteritis in sheep and goats (1,2). First identified in Nairobi, Kenya, in 1910, it is considered endemic in East Africa (1,3). Ganjam virus, a variant of NSDV, is found in India and Sri Lanka (2). NSDV has a limited effect on animals bred in areas to which the virus is endemic but can cause large losses of animals during introduction of new livestock or transport of animals through these areas (4). In humans, NSDV infection can cause febrile illness, headache, nausea, and vomiting (5). Ticks are the main transmission vectors of NSDV and many other viral pathogens and therefore pose a major threat to public health (6,7). Here, we describe a newly discovered NSDV, named NSDV (China), identified by viral metagenomic analysis of ticks collected from the northeast region of the People’s Republic of China (Liaoning, Jilin, and Heilongjiang provinces) during May–July, 2013, and divided into 9 groups according to tick species and sampling sites. Four tick species were morphologically identified: Haemaphysalis longicornis (84.8%); Dermacentor silvarum (7.2%); D. nuttalli (5.5%); and Ixodes persulcatus (2.5%) (Technical Appendix Table 1). Of the 6,427 ticks collected, 3,410 were divided into 9 groups (average 379 ticks/group, range 163–512); each group was homogenized in SM buffer (50 mmol/L Tris, 10 mmol/L MgSO4, 0.1 mol/L NaCl, pH 7.5). Viral RNA extraction, Solexa sequencing, and analysis are described in the online Technical Appendix. Among the sequences annotated to mammalian viruses, 65 contigs were found to target the small (n = 15), medium (n = 27), and large (n = 23) segments of the NSDV genome (Technical Appendix Tables 2–4). To confirm the Solexa results, a 376-nt fragment of the NSDV small gene segment was amplified by reverse transcription PCR (RT-PCR) by using primers P1 (5′-AGCAAAGAGCACATTGACTGGGC-3′) and P2 (5′-CTGTCACACCTGCCTTCCAA-3′). Ticks in 3 H. longicornis groups were positive for NSDV: group 1 from sheep in Jian, Jilin Province (125°34′E, 40°52′N); group 2 from cattle in Jinxing, Jilin Province (130°38′E, 42°25′N); and group 5 from sheep in Dandong, Liaoning Province (124°23′E, 40°07′N). Ticks in the other groups were negative. The obtained sequences shared 92% identity with NSDV from H. intermedia in India. The full-length sequence of NSDV was then obtained from group 2 by RT-PCR by using primers based on the Solexa sequences or the conserved sequences of nairoviruses (Technical Appendix Table 5). The complete sequences of the small, medium, and large segments of NSDV (China) (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KM464724-KM464726","start_term":"KM464724","end_term":"KM464726","start_term_id":"728892682","end_term_id":"728892686"}}KM464724-KM464726) contained 1,590, 5,077, and 12,081 nt, respectively; that is, they were similar to other NSDVs. Sequence comparisons showed 75.1%–89.6% identity with other NSDVs at the nucleotide level and 81.3%–96.7% at the deduced amino acid level (Technical Appendix Table 6). Compared with other member species within the genus Nairovirus (Dugbe, Kupe, Hazara, and Crimean Congo hemorrhagic fever viruses), low identities (37.5%–68.6%) were observed at both nucleotide and amino acid levels (Technical Appendix Table 6). Phylogenetic analysis based on the amino acid sequences grouped the virus together with NSDVs from Africa and South Asia (Figure). Figure Phylogenetic analysis of Nairobi sheep disease virus (China) and other nairoviruses. The phylogenetic trees were generated in MEGA5.2 software (http://www.megasoftware.net). The complete coding regions for nucleocapsid protein in the small segment (A), ... The remaining tick samples of the NSDV-positive groups were used to determine the infection frequency by using RT-PCR to analyze primers P1 and P2. We assayed 104 tick pools (average 15 ticks/pool, range 8–40), 13 pools of 416 ticks in Jian Province and 91 pools of 1,095 ticks in Jinxing Province; 12.5% (13/104) tested positive, 38.5% (5/13) in Jian and 8.8% (8/91) in Jinxing. The higher prevalence in Jian Province may result from more ticks in the pools. Attempts to isolate virus from the positive samples in cell lines (Vero and BHK-21) and suckling mice were unsuccessful; thus, its pathogenicity could not be determined. In Africa, NSDV is primarily transmitted by R. appendiculatus ticks (5). In South Asia (India and Sri Lanka), NSDV has been isolated from ticks (H. intermedia, H. wellingtoni, and R. haemaphysaloides), mosquitoes, sheep and humans; H. intermedia ticks are considered the main vector for the virus (5,8,9). NSDV had not previously been reported from East Asia. The isolate we identified, NSDV (China), is genetically divergent from the NSDVs of South Asia and Africa and is therefore a novel strain, with H. longicornis likely the main vector. Nairobi sheep disease has not been reported in China and East Asia, but our results indicate the risk of its occurrence in these regions, where H. longicornis is widely distributed (10). More extensive investigation to clarifty the natural circulation of NSDV among ticks should be conducted and surveillance of sheep improved to prevent outbreaks of Nairobi sheep disease in China and East Asia. Technical Appendix: Tick collection, RNA extraction and processing, sequencing, and analysis of data resulting in identification of Nairobi sheep disease virus RNA in ixodid ticks, China, 2013. Click here to view.(107K, pdf)

To the Editor: Crimean-Congo hemorrhagic fever (CCHF) is an acute viral infection that causes mild to severe hemorrhagic fever characterized by petechiae, ecchymosis, disseminated intravascular coagulation, and multi-organ failure (1). The etiologic agent, CCHF virus (CCHFV; family Bunyaviridae, genus Nairovirus), is maintained in enzootic cycles involving agricultural and wild animals and the vector, Hyalomma ticks. (2). CCHF predominantly affects persons who have 1) substantial contact with ticks and/or agricultural animals in areas where CCHF is endemic or 2) close contact with infected persons, predominantly close relatives and health care workers. The case-fatality rate for CCHF is generally accepted as 30% (1). CCHF has a wide geographic distribution; cases have been reported in >30 countries across Africa, southeastern Europe, the Middle East, and western Asia. In the western African countries of Nigeria, Mauritania, and Senegal, serologic evidence of CCHFV infections in humans and agricultural animals has been documented frequently (3–5); however, reports of the disease in humans have been limited to Senegal and Mauritania (6,7). In neighboring Mali, where the tick vector is known to be present, little information exists regarding the presence of CCHFV. Thus, to determine if the virus is circulating undetected in Mali, we conducted a study to determine if CCHFV is present in Hyalomma ticks in the country. In November 2011 and March 2012, unfed Hyalomma ticks (adults and nymphs) were collected from 20 cattle at the Daral livestock market (12° 49.855′ N, 08° 05.651′ W) near the town of Kati, Mali, ≈25 km from the capital, Bamako. In the field, ticks were visually identified to genus and pooled accordingly (3–4 ticks per pool, all collected from the same animal). A total of 23 tick pools, representing 80 ticks, were manually homogenized, and RNA was extracted and tested for the presence of CCHFV RNA by using in-house assays that selected for 3 virus genes. Of the 23 tick pools tested, 1 was positive for CCHFV by all 3 assays. Phylogenetic analysis of the complete nucleocapsid protein gene ({"type":"entrez-nucleotide","attrs":{"text":"KF793333","term_id":"648174138","term_text":"KF793333"}}KF793333) showed that the CCHFV strain from Mali most closely resembled a strain from Mauritania (GenBank accession no. ArD39554), sharing 98% sequence identity (Figure, panel A). Figure Phylogenetic analysis of Crimean–Congo hemorrhagic fever virus (CCHFV) was conducted on the complete nucleoprotein (small genomic segment, nt ≈50–1,500) (A), a 900-bp fragment of the glycoprotein precursor (medium genomic segment, ... Further analysis of fragments of the medium segment (pre-Gn coding region, {"type":"entrez-nucleotide","attrs":{"text":"KF793334","term_id":"648174140","term_text":"KF793334"}}KF793334) and large segment (polymerase coding region, {"type":"entrez-nucleotide","attrs":{"text":"KF793335","term_id":"648174141","term_text":"KF793335"}}KF793335) confirmed these findings, showing sequence identities of 91% and 98%, respectively, with ArD39554 (Figure, panels B, C). In a Biosafety Level 4 facility at Rocky Mountain Laboratories, Hamilton, Montana, USA, the original homogenates from the positive pool were passaged in multiple cell lines. After 3 passages, no discernible cytopathic effect was observed and, aside from the initial passage, CCHFV RNA was not detected. Genetic identification of ticks in the CCHFV RNA–positive pool was conducted as described (8,9). Amplified sequences most closely resembled those of H. dromedarii, (97.2%–100% sequence identity), although genetically, we cannot exclude the possibility that H. truncatum and H. rufipes were present with individual sequence identities of >97% to published sequences. The Daral cattle market in Kati is the largest of its kind in Mali, and animals from across the country come into the market every week. Although the market provided a convenient opportunity for collecting ticks, we cannot determine where the infected ticks, and possibly cattle, contracted CCHFV because the animals traversed great distances on foot before arriving at the market. Nevertheless, this study demonstrates the presence of a distinct strain of CCHFV in Hyalomma ticks in Mali, thereby expanding the geographic distribution of this virus in western Africa. Not surprisingly, the highest sequence identity for the CCHFV strain from Mali is to strains known to circulate in neighboring countries (10). We propose Daral 2012 Mali as the temporary designation for this sequence. Unfortunately, our attempts to isolate the virus were unsuccessful, most likely because of processing and storage conditions for homogenates used in these studies. Species of Hyalomma ticks are widely distributed across western Africa, and although reports of CCHF are limited to a few countries, CCHFV is most likely circulating undetected in vast areas of this region. No cases of CCHF have been reported in Mali; however, on the basis of our findings, the potential for human infections exists. Thus, CCHF should be considered in the differential diagnosis of febrile illnesses, with or without hemorrhagic symptoms, in residents of Mali and for persons with a recent history of travel to this country. The ease of CCHFV transmission and the high case-fatality rate associated with infection could have a potentially substantial effect on public health. Future studies in Mali are required to define the geographic distribution of infected ticks and animals and to isolate CCHFV to help focus public health preparedness and countermeasures. In addition, across Mali, operational protocols should be reviewed for persons working at jobs in which the risk for CCHFV transmission is high (e.g., occupations with direct contact with agricultural animals and/or animal blood products), and appropriate countermeasures should be put in place to prevent transmission among such persons.

One-sentence summary for table of contents: A new nairovirus isolated from ticks collected from cattle hides was characterized., We have previously described isolation and preliminary identification of a virus related to Dugbe virus (DUGV), family Bunyaviridae, genus Nairovirus. Six isolates of the virus were obtained from pools of Amblyomma gemma and Rhipicephalus pulchellus ticks collected from hides of cattle in Nairobi, Kenya, in October 1999. We report results of further characterization of this virus, including growth kinetics in cell culture and full-length genome sequencing and genetic characterization, which show it to be distinct from DUGV. We suggest that this is a new virus in the family Bunyaviridae, genus Nairovirus, and we propose that it be designated Kupe virus.

Numerous tickborne viruses, including Dhori virus and foot-and-mouth disease virus, were isolated., To identify tickborne viruses circulating in Kenya and the surrounding region, we conducted surveillance at abattoirs in Nairobi, Kenya. Species of ticks collected included Rhipicephalus pulchellus (56%), Amblyomma gemma (14%), R. appendiculatus (8%), A. variegatum (6%), and others. A total of 56 virus isolates were obtained, 26 from A. gemma, 17 from R. pulchellus, 6 from A. variegatum, and 7 from other species. Virus isolates included Dugbe virus (DUGV), an unknown virus related to DUGV, Thogoto, Bhanja, Kadam, Dhori, Barur, and foot-and-mouth disease (FMDV) viruses. This is the first report of Dhori virus isolation in East Africa and the first known isolation of FMDV associated with tick collection. Our results demonstrate the potential for tickborne dissemination of endemic and emergent viruses and the relevance of A. gemma in the maintenance of tickborne viruses in this region.

To the Editor: Crimean-Congo hemorrhagic fever (CCHF) is a tickborne zoonotic disease caused by a member of the virus family Bunyaviridae, genus Nairovirus. This virus (CCHFV) has caused illness throughout Asia, Europe, Africa, and the Middle East (1). CCHFVs are clustered among 7 genotypes (Asia-1, Asia-2, Euro-1, Euro-2, Africa-1, Africa-2, and Africa-3) on the basis of genetic variation in the small segment (2). These genotypes are well conserved among their regions of origin; however, >1 genotype is prevalent in many countries (2). In Pakistan, the first CCHF case was reported in 1976; multiple sporadic cases and outbreaks have occurred in subsequent years (3). To determine which genotypes were present in Pakistan, we performed molecular analysis of archived serum samples collected during 2008 in Fatima Jinnah General and Chest Hospital, Quetta, Baluchistan, in southwestern of Pakistan. Because of limited diagnostic facilities for CCHFV in this country, samples collected during 1976–2002 were occasionally sent to laboratories in countries such as South Africa and the United States, where genetic analysis showed that all viruses tested from that location belonged to the Asia-1 genotype (4). Data beyond this period are not available; however, because of improved molecular diagnostic facilities at the Department of Virology, National Institute of Health, Pakistan, blood samples collected from patients with suspected cases attending in-country hospitals are now examined by the institute for confirmation. Our findings substantiate the presence of Asia-1 and Asia-2 genotypes in Baluchistan. Thirteen IgM-positive samples collected during 2008 and stored at –70°C were available for study. The samples were processed for amplification of 260 bp of the small segment by using reverse transcription PCR with a previously described protocol (5). The mean age of patients with serology-confirmed CCHF was 31.3 (range 18–40) years; male-to-female IgM positivity ratio was 1:2. Common symptoms were fever, headache, and nosebleeds. Platelet counts ranged from 16,000 to 43,000/μL of blood. Of the 13 samples, viral RNA was detected in 2 (CCHF-65–2008PAK and CCHF-43–2008PAK); the amplicons were subjected to bidirectional sequencing by using the BigDye Terminator v3.1 cycle sequencing kit (Applied BioSystems, Foster City, CA, USA). Sequences were analyzed with Sequencher (GeneCodes Corp., Ann Arbor, MI, USA) and MEGA v4.0 (http://megasoftware.net/). The 2 viruses were phylogenetically clustered into Asia-1 and Asia-2 genotypes, with 7% nucleotide divergence, although both samples were collected during September–October, 2008. The closest nucleotide identity (99%–100%) for CCHF-65–2008PAK was found with the previously reported Asia-1 strains from Pakistan, Afghanistan, and Iran; CCHF-43–2008PAK had 96%–97% similarity to viruses from Dubai and Tajikistan (Figure). The sequences reported from United Arab Emirates, Pakistan, Afghanistan, Iran, and Iraq belong to the Asia-1 genotype; the Asia-2 genotype sequences were mostly from China and Central Asian countries such as Uzbekistan, Tajikistan, and Kazakhstan (6). All viruses detected intermittently in Pakistan during 1976–2002 were of the Asia-1 genotype (4). However, the analysis of the 2 samples reported here enhances our knowledge of CCHFV genetic diversity in Pakistan. Figure Phylogenetic analysis of partial small gene fragment (220 bp) obtained from Crimean-Congo hemorrhagic fever virus strains analyzed in this study (black circles). Reference strains belong to different genogroups as retrieved from GenBank. Diamonds indicate ... The closest phylogenetic positioning of CCHF-43–2008PAK with Asia-2 strain Dubai-616 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JN108025","term_id":"354990643","term_text":"JN108025"}}JN108025) indicates that the probable route of CCHFV transmission was through animal trade between the United Arab Emirates and Pakistan. This finding supports the proposition that animals imported from Pakistan were the probable source of a 1979 outbreak in the United Arab Emirates (7). However, we cannot determine the direct source of the Asia-2 genotype in Pakistan, nor confirm the transmission link between the 2 countries. We attribute this to a lack of consistent, contemporary viral genetic information of CCHFV strains in Pakistan and the United Arab Emirates. This lack of data necessitates intensive surveillance and epidemiologic investigations in animal and human populations because geographic factors alone do not provide comprehensive information about the diversity of CCHFV strains circulating in Asia (6). The presence of geographically distant, but genetically similar, strains suggests that the viruses are dispersed either through animal trade or migratory birds (8). No clear evidence of CCHFV infection in migratory birds has been found, but they may play a major role in translocation of infected ticks to distant areas (9). Birds are known to be parasitized by these vectors of CCHFV in eastern Europe and Asia and disseminate the virus by transporting infected immature ticks between continents (4). It is therefore highly advisable to develop an active surveillance system with appropriate laboratory facilities to conduct the seroepidemiologic surveys and screening of household animals and vectors for CCHFV to rule out potential risks. Our study was limited by a low number of samples, resulting in availability of only a short fragment of the small gene for analysis. However, similar partial small gene sequencing has been used in previous studies (2) and has supported the classification of CCHFV strains correctly into 7 genotypes. In conclusion, because tick control is not feasible, surveillance activities and laboratory facilities should be improved. Health care workers should also be aware of proper patient management and standard prophylactic and preventive measures, particularly in areas where CCHFV is endemic, such as Baluchistan, where many deaths associated with nosocomial transmission have been reported (10).