IntroductionWe evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compar... more IntroductionWe evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays.MethodsForty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach.ResultsEleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted...
Currently, next generation sequencing (NGS) is the mainly used approach for identification and mo... more Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA ...
Chaphamaparvoviruses, a divergent group of parvoviruses (family Parvoviridae) infect both domesti... more Chaphamaparvoviruses, a divergent group of parvoviruses (family Parvoviridae) infect both domestic and wild mammalian and avian species, including bats, chickens and pigs. They have recently been identified using metagenomic analysis from tissue and stool samples of healthy and sick animals. Here, dogs (60 healthy and 43 diarrheal) were investigated for the presence of canine chaphamaparvovirus, canine coronavirus (CCoV), canine adenovirus (CAV), canine distemper virus (CDV) and canine parvovirus 2 (CPV2) by rectal swabs using polymerase chain reaction (PCR). For chaphamaparvovirus, all the healthy dogs tested negative whereas three of the diarrheal dogs tested positive (3 /103; 2.9%). CPV-2 was also detected in diarrheal dogs (28/43; 65%) but no other viruses were found in the rectal samples of the sick dogs. The three chaphamaparvovirus-positive dogs were also positive for CPV-2. Phylogenetic analysis showed that the chaphamaparvoviruses were all related to American strains, formi...
This study aimed to determine the presence and prevalence of viral and parasitic infections causi... more This study aimed to determine the presence and prevalence of viral and parasitic infections causing high rates of colony loss in honey bee colonies in Van province, eastern Turkey. Twenty-six different apiaries were collected from five counties in Van province. These samples were tested by Reverse-Transcriptase PCR (RT-PCR) for acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), black queen cell virus (BQCV) and deformed wing virus (DWV). Selected positives were sequenced, phylogenetically analyzed and investigated in terms of Varroa. DWV and BQCV were identified in 69.23% (18/26) and 88.46% (23/26) of the bees respectively whereas ABPV and CBPV were not detected in the sampled apiaries. Results of the phylogenetic analysis of DWV and BQCV sequences showed 94–100% similarity to DWV and BQCV isolates obtained from Genbank. Prevalence of varroasis was 89% (23/26) in Van. The obtained samples were identified as V. Varroa destructor by morphological investigation. The ...
We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detect... more We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. We subsequently screened 7223 ticks, representing 15 species in five genera, collected from various regions in Anatolia and eastern Thrace, Turkey. Moreover, we tested specimens from various patient cohorts (n = 103), and canine (n = 60), bovine (n = 20) and avian specimens (n = 65). JMTV nucleic acids were detected in 3.9% of the tick pools, including those from several tick species from the genera Rhipicephalus and Haemaphysalis, and Hyalomma marginatum, the main vector of CCHFV in Turkey. Phylogenetic analysis supported two separate clades, independent of host or location, suggesting ubiquitous distribution in ticks. JMTV was not recovered from any human, animal or bird specimens tested. Near-complete viral genomes were sequenced from the prototype isolate and from three infected tick pools. Genome topology and functional organiz...
We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and ... more We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kırklareli, and Tekirdağ (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Şanlıurfa (Southeastern Anatolia) provinces from 2013–2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequen...
Vector borne and zoonotic diseases (Larchmont, N.Y.), Oct 1, 2017
Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts fo... more Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts for viruses, dissemination vehicles for the vectors, and sources of emerging strains in cross-species transmission. Turkey provides diverse habitats for a variety of wild birds and is located along major bird migration routes. This study was undertaken to provide a cross-sectional screening of avian specimens for a spectrum of vector-borne viruses. The specimens were collected in Hatay province, in the Mediterranean coast of the Anatolian peninsula, located in the convergence zone of the known migration routes. Generic PCR assays were used for the detection of members of Nairovirus, Flavivirus, and Phlebovirus genera of Flaviviridae and Bunyaviridae families. The circulating viruses were characterized via sequencing and selected specimens were inoculated onto Vero cell lines for virus isolation. Specimens from 72 wild birds belonging in 8 orders and 14 species were collected. A total of 15...
We employed a direct metagenomic approach via next-generation sequencing for a cross-sectional in... more We employed a direct metagenomic approach via next-generation sequencing for a cross-sectional investigation of viruses in 10 tick pools, collected from Aegean, Mediterranean and central Anatolian locations in Turkey. Sequences from all genome segments of Tamdy orthonairovirus (family Nairoviridae) were characterized in ticks collected from a Meriones tristrami. We further obtained near-complete L and partial S segments of several tick-associated phleboviruses (family Phenuiviridae), including Tacheng tick virus 2 and a novel virus, tentatively named as the tick phlebovirus Anatolia. Partial NS5-coding region of recently-described flavi-like virus (Tacheng tick virus 8) was further detected. Moreover, near-complete and polymerase-coding regions of arthropod-associated rhabdoviruses as well as sequences closely-related to the members of the newly-proposed virus family, the Chuviridae, were characterized. Despite origins of the viral sequences could not be fully elucidated, the findin...
Ticks are involved with the transmission of several viruses with significant health impact. As in... more Ticks are involved with the transmission of several viruses with significant health impact. As incidences of tick-borne viral infections are rising, several novel and divergent tick- associated viruses have recently been documented to exist and circulate worldwide. This study was performed as a cross-sectional screening for all major tick-borne viruses in several regions in Turkey. Next generation sequencing (NGS) was employed for virus genome characterization. Ticks were collected at 43 locations in 14 provinces across the Aegean, Thrace, Mediterranean, Black Sea, central, southern and eastern regions of Anatolia during 2014-2016. Following morphological identification, ticks were pooled and analysed via generic nucleic acid amplification of the viruses belonging to the genera Flavivirus, Nairovirus and Phlebovirus of the families Flaviviridae and Bunyaviridae, followed by sequencing and NGS in selected specimens. A total of 814 specimens, comprising 13 tick species, were collected...
The American journal of tropical medicine and hygiene, 2015
Toscana virus (TOSV), a sandfly-borne phlebovirus, is an important agent of human meningoencephal... more Toscana virus (TOSV), a sandfly-borne phlebovirus, is an important agent of human meningoencephalitis in the Mediterranean region, for which vertebrates acting as reservoirs have not yet been determined. This study investigates TOSV and Leishmania infections in dogs, cats, sheep, and goats from Adana and Mersin provinces in southeastern Turkey. TOSV neutralizing antibodies were demonstrated in 40.4% of the dog and 4% of the goat samples. TOSV RNA was detected in 9.9% of the 252 samples that mainly comprise dogs (96%). Thus, canine species can be suggested as the candidate reservoirs of TOSV. Partial sequences revealed the activity of TOSV genotypes A and B. In two dogs presenting with symptoms of canine leishmaniasis, infections of TOSV genotype B and Leishmania infantum have been documented, describing the first report of coinfections with these agents.
A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. ( Diptera ; Psychodi... more A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. ( Diptera ; Psychodidae ) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae . Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad s...
IntroductionWe evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compar... more IntroductionWe evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays.MethodsForty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach.ResultsEleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted...
Currently, next generation sequencing (NGS) is the mainly used approach for identification and mo... more Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA ...
Chaphamaparvoviruses, a divergent group of parvoviruses (family Parvoviridae) infect both domesti... more Chaphamaparvoviruses, a divergent group of parvoviruses (family Parvoviridae) infect both domestic and wild mammalian and avian species, including bats, chickens and pigs. They have recently been identified using metagenomic analysis from tissue and stool samples of healthy and sick animals. Here, dogs (60 healthy and 43 diarrheal) were investigated for the presence of canine chaphamaparvovirus, canine coronavirus (CCoV), canine adenovirus (CAV), canine distemper virus (CDV) and canine parvovirus 2 (CPV2) by rectal swabs using polymerase chain reaction (PCR). For chaphamaparvovirus, all the healthy dogs tested negative whereas three of the diarrheal dogs tested positive (3 /103; 2.9%). CPV-2 was also detected in diarrheal dogs (28/43; 65%) but no other viruses were found in the rectal samples of the sick dogs. The three chaphamaparvovirus-positive dogs were also positive for CPV-2. Phylogenetic analysis showed that the chaphamaparvoviruses were all related to American strains, formi...
This study aimed to determine the presence and prevalence of viral and parasitic infections causi... more This study aimed to determine the presence and prevalence of viral and parasitic infections causing high rates of colony loss in honey bee colonies in Van province, eastern Turkey. Twenty-six different apiaries were collected from five counties in Van province. These samples were tested by Reverse-Transcriptase PCR (RT-PCR) for acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), black queen cell virus (BQCV) and deformed wing virus (DWV). Selected positives were sequenced, phylogenetically analyzed and investigated in terms of Varroa. DWV and BQCV were identified in 69.23% (18/26) and 88.46% (23/26) of the bees respectively whereas ABPV and CBPV were not detected in the sampled apiaries. Results of the phylogenetic analysis of DWV and BQCV sequences showed 94–100% similarity to DWV and BQCV isolates obtained from Genbank. Prevalence of varroasis was 89% (23/26) in Van. The obtained samples were identified as V. Varroa destructor by morphological investigation. The ...
We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detect... more We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. We subsequently screened 7223 ticks, representing 15 species in five genera, collected from various regions in Anatolia and eastern Thrace, Turkey. Moreover, we tested specimens from various patient cohorts (n = 103), and canine (n = 60), bovine (n = 20) and avian specimens (n = 65). JMTV nucleic acids were detected in 3.9% of the tick pools, including those from several tick species from the genera Rhipicephalus and Haemaphysalis, and Hyalomma marginatum, the main vector of CCHFV in Turkey. Phylogenetic analysis supported two separate clades, independent of host or location, suggesting ubiquitous distribution in ticks. JMTV was not recovered from any human, animal or bird specimens tested. Near-complete viral genomes were sequenced from the prototype isolate and from three infected tick pools. Genome topology and functional organiz...
We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and ... more We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kırklareli, and Tekirdağ (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Şanlıurfa (Southeastern Anatolia) provinces from 2013–2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequen...
Vector borne and zoonotic diseases (Larchmont, N.Y.), Oct 1, 2017
Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts fo... more Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts for viruses, dissemination vehicles for the vectors, and sources of emerging strains in cross-species transmission. Turkey provides diverse habitats for a variety of wild birds and is located along major bird migration routes. This study was undertaken to provide a cross-sectional screening of avian specimens for a spectrum of vector-borne viruses. The specimens were collected in Hatay province, in the Mediterranean coast of the Anatolian peninsula, located in the convergence zone of the known migration routes. Generic PCR assays were used for the detection of members of Nairovirus, Flavivirus, and Phlebovirus genera of Flaviviridae and Bunyaviridae families. The circulating viruses were characterized via sequencing and selected specimens were inoculated onto Vero cell lines for virus isolation. Specimens from 72 wild birds belonging in 8 orders and 14 species were collected. A total of 15...
We employed a direct metagenomic approach via next-generation sequencing for a cross-sectional in... more We employed a direct metagenomic approach via next-generation sequencing for a cross-sectional investigation of viruses in 10 tick pools, collected from Aegean, Mediterranean and central Anatolian locations in Turkey. Sequences from all genome segments of Tamdy orthonairovirus (family Nairoviridae) were characterized in ticks collected from a Meriones tristrami. We further obtained near-complete L and partial S segments of several tick-associated phleboviruses (family Phenuiviridae), including Tacheng tick virus 2 and a novel virus, tentatively named as the tick phlebovirus Anatolia. Partial NS5-coding region of recently-described flavi-like virus (Tacheng tick virus 8) was further detected. Moreover, near-complete and polymerase-coding regions of arthropod-associated rhabdoviruses as well as sequences closely-related to the members of the newly-proposed virus family, the Chuviridae, were characterized. Despite origins of the viral sequences could not be fully elucidated, the findin...
Ticks are involved with the transmission of several viruses with significant health impact. As in... more Ticks are involved with the transmission of several viruses with significant health impact. As incidences of tick-borne viral infections are rising, several novel and divergent tick- associated viruses have recently been documented to exist and circulate worldwide. This study was performed as a cross-sectional screening for all major tick-borne viruses in several regions in Turkey. Next generation sequencing (NGS) was employed for virus genome characterization. Ticks were collected at 43 locations in 14 provinces across the Aegean, Thrace, Mediterranean, Black Sea, central, southern and eastern regions of Anatolia during 2014-2016. Following morphological identification, ticks were pooled and analysed via generic nucleic acid amplification of the viruses belonging to the genera Flavivirus, Nairovirus and Phlebovirus of the families Flaviviridae and Bunyaviridae, followed by sequencing and NGS in selected specimens. A total of 814 specimens, comprising 13 tick species, were collected...
The American journal of tropical medicine and hygiene, 2015
Toscana virus (TOSV), a sandfly-borne phlebovirus, is an important agent of human meningoencephal... more Toscana virus (TOSV), a sandfly-borne phlebovirus, is an important agent of human meningoencephalitis in the Mediterranean region, for which vertebrates acting as reservoirs have not yet been determined. This study investigates TOSV and Leishmania infections in dogs, cats, sheep, and goats from Adana and Mersin provinces in southeastern Turkey. TOSV neutralizing antibodies were demonstrated in 40.4% of the dog and 4% of the goat samples. TOSV RNA was detected in 9.9% of the 252 samples that mainly comprise dogs (96%). Thus, canine species can be suggested as the candidate reservoirs of TOSV. Partial sequences revealed the activity of TOSV genotypes A and B. In two dogs presenting with symptoms of canine leishmaniasis, infections of TOSV genotype B and Leishmania infantum have been documented, describing the first report of coinfections with these agents.
A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. ( Diptera ; Psychodi... more A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. ( Diptera ; Psychodidae ) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae . Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad s...
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