The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Wide distribution of Mediterranean and African spotted fever agents and the first identification of Israeli spotted fever agent in ticks in Uganda.

Rickettsia microorganisms are causative agents of several neglected emerging infectious diseases in humans transmitted by arthropods including ticks. In this study, ticks were collected from four geographical regions of Uganda and pooled in sizes of 1-179 ticks based on location, tick species, life stage, host, and time of collection. Then, they were tested by real-time PCR for Rickettsia species with primers targeting gltA, 17kDa and ompA genes, followed by Sanger sequencing of the 17kDa and ompA genes. Of the 471 tick pools tested, 116 (24.6%) were positive for Rickettsia spp. by the gltA primers. The prevalence of Rickettsia varied by district with Gulu recording the highest (30.1%) followed by Luwero (28.1%) and Kasese had the lowest (14%). Tick pools from livestock (cattle, goats, sheep, and pigs) had the highest positivity rate, 26.9%, followed by vegetation, 23.1%, and pets (dogs and cats), 19.7%. Of 116 gltA-positive tick pools, 86 pools were positive using 17kDa primers of which 48 purified PCR products were successfully sequenced. The predominant Rickettsia spp. identified was R. africae (n = 15) in four tick species, followed by R. conorii (n = 5) in three tick species (Haemaphysalis elliptica, Rhipicephalus appendiculatus, and Rh. decoloratus). Rickettsia conorii subsp. israelensis was detected in one tick pool. These findings indicate that multiple Rickettsia spp. capable of causing human illness are circulating in the four diverse geographical regions of Uganda including new strains previously known to occur in the Mediterranean region. Physicians should be informed about Rickettsia spp. as potential causes of acute febrile illnesses in these regions. Continued and expanded surveillance is essential to further identify and locate potential hotspots with Rickettsia spp. of concern.

A custom hepatitis A virus assay for whole-genome sequencing.

Since 2016, the United States has experienced a resurgence in the number of hepatitis A virus (HAV) cases and outbreaks. These outbreaks have been sustained by person-to-person transmission with cases occurring predominantly in high-risk populations including intravenous drug users, individuals experiencing homelessness, and men who have sex with men. To investigate HAV transmission, a molecular-surveillance system consisting of real-time RT-PCR (rRT-PCR) for detection, and a conventional RT-PCR assay for genotyping of HAV, was established in New York State (NYS) in 2019. Since then, a total of 271 HAV-positive serum samples collected from cases across NYS between 2019 and 2021 were identified by rRT-PCR. To rapidly and efficiently generate HAV whole-genome sequences, a custom AmpliSeq™ panel was designed in collaboration with Thermo Fisher. To streamline the process, sample preparation was performed on an Ion Chef and sequencing on an Ion S5XL. Of the 271 HAV-positive samples, the whole-genome sequencing (WGS) assay successfully generated 134 near-complete, high-quality HAV sequences. Phylogenetic analysis of the VP1-2A region identified 216 IB, 48 IA, and 2 IIIA genotypes, while 5 were unable to be typed due to poor sequence in this key region. The HAV whole-genome sequencing approach provided a more efficient and streamlined approach for genotyping HAV compared to previous methods and resulted in phylogenetic trees with enhanced resolution compared to the HAV VP1-2A region alone.