First isolation and characterisation of Alongshan virus in Russia

In recent decades, many new flavi-like viruses have been discovered predominantly in different invertebrates and, as was recently shown, some of them may cause disease in humans. The Jingmen tick virus (JMTV) group holds a special place among flavi-like viruses because, in contrast to the “classic” flaviviruses and other flavi-like viruses, they have a segmented ssRNA(+) genome. Two segments of the JMTV genome have homology with regions of the flavivirus genome encoding polymerase and helicase-protease proteins. JMTVs have several open reading frames (ORF) in segments encoding glycoprotein(s) and capsid protein and these ORF are specific only to them. JMTVs greatly differ in virion size.We isolated three strains of Alongshan virus (ALSV), which is a representative of the JMTV group, from adult Ixodes persulcatus ticks collected in two geographically-separated Russian regions in the tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Most virions purified from tick cells were spherical with a diameter of approximately 40.5 nm. In addition, we found smaller particles of approximately 13.1 nm in diameter. We obtained full genome sequences of all four segments of two of the isolated ALSV strains, and partial sequences of one segment from the third strain. Phylogenetic analysis on genome segment 2 of the JMTV group clustered our novel strains with other ALSV strains. We found evidence for the existence of a novel upstream ORF in the glycoprotein-coding segment of ALSV and other members of the JMTV group.Significance StatementWe isolated three strains of Alongshan virus (ALSV) from adult Ixodes persulcatus ticks from two geographically separate areas of Russia in the Ixodes ricinus tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Our study confirmed the value of tick cell lines in virus isolation and maintenance of persistent infection. The majority of virions of the ALSV strain Miass527 were enveloped spherical particles with a diameter of 40.5±3.7 nm. We found evidence for the existence of a novel upstream ORF in the glycoprotein-coding segment of ALSV and other members of the Jingmen tick virus group.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale

Background Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes, causing anemia and impairing milk production and weight gain. In addition, infection can cause abortion and high mortality in areas of enzootic instability. The immunization with live and inactivated vaccines are employed to the control acute bovine anaplasmosis. However, they do not prevent persistent infection. Therefore, infected animals, even if immunized, are reservoirs of the bacterium and contribute to the dissemination of the disease. Antimicrobials are also largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in subtlethal doses, what can select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, the standardization of an in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials is important to allow the prescription of the more effective treatment, preventing both the selection and spread of resistant strains. Results Initially the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines was evaluated. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 μg/mL and oxytetracycline (OTC) at 4 or 16 μg/mL are the most efficient treatments, followed by OTC at 1 μg/mL and imidocarb dipropionate (IMD) at 1 or 4 μg/mL. Conclusion In the current study, we present a new in vitro assay using a tick cell line to evaluate the susceptibility of A. marginale to antimicrobials. The maintenance of such culture is much easier than the maintenance of bovine erythrocyte culture, which depends on continuous cell replacement. This assay may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, preventing the spread of resistant strains and helping in the control of bovine anaplasmosis.