First isolation and characterisation of Alongshan virus in Russia

Abstract

<jats:title>Abstract</jats:title><jats:p>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.</jats:p><jats:p>We isolated three strains of Alongshan virus (ALSV), which is a representative of the JMTV group, from adult <jats:italic>Ixodes persulcatus</jats:italic> 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.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p>We isolated three strains of Alongshan virus (ALSV) from adult <jats:italic>Ixodes persulcatus</jats:italic> ticks from two geographically separate areas of Russia in the <jats:italic>Ixodes ricinus</jats:italic> 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.</jats:p></jats:sec>

qPCR System for Alongshan Virus Detection

“…Previously, Miass_gly_3F/Miass_gly_3R oligonucleotide pair (Table 4) was first designed to detect the ALSV strain Miass527 in the IRE/CTVM19 cell culture [14]. It was targeting the VP1a putative envelope protein [11] and amplified a sequence of 333 nt in length.…”

“…The JMV group members, the ALSV strains Miass519 and Miass527 [14] and YGTV strains Plast-T22438 and Bredy-T22181 [26], described previously were used in the current work. Several members of the genus Flavivirus from the institute collection were used: TBEV (strains Sofjin, EK-328, Absettarov), LIV strain S1, POWV strain Pow-24, West Nile virus (WNV) strain SHUA-3, Japanese encephalitis virus (JEV) strain Gagar, and Dengue-4 strain Cambodja.…”

“…The genome of the JMV group members is segmented, and it has proteins with homology to genus Flavivirus polymerase [9] and helicase [9,10], and little homology to envelope protein [9,11]. JMV members include Jingmen tick virus (JMTV) [9], Yanggou tick virus (YGTV) [12], Guaico Culex virus [13], Alongshan virus (ALSV) [14,15], and several other viruses [16,17]. The geographic distribution of the JMV group is very wide, encompassing Europe, Asia, America, and Africa [9,[12][13][14][15][18][19][20].…”

“…JMV members include Jingmen tick virus (JMTV) [9], Yanggou tick virus (YGTV) [12], Guaico Culex virus [13], Alongshan virus (ALSV) [14,15], and several other viruses [16,17]. The geographic distribution of the JMV group is very wide, encompassing Europe, Asia, America, and Africa [9,[12][13][14][15][18][19][20]. JMV group members are considered to be arboviruses.…”

“…ALSV has been detected in Eurasia, including France [25], Germany [24], Finland [19], Switzerland [20], China [15], and several regions across Russia [12,14,26]. In Russia alone, ALSV is circulating across territories where more than ten million people live.…”