Integrated Jingmenvirus Polymerase Gene in Ixodes ricinus Genome

Abstract

Members of the jingmenviruses group have been found in arthropods and mammals on all continents except Australia and Antarctica. Two viruses of this group were isolated from patients with fever after a tick bite. Using a nested RT-PCR assay targeting a jingmenvirus polymerase gene fragment, we screened ticks collected in seven regions of Russia and found that the abundant jingmenvirus-positive were of Ixodes ricinus species, with the prevalence ranging from 19.8% to 34.3%. In all cases, DNase/RNase treatment suggested that the detected molecule was DNA and subsequent next generation sequencing (NGS) proved that the viral polymerase gene was integrated in the I. ricinus genome. The copy number of the integrated polymerase gene was quantified by qPCR relative to the ITS2 gene and estimated as 1.32 copies per cell. At least three different genetic variants of the integrated polymerase gene were found in the territory of Russia. Phylogenetic analysis of the integrated jingmenvirus polymerase gene showed the highest similarity with the sequence of the correspondent gene obtained in Serbia from I. ricinus.

Keywords: Alongshan virus; Ixodes; endogenous viral elements; ixodid ticks; jingmenviruses; tick cell line.

Figure 1

Study design diagram. The flow chart demonstrates different types of treatment and analysis performed to investigate the presence of JMV polymerase gene in ixodid ticks.

Figure 2

European part of Russia with depicted Ixodes ricinus collection sites.

Figure 3

Graphical alignment of a 4005 bp contig of a JMV-positive sample obtained by HiSeq (specimen 264), two cloned samples (id 180 and 288), and a reference sample-segment 1 of Alongshan virus, strain Miass519 (GenBank accession number MN648774). The dark blue boxes represent the coding sequence of the jingmenvirus polymerase gene, the hatched box corresponds to the fragment of the Ixodes scapularis genome (GenBank accession number XM_042293990). The light blue box represents the fragment with an unknown sequence. The horizontal axis shows the length of the nucleotide sequences.

Figure 4

The jingmenvirus polymerase gene copy number estimated by two qPCR assays. Histograms represent the distribution of the obtained copy number values. ITS2 is the internal transcribed spacer of the I. ricinus nuclear genome. Dashed vertical lines show median for each plot. Outliers shown by asterisks were excluded from calculating the median.

Figure 5

Phylogenetic tree of the integrated jingmenvirus polymerase gene (826 aa fragment) in comparison with jingmenviruses. The phylogenetic analyses were inferred by using the maximum likelihood method with 1000 pseudoreplicates. The Akaike information criterion was chosen as a model selection framework and the general time-reversible model as the best model. Maximum likelihood method bootstrap replicates (≥70%) are indicated. The sequences from the Moscow region generated during this study are indicated with an asterisk and blue filled text background. Scale bar indicates the mean number of nucleotide substitutions per site. The filled circles on branches indicate the bootstrap value greater than 0.9.

Figure 6

Phylogenetic analysis of the integrated jingmenvirus polymerase gene based on a 509 bp fragment. The phylogenetic analyses were inferred by using the maximum likelihood method with 1000 pseudoreplicates. The Akaike information criterion was chosen as a model selection framework and the Tamura-Nei model as the best model. Maximum likelihood method bootstrap replicates (≥70%) are indicated by filled circles. The sequences obtained in this study are indicated with asterisks and filled text background. Scale bar indicates the mean number of nucleotide substitutions per site.