Genetic diversity of Kemerovo virus and phylogenetic relationships within the Great Island virus genetic group

Journal Pre-proof GENETIC DIVERSITY OF KEMEROVO VIRUS AND PHYLOGENETIC RELATIONSHIPS WITHIN THE GREAT ISLAND VIRUS GENETIC GROUP Marina V. Safonova, Anatoly P. Gmyl, Alexander N. Lukashev, Anna S. Speranskaya, Alexey D. Neverov, Gennady G. Fedonin, Ekaterina V. Pimkina, Alina D. Matsvay, Kamil F. Khafizov, Galina G. Karganova, Lubov I. Kozlovskaya, Anna V. Valdokhina, Victoria P. Bulanenko, Vladimir G. Dedkov

PII:

S1877-959X(19)30246-8

DOI:

https://doi.org/10.1016/j.ttbdis.2019.101333

Reference:

TTBDIS 101333

To appear in:

Ticks and Tick-borne Diseases

Received Date:

6 June 2019

Revised Date:

29 October 2019

Accepted Date:

15 November 2019

Please cite this article as: Safonova MV, Gmyl AP, Lukashev AN, Speranskaya AS, Neverov AD, Fedonin GG, Pimkina EV, Matsvay AD, Khafizov KF, Karganova GG, Kozlovskaya LI, Valdokhina AV, Bulanenko VP, Dedkov VG, GENETIC DIVERSITY OF KEMEROVO VIRUS AND PHYLOGENETIC RELATIONSHIPS WITHIN THE GREAT ISLAND VIRUS GENETIC GROUP, Ticks and Tick-borne Diseases (2019), doi: https://doi.org/10.1016/j.ttbdis.2019.101333

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GENETIC DIVERSITY OF KEMEROVO VIRUS AND PHYLOGENETIC RELATIONSHIPS WITHIN THE GREAT ISLAND VIRUS GENETIC GROUP Marina V. Safonova1*, Anatoly P. Gmyl2,3 , Alexander N. Lukashev4, Anna S. Speranskaya5, Alexey D. Neverov5, Gennady G. Fedonin5, Ekaterina V. Pimkina5, Alina D. Matsvay5, Kamil F. Khafizov5, Galina G. Karganova2,3, Lubov I. Kozlovskaya2,3, Anna V. Valdokhina5, Victoria P. Bulanenko5, Vladimir G. Dedkov4,6 1

Plague Control Center, Federal Service on Consumers’ Rights Protection and Human Well-Being

Surveillance, Moscow, Russia Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological

Products of Russian Academy of Sciences, Moscow, Russia

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2

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Sechenov First Moscow State Medical University, Moscow, Russia

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Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov

First Moscow State Medical University, Moscow, Russia

Central Research Institute for Epidemiology, Federal Service on Consumers’ Rights Protection

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and Human Well-Being Surveillance, Moscow, Russia

Saint-Petersburg Pasteur Institute, Federal Service on Consumers’ Rights Protection and Human

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*

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Well-Being Surveillance, Saint-Petersburg, Russia

Corresponding author: Marina V. Safonova, 127490, Anti-Plague Center, Musorgskogo

street 4, Moscow, Russian Federation.

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Tel.: +74992029934; fax: +74997452848

Email address: [email protected] (M.V. Safonova)

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Abstract

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group,

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belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae.

Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group. For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 1

and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study. Key words: Kemerovo virus, Orbivirus, Reoviridae, Tribeč virus, Great Island virus genetic group

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Introduction The genus Orbivirus of the family Reoviridae consists of 22 species and ten

nonclassified isolates that are candidate novel species based on their serological and phylogenetic characteristics (Belaganahalli et al., 2015; King et al., 2012). From an

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ecological point of view, all orbiviruses are arboviruses and are capable of infecting a wide range of vertebrate hosts, including wild and domestic animals, bats, birds, and

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humans. In addition, mosquitoes, midges and ticks are known as Orbivirus vectors (Belaganahalli et al., 2015; Karabatsos, 1985; Attoui et al., 2005; Belaganahalli et al.,

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2012; Maan et al., 2012). Traditionally, most attention has been paid to four representatives of the genus Orbivirus that are known to cause economically

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significant diseases of farm animals: bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), African horse sickness virus (AHSV) and equine encephalosis

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virus (EEV) (Attoui et al., 2009). However, the role of orbiviruses in human pathology is still poorly understood.

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The potential pathogenicity of the tick-borne orbiviruses of the Great Island genetic group is of particular interest. Currently, this group, apart from the Great Island virus (GIV) itself, includes Kemerovo virus (KEMV), Tribeč virus (TRBV), Muko virus (MUV), Lipovník virus (LIPV), Broadhaven virus (BRDV) and Nugget virus (NUGV) (King et al., 2012). Some members of this group have been shown to cause febrile illness and neurological disorders (Chumakov et al., 1963; Libikova et al., 1970). However, the medical and social impact of these viruses remains unknown. Studies of the phylogenetic relationships within the GIV genetic group and the 2

intraspecific genetic variability among its members are still limited by the small number of complete genome sequences available for these viruses. KEMV is a GIV genetic group member reported to cause encephalitis in humans (Chumakov et al., 1963; Cherkasskij, 1994). KEMV was first isolated in 1962 by a team of Soviet and Czechoslovak virologists led by M.P. Chumakov (Chumakov et al., 1963). The virus was isolated from Ixodes persulcatus ticks and from the cerebrospinal fluid of a patient with encephalitis that developed after a tick bite. Until recently, KEMV was believed to be prevalent only in Western Siberia (Cherkasskij, 1994). However, studies using molecular methods have shown that the

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area of KEMV circulation is much broader than previously estimated and includes not only Siberia but also the Urals and certain areas in the European part of the

Russian Federation (Dedkov et al., 2014b; Tkachev et al., 2014; Kozlova et al.,

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2018). KEMV has also been found in Dermacentor reticulatus ticks (Dedkov et al., 2014b), Ixodes pavlovskyi ticks (Rar et al., 2017) and Ixodes ricinus ticks (Dedkov et

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al., 2014b), which are known as a source of the closely related TRBV, which is prevalent in Eastern Europe, Moldova and the south of Ukraine (Odessa region)

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(Dedkov et al., 2014a; Belhouchet et al., 2010; Dilcher et al., 2012). Currently, the complete genomes of only two strains of KEMV (HQ266591-HQ266660 (Belhouchet

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et al., 2010) and KC288130-KC288139) and several genome fragments are available for public access. For a more detailed understanding of both the intraspecific diversity of KEMV and the interspecific relationships among viruses in the GIV

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genetic group, it was necessary to sequence more genomes. Here, the genomes of nine additional KEMV strains from Russia were sequenced and

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compared with the previously published sequences of KEMV and other representatives of the GIV genetic group. Materials and methods Virus propagation KEMV strains were obtained from the collection of the Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products (Table 1). Regions of isolation are shown in Figure 1. The strains were propagated in Syrian 3

hamster embryo kidney primary cell culture (BHK-21) or in porcine embryo kidney cell culture (PEK) (Poljanskaya et al., 2014). PEK cell culture is commonly used in Russia for the propagation of arboviruses and is available in Russian cell culture collections. The virus was grown for 48-72 hours at 37°C in the absence of CO2. After propagation, the cell culture medium was used for RNA isolation. RNA extraction The cell culture medium (50 ml) was purified from the cell debris by centrifugation at 10,000 rpm for 30 min at 4°C. The supernatant was further centrifuged at 25,000 rpm

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for 4 hours at 4°C. Then, the supernatant was discarded, and the precipitate of the virus particles was carefully resuspended in 600 μl of Tris-Sodium EDTA buffer (10 mM Tris, 100 mM sodium chloride, 1 mM EDTA, pH 8.0). Then, 10% (w / v)

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sodium dodecyl sulfate was added to a final concentration of 1%. RNA was extracted twice with a mixture of phenol and chloroform (1:1 by volume). Furthermore, the

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aqueous phase was separated, 5 M ammonium acetate was added to a final concentration of 1 M, and three volumes of 96% ethanol were added. The resulting

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mixture was shaken thoroughly and incubated for 1 hour at -80 °C, and the RNA was precipitated by centrifugation.

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The precipitate was washed with 80% ethanol, dried in a desiccator under vacuum and dissolved in RNase-free water (Qiagen, Germany). The quality of the obtained RNA was evaluated by the electrophoretic method using 1% agarose gel with the

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addition of ethidium bromide.

To remove the ballast DNA, 9 μl of the original RNA was treated with 2 units of

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DNase I (Fermentas, Latvia) with the addition of 1.2 μl of 10x reaction buffer (Fermentas, Latvia) at 37°C for 30 min. Inactivation of the DNase was performed by the addition of 25 mM EDTA (Fermentas, Latvia), followed by incubation of the mixture for 10 min at 65°C. The product was purified using a QIAmp MinElute PCR Purification Kit (Qiagen, Germany) according to the manufacturer's protocol. Purified RNA was eluted in 10 μl of RNase-free water (Qiagen, Germany).

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Viral RNA was recovered by precipitation in 2 M lithium chloride. Then, 20 μl of 8 M lithium chloride was added to 5 μl of DNase I-treated purified RNA. The resulting reaction mixture was adjusted to 80 μl by adding RNase-free water (Qiagen, Germany) and incubated on ice for 16 hours. The reaction mixture was then centrifuged for 30 min at 13,000 rpm. Supernatant containing dsRNA was purified using the QIAamp MinElute PCR Purification Kit (Qiagen, Germany) according to the manufacturer's protocol. Purified RNA was eluted in 10 μl of RNase-free water (Qiagen, Germany). Preparation of viral cDNA libraries

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To obtain libraries of viral cDNA, a previously described approach (Maan et al., 2007) was implemented with some modifications, which are schematically shown in Figure 2.

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At the first stage, the loop adapters Reo-Sp-Loop, i.e., "anchor primers", were ligated to RNA with T4 RNA ligase (Fermentas, Latvia). The 20-μl reaction mixture

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contained 10 μl of purified dsRNA, 25 pmol of the Reo-Sp-Loop adapter (5'pGACCTCTGAGGATTCTAAAC_Sp9_TCCAGTTTAGAATCC-3'), 2 μl of 10x T4

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RNA ligase reaction buffer (Fermentas, Latvia), 4 μl of 50% polyethylene glycol (PEG4000, Fermentas, Latvia), and 10 units of T4 RNA ligase (Fermentas, Latvia).

Germany).

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The reaction volume was adjusted to 20 μl using RNase-free water (Qiagen,

The mixture was incubated at 25°C in a thermoshaker at 400 rpm. The ligation

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product was then purified using a QIAamp MinElute PCR Purification Kit (Qiagen, Germany) according to the manufacturer's protocol. RNA was eluted in 10 μl of

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RNase-free water (Qiagen, Germany). Five microliters of dsRNA with ligated adapters was denatured under a layer of mineral oil upon the addition of 3 μl of DMSO (Fermentas, Latvia) at 90°C for 2 min. The samples were then removed from the thermostat and immediately placed on ice. Twelve μl of the reaction mixture consisting of 4 μl of 5x AMV reverse transcriptase buffer (Fermentas, Latvia), 1 μl of 10 mM dNTP mix (Thermo Fisher Scientific Inc., US), 6 μl of RNase-free water (Qiagen, Germany), and 20 units of AMV reverse 5

transcriptase (Fermentas, Latvia) was added under the layer of mineral oil. The reaction was carried out in a thermo shaker at 37°C for 30 min at 580 rpm. Then, an additional 20 units of AMV reverse transcriptase (Fermentas, Latvia) were added, and the mixture was incubated for another 30 min at 42°C. The reverse transcription product was purified using the QIAamp MinElute PCR Purification Kit (Qiagen, Germany) according to the manufacturer's protocol. The resulting first strand cDNA was eluted in 10 μl of RNase-free water (Qiagen, Germany). Completion of the cDNA second strand and amplification were carried out in a onestep mode with the pReo primer complementary to the adapter (5'p-

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GTTTAGAATCCTCAGAGGTC-3') in a 25 μl reaction mixture containing 2 μl of cDNA, 10 pmoles of pReo primer, 2.5 μl of dNTPs (1.76 mM, Amplisens, Russia), 10 μl of PCR mix-2-blue (Amplisens, Russia) that included thermostable DNA

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polymerase. The reaction volume was adjusted to 25 μl using RNase-free water (Amplisens, Russia). The reaction was performed in a Tercyc PCR Thermal Cycler

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(DNA-Technology, Russia) with the following thermal cycling parameters: 95°С-3

cycles; 72°C-3 min.

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min; (95˚С-20 s, 72°С-15 min) x 2 cycles; (95°C-20 s, 50°C-20 s, 72°C-1 min) x 45

The resulting PCR product was purified using a QIAamp MinElute PCR Purification

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Kit (Qiagen, Germany) according to the manufacturer's protocol and eluted in 15 μl of RNase-free water (Qiagen, Germany). The library quality was evaluated by electrophoresis using 1.5% agarose gel with the addition of ethidium bromide.

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Preparation of libraries for high-throughput sequencing was performed using the Nextera XT DNA Library Preparation Kit (Illumina, Inc., USA). The quality of the

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final libraries was assessed using a 2100 Electrophoresis Bioanalyser (Agilent Technologies, Inc., USA). High-throughput sequencing was performed using the MiSeq (Illumina) platform according to the manufacturer’s instructions. In silico analysis Sequences were mapped to the reference genome sequence of KEMV strain 21/10 (GenBank accession numbers KC288130-KC288139).

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Alignments of nucleotide and amino acid sequences were performed in MEGA v.5.2 (Tamura et al., 2011) using the MUSCLE algorithm (Edgar, 2004). Phylogenetic trees were reconstructed using maximum likelihood estimation based on the general time-reversible (GTR) parametric model allowing gamma-distributed frequency variation between sites and a proportion of invariant sites in the sequence (Nei and Kumar, 2001; Tamura et al., 2011). The GTR substitution model evaluated 24 models with various combinations of parameters of nucleotide substitution based on Maximum Likelihood fits and selected the best model of them. Genome sequences of other members within the GIV genetic group were also used for the phylogenetic

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analysis (see Table 2).

The open reading frames (ORFs) were identified using Vector NTI Advance 11.0 software (Invitrogen, USA).

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The nucleotide sequence identities of the complete KEMV genomes and segment 2 of the Great Island group members were calculated using CLC Genomics Workbench

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version 3.6.5 (CLC Bio, Denmark).

A test for probable recombination was performed using the Recombination Detection

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Program (RDP) 4 beta 80 using eight methods provided by the software with the default settings (Martin et al., 2015).

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Results

Sequencing and genetic characterization The complete genomes of 9 strains of KEMV were sequenced and annotated. The

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nucleotide sequences were deposited in NCBI GenBank (Table 1). The genome segment lengths were identical in all strains studied, with the exception

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of segment 5, which encodes the nonstructural protein NS1. An insertion of 1 base pair (bp) was found in the 5' noncoding region of the KEMV 61 strain, and a 1-bp deletion was observed in KEMV strain k10. Thus, the lengths of segment 5 in these strains were 1720 bp and 1718 bp, respectively. Basic genetic features, such as the G+C composition, the length and structure of the terminal fragments in all of the segments, and the nucleotide identity values were highly uniform among all the KEMV genomes studied (data not shown). Individual 7

KEMV segments of distinct isolates had nucleotide identity values in the range of 88.61% to 99.77% (Table 3). The nucleotide sequence identities in the genome segment encoding the VP3 protein (T2) that is commonly used for reovirus identification were compared among the KEMV strains and among members of the Great Island genetic group. The KEMV sequences obtained here shared 94.91 to 99.53% identity with each other and 95.74 to 99.32% identity with the reference strain EgAn-1169. The TRBV strains showed 93.2 to 98.62% nucleotide sequence identity with each other and 74.39 to 76.44% identity

this segment ranged from 81.78 to 83.06% (Table 4).

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with the KEMV strains. The nucleotide sequence identities of MUV and TRBV for

Another genome segment of particular interest was segment 9 which encodes the VP6 helicase. In addition to this ORF, all KEMV strains except for strains 106, k10

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and 101, had an alternative 589 bp ORF in segment 9 encoding a putative VP6a protein. In KEMV strain 101, this alternative reading frame was 33 bp longer (615

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bp) than that of most other strains. In KEMV strains 106 and k10, the alternative segment 9 reading frame encoding the VP6a protein contained the amber stop codon

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TAG and the opal stop codon TGA, respectively, at position 307-309 bp (Figure 3). Interestingly, in strain k10 (but not in strain 106) high-throughput sequencing

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revealed heterogeneity of the viral population with regard to the VP6a stop codon configuration (Table 5): 53.8% of the reads had the opal stop codon TGA at nucleotide positions 489–491, while in 46.1% of the reads, the adenosine at position

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491 was replaced with thymidine. As a result, the opal stop codon was replaced by the tryptophan codon, and a full-fledged 589-bp alternative reading frame, i.e., VP6a,

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appeared (Figure 3) in a large fraction of the virus population. Effect of an alternative ORF on the cytopathic properties of KEMV It has been suggested previously that segment 9 alternative ORFs may play a role in determining the cytopathic properties of KEMV (Attoui et al., 2009). To test this hypothesis, KEMV strain suspensions were inoculated into two cell cultures, BHK21 and PEK (porcine embryo kidney), which were used in this study to replicate the virus for sequencing. The reference strain KEMV EgAn-1169, with the two short 8

alternative reading frames ORF a and ORF b, different from the ORFs found in strains KEMV 106 and KEMV k10, was reportedly grown in green monkey kidney cells Vero E6 (Belhouchet et al., 2010), but unfortunately was not available to us; therefore, the previously reported profile of KEMV EgAn-1169 could not be compared to the KEMV strains studied here. The virus from the original frozen stocks was grown for 48 hours at 37°C. After multiplication, the cytopathic effect was assessed visually. The input virus stock and the cell culture medium after virus replication were used to determine the concentration of RNA using quantitative PCR with RNA calibrators (by modification

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of the method described by Dedkov et al., 2012). When comparing the concentration of viral RNA before inoculation and after 48 hours of propagation, no connection between the replication efficiency of the KEMV strains in the cell cultures and the

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configuration of the VP6a ORF was observed (Figure 6). The heterogeneity of the KEMV strain k10 population was also not related to its replication properties.

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Phylogenetic analysis

Phylogenetic analysis was performed for distinct virus segments (Figure 4) using

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genome sequences of other members of the Great Island group available in NCBI GenBank (Table 2). Multiple reassortment events involving all ten segments were

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found within the KEMV strains clade. Only the grouping of strains 61 and k37, which were 99.53% identical in segment 2 (Table 4), was conserved in all genome segments. Other virus pairs and clusters, which were up to 99.35% identical in

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segment 2, were all reassortant. There were no identical (or, even highly congruent) phylogenetic trees among different genome segments, and thus no evidence of any

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cosegregating segment pairs. Strains from very distant regions (e.g. Vologda and Altai, distance ca. 2700 km) were equally involved in the reassortment. However, strains of TRBV and MUV always grouped separately from the KEMV strains. There was also evidence of reassortment within TRBV, which has been reported previously (Dedkov et al., 2014a). The topology of the tree for segment 10 differed from those for the other segments for the clade containing MUV and TRBV. In particular, the TRBV reference strain 9

(TRBV ref) was moved to the clade formed by MUV (Figure 5). This fact suggests reassortment between these viruses. No within-segment recombination events could be detected among the KEMV strains using RDP 4 software.

Discussion Phylogenetic analysis of all genome segments confirmed that the KEMV found in distant regions of Russia, from the Northwest to East Siberia, formed a well-

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supported clade within the GIV genetic group, which was distinct from TRBV (found in Slovakia, Ukraine and Moldova (Dedkov et al., 2014a, Libikova et al., 1965)) and MUV (isolated and described in Japan by Ejiri et al., 2015).

Among viruses within the GIV genetic group, segment reassortment was previously

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described for TRBV (Dedkov et al., 2014a). Notably, a possible reassortment among KEMV isolates was earlier demonstrated using short genome fragments of the virus

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and the same approach (Tkachev et al., 2017). Our analysis found multiple reliably supported segment reassortment events in KEMV. The ability to exchange genome

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segments is inherent in RNA viruses and is one of the most important sources of genetic variability. Almost all KEMV isolates were reassortant, even viruses that

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differed by as little as 0.65% of the nucleotide sequence. Extrapolating the substitution rates inferred for bluetongue virus (0.5 – 7 x 10-4 substitutions/site/year in different segments (Carpi et al., 2010), this nucleotide sequence distance

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corresponds to 10 – 130 years of circulation. Strains from different regions were

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equally involved in this ubiquitous reassortment. Therefore, the global population of KEMV exists as a highly interconnected gene pool of reassorting genome segments. Common reassortment implies the genetic compatibility of virus segments within KEMV, the regular mixing of virus populations within its distribution area and the common coinfection of either arthropods or vertebrates with diverse virus variants. Some reassortant strains originated from geographically remote regions (Table 1, Figures 1, 4), suggesting common virus trafficking within its areal. This conclusion may have significant practical implications. One of the early KEMV isolates 10

originated from a patient with encephalitis. Despite the widespread prevalence of the virus in tick vectors in Russia (Dedkov et al., 2014b; Kozlova et al., 2018, Tkachev et al., 2014), no incidence of KEMV infection has been recorded officially, and there is no recent data on KEMV pathogenicity for humans. In this regard, it can be assumed that the ability of KEMV to cause encephalitis and febrile illness might have been lost, or has never been a general property of KEMV. It is possible that human cases of Kemerovo fever are misdiagnosed, because specific testing for KEMV is not routinely performed. However, the inherent ability of KEMV to change by reassortment and the ample modification of alternative ORFs may lead to the

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appearance of viruses with novel pathogenic properties, which have the potential to cause disease in humans.

The phylogenetic position of KEMV strain EgAn-1169, which was isolated from a

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redstart (Phoenicurus ochruros) in Egypt in 1961 (Smidt and Shope, 1971), is of particular interest. Its location in the phylogenetic trees within the KEMV clade,

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which includes strains KEMV 205, KEMV 101, KEMV 106, KEMV K10, KEMV 21/10 and KEMV 483 obtained from I. persulcatus and I. ricinus in the Kemerovo,

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Vologda regions and the Altai Territory in the 1970s, suggests that the EgAn-1169 strain could have been introduced to Egypt from Western Siberia during the

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migration of small passerines, since their migration routes pass through these territories (Judin, 2002; Rjabicev, 2008). This observation indicates that migratory birds may provide the mechanism that supports virus trafficking within the global

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KEMV reservoir. It is known that the KEMV virus is not tied to a particular type of tick, and both ornithophilous (Ixodes spp. ticks) and polyphagous ticks (Ixodes spp.

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ticks and D. reticulatus ticks) are among the vectors of KEMV, TRBV and MUV, while birds act as natural feeders for those vectors (Dedkov et al., 2014b; Gresikova et al., 1965; Semashko, 1971; Tkachev et al., 2014; Ejiri et al., 2015). Birds can translocate infected ticks during their natural migration and thus aid the spread of KEMV, TRBV and MUV. In addition, there is evidence that birds can be reservoirs for those viruses (Semashko, 1971 ). This assumption is in good accordance with the data available for other orbiviruses, including members of the GIV genetic group 11

(Belaganahalli et al., 2015; King et al., 2012). Additionally, this transmission route may explain recombination events among viruses isolated thousands of kilometers apart. In accordance with the IX Report of the International Committee on the Taxonomy of Viruses (ICTV), there are several criteria for members of the Orbivirus genus to be recognized as the same species. One criterion is a nucleotide sequence identity of the segment encoding the subcore protein T2 (VP3) above 76% for members of the same species and less than 74% for representatives of different species (King et al., 2012). TRBV and MUV share more than 82% nucleotide identity in the VP3 protein

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sequence. Therefore, according to the nucleotide sequence criteria of species identity (King et al., 2012), they should be attributed to the same species. The close

relationship between MUV and TRBV has been described previously (Ejiri et al.,

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2015). We found that in segment 10, the reference strain of TRBV (TRBV ref)

belonged to the clade of MUV (Figure 5), suggesting segment reassortment between

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MUV and TRBV. The capacity for reassortment can be viewed as an analog of sexual reproduction in higher organisms and an additional species criterion that supports the

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assignment of viruses to the same species (Lukashev, 2010); however more sequences of TRBV and MUV are required to understand their genetic relationships

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and make a conclusion on the frequency of reassortment. According to the nucleotide identity values for the second segment coding the VP3 protein, KEMV and TRBV also belong to the same species. Previously published

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data on the antigenic similarity of KEMV and TRBV (Belhouchet et al., 2010; Gresikova et al., 1965; Semashko, 1971) and the presence of a common vector also

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suggest that KEMV, TRBV and MUV are not separate species but are representatives of the same species (Dedkov et al., 2014b). Therefore, the nucleotide identity of these viruses, currently considered to be independent representatives of the Great Island group, suggests that they may be a single polymorphic species widely distributed in Eurasia from Eastern Europe (TRBV) through the territory of Russia and the Far East (KEMV) to Japan (MUV). On the other hand, evidence of common reassortment was found within KEMV and within TRBV, but not between them, suggesting the 12

assignment of these viruses to distinct species. Therefore, the final conclusion on the taxonomic affiliation of KEMV and TRBV requires the sequencing of more strains to characterize the feasibility and frequency of natural reassortment. The role of the KEMV alternative ORF in segment 9 (VP6a protein) in determining cell tropism and pathogenicity in mammals was suggested previously (Attoui et al., 2009). Alternative reading frames in segment 9 have also been described in other representatives of the GIV genetic group (Belhouchet et al., 2010), but their function is not yet fully understood. Distinct VP6a stop codons in two KEMV strains and two different types of VP6a

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alternative reading frames in the KEMV strain k10 RNA population are compatible with a hypothetical role of VP6a as a dynamic switch that controls virus replication properties. A similar phenomenon has been described in reoviruses, when an opal

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stop codon (TGA) in segments encoding regulatory proteins can be read-through (Napthine et al., 2012). It is not obvious how exactly this adaptation could work

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mechanistically, as a stop codon does not necessarily imply the termination of translation. As a result, clones with the TGA opal stop codon in alternative reading

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frame VP6a can produce a full-length VP6a protein. It should be noted that a similar modulation of virulence by a read-through stop codon has been described in the

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Semliki Forest virus (genus Alphavirus) (Lancotti et al., 1998; Tuittila et al., 2000). Additionally, the opal stop codon is present in the O'Nyong-Nyong virus and can be replaced with an amino acid during several passages in cell cultures (Lancotti et al.,

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1998 ). Thus, read-through and emerging/disappearing stop codons are not uncommon in RNA viruses, and the VP6a stop codon could in fact be a read-through

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codon. No evidence of the role of VP6a ORF in modulating virulence could be observed in this study; however, the experimental setup was basic, and further studies are suggested to test this hypothesis.

Conclusions Full genome sequences of 9 KEMV strains were characterized, enriching knowledge of the genetic diversity of this virus and its relationship with closely related viruses 13

of the Great Island genetic group. For the first time, multiple reassortment events within KEMV were demonstrated. A high level of nucleotide similarity among KEMV, TRBV and MUV was shown, suggesting close common ancestry and potential affiliation with the same species. However, a more detailed understanding of the complex genetic relationship of viruses within this genetic group is required for a reliable conclusion, and will require the production of more genome sequences of modern strains of KEMV and TRBV and further research on the ecological aspects of the circulation of these viruses. A wide distribution area covering most of the Eurasian continent, high genetic flexibility, the capacity to use various vectors

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and the ability to move over long distances, which is likely assisted by bird

migration, justify further studies of KEMV as a potential source of new and

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recurring infections that may become epidemiologically significant in the future.

Competing interests

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The authors declare that they have no competing interests

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Foundation

The reported study was funded by RFBR according to the research project N. 18-31-

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00001 and RSF grant N. 17-74-20096.

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Belaganahalli M.N., Maan S., Maan N.S., Brownlie J., Tesh R., Attoui H., Mertens P., 2015. Genetic characterization of the tick-borne orbiviruses. Viruses.;7:2185-209. doi: 10.3390/v7052185

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Belhouchet M., Mohd Jaafar F., Tesh R., Grimes J., Maan S., Mertens P. P. C., Attoui H., 2010. Complete sequence of Great Island virus and comparison

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with the T2 and outer-capsid proteins of Kemerovo, Lipovník and Tribeč viruses (genus Orbivirus, family Reoviridae). J. Gen. Virol.; 91:2985–2993

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doi: 10.1099/vir.0.024760-0 Carpi G., Holmes E.C., Kitchen, A., 2010. The Evolutionary Dynamics of Bluetongue Virus. J. Mol. Evol.; 70:583–592. doi:10.1007/s00239-010-9354-y

Cherkasskij B.L., 1994. Infectious and parasitic diseases of human. Handbook of epidemiology. Moscow: Medicinskaja gazeta.; 617p. (in Russian) Chumakov M.P., 1963. Report on Isolation from Ixodes persulcatus ticks and from patients in Western Siberia of a virus differing from agent of tick-borne encephalitis. Acta Virol.;7:82–83 15

Dedkov,V.G., Markelov,M.L., Voitsekhovskaya,Ya.A., Karganova,G.G., Gmyl,A.P., Kozlovskaya, L.I.,Pivanova, G.P., Shipulin,G.A., 2012. Development of a real-time PCR-based test system to detect Kemerovo virus RNA. J.Epidemiol.Infect.Dis.Curr.Items., 3:38-41 (in Russian) Dedkov V. G., Dubina D. A., Yurchenko O. A., Bekova M. V., Valdokhina A. V., Shipulin, G. A., 2014. Characterization of two strains of Tribeč virus isolated in Ukraine. Vector Borne Zoonotic Dis.; 14:808-16 doi: 10.1089/vbz.2014.1683 Dedkov V.G., Markelov M.L., Gridneva K.A., Bekova M.V., Gmyl A.P.,

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Edgar R.C., 2001. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res.; 32:1792-7. doi: 10.1093/nar/gkh340 Ejiri H., Lim C.K., Isawa H., Kuwata R., Kobayashi D., Yamaguchi Y.,

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Sawabe K., 2015. Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan. Arch Virol.; 160:2965-77. doi: 10.1007/s00705-015-2588-729.

Shmidt J.R., Shope, R.E., 1971. Kemerovo virus from a migrating common redstart of Eurasia. Acta Virol.;15:112

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Gresikova M., Nosek J., Kozuch O., Ernek E., Lichard M., 1965. Study on the ecology of Tribeč virus. Acta Virol.;9:83–88. Judin K.A., 2002. Fauna of Russia and adjacent countries. Birds. T II, vyp.2. S-P: Nauka; 667p (in Russian) Karabatsos N., 1985. International Catalogue of Arboviruses Including Certain Other Viruses of Vertebrates, 3rd ed. American Society of Tropical Medicine and Hygiene: San Antonio, TX, USA. King A., Adams M.J., Carstens E.B., Lefkowitz E.J., 2012. Virus Taxonomy, Classification and Nomenclature of Viruses. Ninth Report of the International

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characterization of the virus. Virology.; 252:258-68. doi: 10.1006/viro.1998.9437

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Libikova H, Rehacek J, Somogyiova J., 1965. Viruses related to Kemerovo virus in Ixodes ricinus ticks in Czechoslovakia. Acta Virol.; 9:76–82

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Characterization of the stop codon read through signal of Colorado tick fever

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Poljanskaya G.G., Sakuta G.A., Eropkin M.Ju., Smirnova T.D., Podchernaeva

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Rjabicev V.K., 2008. Birds of the Urals and Western Siberia. Ekaterinburg: Izd-vo Ural;2008. 634p (in Russian) Semashko, I.V., 1971. Comparative Investigation of the Kemerovo Serological GroupMembers (Ph.D. Thesis) Moscow (in Russian) 18

Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol.; 28:2731-2739. doi: 10.1093/molbev/msr121 Tkachev S., Panov V., Dobler G., Tikunova N., 2014. First detection of Kemerovo virus in Ixodes pavlovskyi and Ixodes persulcatus ticks collected in Novosibirsk region, Russia. Ticks Tick Borne Dis.; 5:494-6. doi: 10.1016/j.ttbdis.2014.03.003 Tkachev, S.E., Tikunov, A.Y., Babkin, I.V., Livanova, N.N., Livanov, S.G.,

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Tuittila M.T., Santagati M.G., Röyttä M., Määttä J.A., Hinkkanen A.E., 2000.

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J Virol.; 74:4579-89.

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Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

19

Table 1. Description of KEMV strains Strain

Isolation source

Region

Time

References

1

61

I. persulcatus

Altai region

1973

MF939485 – MF939494

2

37

I. persulcatus

Altai region

1973

MF939545 – MF939554

3

101

I. persulcatus

Kemerovo region

1970

MF939495 – MF939504

4

5/1

I. persulcatus

Kemerovo region

1969

MF939475 – MF939484

5

L75

Human

Kemerovo region

1962

MF939555 – MF939564

6

205

I. persulcatus

Vologda region

1974

7

483

I. ricinus

Vologda region

1975

8

106

I. persulcatus

Altai region

9

k10

I. persulcatus

Kemerovo region

ro of

№

MF939515 – MF939524

-p

MF939525 – MF939534

re

1973

MF939535 – MF939544

Jo

ur

na

lP

1966

MF939505 – MF939514

20

Table 2. GenBank sequences used for phylogenetic analysis Virus

Acronym

Strain

References

1

Kemerovo virus

KEMV

21/10

KC288130 – KC288139

2

Kemerovo virus

KEMV

EgAn-1169

HQ266591 – HQ266600

3

Great Island virus

GIV

CanAr 42

NC_014522 – NC_014531

4

Tribeč virus

TRBV

Tr19

KJ010789 – KJ010797; KJ574045

5

Tribeč virus

TRBV

Tr35

KJ010798 – KJ010806; KJ574044

6

Tribeč virus

TRBV

-

HQ266581 – HQ266590

8

Muko virus

MUV

MUV-hay

LC158842 – LC158851

9

Muko virus

MUV

Ix7-S1

LC019131 –LC019140

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№

Table 3 Nucleotide identities of all known KEMV genomes by segments*

Range of nucleotide identity values, %

Protein

Function

1

VP1 (Pol)

RNA-dependent RNA polymerase

99,69 – 94,61

2

VP3 (T2)

Forms the subcore layer of the capsid

99,53 – 94,91

3

VP4 (Cap)

Capturing enzyme

4

VP 2

Forms the outer layer of the particle, ensures the penetration of the virus into the host cell, is a serotype specific antigen

99,77 – 88,61

5

NS1 (TuP)

The nonstructural protein forms tubular structures in the cytoplasm of the host cell

99,36 – 95,76

6

VP5

7

NS2 (ViP)

10

re

lP

na

VP6 (Hel)

Jo

9

VP7 (T13)

NS3

99,59 – 90,12

Forms the outer capsid

99,58 – 95,14

Unstructured protein forms viral inclusion bodies

99,67 – 92,57

ur

8

-p

Segment №

Forms the outer core layer Helicase Unstructured proteins involved in the release of viral particles from the host cell

99,41 – 92,81 99,33 – 92,47 99,58 – 92,64

*The correspondence between the segments of the KEMV genome and the encoded viral proteins are given according to (Belaganahalli et al., 2015).

21

Table 4 Nucleotide sequence identity of the structural subcore protein VP3 (T2) among the Great Island group viruses (in %) KEMV 101

KEMV 5/1

KEMV L75

KEMV 205

KEMV 106

KEMV k10

KEMV k37

KEMV 61

KEMV 21/10

KEMV 483

TRBV 35

TRBV 19

TRBV ref

MUV Ix7-S1

MUV MUVhay

№

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

KEMV EgAr

1

–

–

–

–

–

–

–

–

KEMV 101

2

99,35

–

–

–

–

–

–

–

3

98,50

98,46

–

–

–

–

–

–

KEMV L75

4

96,92

96,96

96,60

–

–

–

–

KEMV 205

5

97,10

97,21

96,81

96,81

–

–

KEMV 106

6

96,74

96,67

96,24

96,28

96,56

–

KEMV k10

7

96,81

96,74

96,45

96,42

KEMV k37

8

96,60

96,56

96,13

96,24

KEMV 61

9

96,45

96,45

96,02

96,13

KEMV 21/10

10

95,67

95,52

KEMV 483

11

95,74

95,74

TRBV 35

12

75,11

75,04

TRBV 19

13

TRBV ref

14

MUV Ix7-S1

15

MUV MUV-hay

16

GIV CanAr 42

17

oo

–

e-

5/1

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

pr

KEMV

f

KEMV EgAr

Virus, strain

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

Pr

–

98,93

–

–

–

–

–

–

–

–

–

–

96,63

98,96

98,96

–

–

–

–

–

–

–

–

–

96,49

98,78

98,78

99,53

–

–

–

–

–

–

–

–

na l

96,70

95,63

95,74

95,52

95,67

95,59

95,52

–

–

–

–

–

–

–

95,31

95,20

95,74

95,63

95,70

95,52

95,49

94,91

–

–

–

–

–

–

74,71

74,43

74,50

74,79

74,75

74,61

74,68

75,07

74,64

–

–

–

–

–

Jo ur

95,24

75,11

75,04

74,71

74,50

74,61

74,79

74,75

74,68

74,68

75,07

74,64

98,62

–

–

–

–

76,37

76,33

76,01

76,26

76,08

76,15

75,90

75,90

75,94

76,41

75,94

93,20

93,20

–

–

–

75,94

75,90

76,01

75,98

75,47

75,76

75,73

75,69

75,80

75,22

75,30

81,85

82,03

82,78

–

–

75,90

75,87

75,83

76,08

75,58

75,83

75,65

75,76

75,94

75,55

75,47

81,78

82,03

82,81

98,14

–

73,19

73,05

72,80

72,48

72,87

72,80

72,98

72,80

72,80

72,80

72,58

71,58

71,76

72,33

71,80

71,65

22

Table 5. Heterogeneity in the KEMV strain k10 genome (MF939535 – MF939544).

5

6 7 8

9

10

E

64.71%

35.29%

Nonsynonymous substitution

E

88.7%

11.3%

Synonymous substitution

69.72%

30.28%

Reads alteration

557

G

A

1316

1133

2709

C

A

51

33

18

A

60

G

A

1531

1358

173

E

1737

C

CA

327

228

99

1740

C

G

555

253

302

92

A

G

4313

3715

598

101

T

C

2668

1812

856

1692

T

C

2433

2148

379

C

T

4714

1364

Nonsynonymous substitution

D

E

45.59%

54.41%

K

K

86.13%

13.87%

Synonymous substitution

I

I

67.92%

32.08%

Synonymous substitution

285

-

-

88.29%

11.71%

Noncoding region

3350

I

I

28.94%

71.06%

Synonymous substitution

765

E

K

76.95%

23.05%

Nonsynonymous substitution

2623

341

Q

Q

88.5%

11.5%

Synonymous substitution

1908

235

R

R

89.03%

10.97%

Synonymous substitution

908

383

C

R

70.33%

29.67%

Nonsynonymous substitution

pr

L

Notes

Synonymous substitution; Shift of ORF Nonsynonymous substitution

2554

L

oo

Reads reference

f

% of alteration reads 13.91%

All reads

e-

3

% of reference reads 86.09%

Alteration nucleotide

542

G

A

3319

1513

A

G

2964

736

C

A

2143

340

T

C

1291

851

G

A

2836

2522

314

G

D

88.93%

11.07%

Nonsynonymous substitution

908

A

G

2374

1444

930

H

R

60.83%

39.17%

Nonsynonymous substitution

297

T

C

3513

2731

782

W

R

77.74%

22.26%

Nonsynonymous substitution

491

A

G

879

473

406

W

53.81%

46.19%

522

G

A

890

657

233

Opal stop codon D

N

73.82%

26.18%

Nonsynonymous substitution

100

A

G

3841

2934

907

S

G

76.39%

23.61%

Nonsynonymous substitution

Pr

2

183

Alteration amino acid I

Reference nucleotide

na l

1

Reference amino acid V

Nucleotide position

Jo ur

Segment

Read-through opal stop codon

23

na

lP

re

-p

ro of

Figure legends Figure 1. Isolation regions of the KEMV strains and other representatives of the GIV genetic group viruses.

Jo

ur

Figure 2. Principal scheme of the method for dsRNA nonspecific amplification

24

25

ro of

-p

re

lP

na

ur

Jo

26

ro of

-p

re

lP

na

ur

Jo

Jo

ur

na

lP

re

-p

ro of

Figure 3. Map of KEMV genome segment 9 with the locations of ORFs

27

28

ro of

-p

re

lP

na

ur

Jo

Figure 4. Phylogenetic trees of the Great Island group viruses. The phylogenetic

Jo

ur

na

lP

re

-p

ro of

analysis of nucleotide sequences was performed by maximum likelihood estimation using the GTR parametric model allowing gamma distribution of the frequency variation between sites and a proportion of invariant sites with MEGA software version 5.2. Trees were rooted by the genome of the Great Island virus (GIV, strain CanAr 42; NC_014522-NC_014531) (not shown in the figure). The reliability of the topology was estimated using 1000 bootstraps pseudoreplicates. The locations of KEMV strains are marked by colored dots; topology incongruences between adjacent phylogenetic trees are indicated by continuous and dotted lines. KEMV, Kemerovo virus; TRBV, Tribeč virus; MUV, Muko virus.

Figure 5. Phylogenetic tree of the Tribeč virus (TRBV) and Muko virus (MUV) built on the basis of nucleotide sequences. The phylogenetic analysis was carried out by maximum likelihood estimation using the GTR parametric model allowing the gamma distribution of the frequency variation between sites and a proportion of invariant sites with MEGA software version 5.2. Trees were rooted by the genome of the Great Island virus (GIV, CanAr 42 strain, NC_014522-NC_014531). The reliability of the topology was estimated using 1000 bootstrap pseudoreplicates. The positions of the TRBV and MUV strains on the 29

Jo

ur

na

lP

re

-p

ro of

phylogenetic trees are marked by dots, phylogenetic incongruences among the Tribeč virus strains are indicated by dotted lines, and phylogenetic incongruences that included TRBV Ref virus strain and the Muko virus are indicated by a red solid line. TRBV, Tribeč virus; MUV, Muko virus; GIV, Great Island virus.

30

31

ro of

-p

re

lP

na

ur

Jo

Jo

ur

na

lP

re

-p

ro of

Figure 6. Concentrations of viral RNA before inoculation and after 48-hours of growth in various cell cultures.

32