Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Abstract

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Fig. 1. conserved motifs in nonstructural (NS)3 of Mogiana tick virus (MGTV). Multiple sequence alignment of NS3 from MGTV and representative of tick-borne encephalitis virus (TBEV), mosquito-borne [dengue virus type 2 (DENV-2) and yellow fever virus (YFV)], insect-only [cell fusing agent virus (CFAV)] and not known vector [Apoi virus (APOIV) and Tamana bat virus (TABV)] flavivirus groups. For better visualisation, only blocks of conserved regions along the alignment are displayed. Motifs are delimited accordingly. A: N-terminal regions of NS3 contain a serine protease, amino acids (aa) of the catalytic triad of which are highlighted; B: C-terminus portion of NS3 contain a helicase/nucleotidases (NTPases) where seven conserved motifs (I, Ia and II-VI) are found. Numbers at the bottom of the alignments refer to MGTV sequence. The threshold for shading colours of aa similarity was 50%.

Fig. 2. conserved motifs in nonstructural (NS)5 of Mogiana tick virus (MGTV). Multiple sequence alignment of NS5 from MGTV and representative of tick-borne encephalitis virus (TBEV), mosquito-borne [dengue virus type 2 (DENV-2) and yellow fever virus (YFV)], insect-only [cell fusing agent virus (CFAV)] and no known vector [Apoi virus (APOIV) and Tamana bat virus (TABV)] flavivirus groups. To better visualisation, only blocks of conserved regions along the alignment are displayed. Motifs are accordingly delimited. A: N-terminal region of NS5 presents methyltransferase activity; two conserved motifs are found (I and II); B: C-terminus portion of NS5 contains an RNA-dependent RNA polymerase; four conserved motifs (A-D) are found; asterisk indicates the conserved aspartate residues important for enzyme activity. Numbers at the bottom of the alignments refer to MGTV sequences. The threshold for shading colours of amino acid similarity was 50%.

Fig. 3. phylogenetic analysis of nonstructural (NS)3 and NS5 sequences of Mogiana tick virus (MGTV) virus within the Flaviviridae family. Maximum likelihood analyses were performed with 1,000 bootstrap replicates. The evolutionary distances were computed using the JTT matrix-based method. A: NS3 tree; B: NS5 tree. Virus groups were condensed for better visualisation and all viruses used in trees construction are listed in Supplementary data (Table SIII). The bar at the bottom indicates 50% amino acid divergence. GBV: GB virus; HCV: hepatitis C virus; TABV: Tamana bat virus (TABV).

Fig. 4. hydropathy profile. Hydropathy values were determinate by Kyte and Doolittle scale using ProstScale web-based tool. Plot of nonstructural (NS)3 (A) and NS5 (B) of Mogiana tick virus (MGTV). Both are compared with dengue virus type 2 (DENV-2) proteins. Negative scores mean a hydrophilic residue. Dashed line is the upper threshold for hydrophilicity.

Fig. 5. codon usage pattern in Mogiana tick virus (MGTV) sequences. Codon bias in MGTV sequences were analysed with CAIcal Server. Codon usage adaptation index (CAI) calculation used Flavivirus sp. codon usage table as reference. The values were normalised with the expected CAI value (eCAI) for codon usage pattern in Flavivirus, calculated with input sequences. The dashed line indicates the threshold for which equal or higher values are interpreted as a codon usage adaptation towards Flavivirus codon usage pattern. APOIV: Apoi virus; CFAV: cell fusing agent virus; CxFV: Culex flavivirus; DENV: dengue virus; NKV: no known vector; NS: nonstructural; RBV: Rio Bravo virus; YFV: yellow fever virus.

Fig. 6. molecular detection of viral transcripts in total RNA of tick samples collected in farms from seven Brazilian states. Detections were done through reverse-transcription-polymerase chain reaction using primers that target the 317 contig sequence [nonstructural (NS)3 fragment] or primers that target the 401 contig sequence (NS5 fragment). A: tick samples were collected from seven Brazilian states: Mato Grosso do Sul (MS) and Goiás (GO) (Central-West Region), São Paulo (SP), Minas Gerais (MG) and Rio de Janeiro (RJ) (Southeast Region), Paraná (PR) and Rio Grande do Sul (RS) (Southeast Region); B: panel for molecular detection linking farms to results for primers 317 (light grey), 401 (medium grey), both (dark grey) or no detection (ND) using any of the primers. Life stages of ticks: EF: engorged female (F); F _<4 : F less than 4 mm (before the rapid engorgement phase of feeding); M: male; UL: unfed larvae; *: samples from farm X (Ribeirão Preto, SP) were the only ones that presented positive detection when 2,743 primers were tested. Other Brazilian states: AC: Acre; AL: Alagoas; AP: Amapá; AM: Amazonas; BA: Bahia; CE: Ceará; DF: Distrito Federal; ES: Espírito Santo; MA: Maranhão; MT: Mato Grosso; PA: Pará; PB: Paraíba; PE: Pernambuco; PI: Piauí; RN: Rio Grande do Norte; RO: Rondônia; RR: Roraima; SC: Santa Catarina; SE: Sergipe; TO: Tocantins;

Fig. 7. phylogenetic trees of nonstructural (NS)3/NS5 sequences from positive tick samples. Sequences are named according to detection primers (317 or 401) followed by tick life stage [UL: unfed larvae; EF: engorged female (F) less than 4 mm; M: male] and the farm alphabetic label (see Table I for corresponding location). A: 36 sequences positive for detection using primers 317 (NS3 fragment) plus original contig sequence from the complementary DNA (cDNA) library (named ContLib highlighted in closed circle); B: 21 sequences positive for detection using primers 401 (NS5 fragment) plus original contig sequence from the cDNA library (named ContLib highlighted in closed circle); I-V: branches composed of samples from same farm. Neighbour-joining analysis was performed with 1,000 bootstrap replicates. The bar at the bottom indicates 1% nucleotide substitution.

Fig. S1. molecular detection of Mogiana tick virus (MGTV) partial sequences in tick samples from different farms located in Mogiana region (Line: 1 Kb plus DNA ladder; C-: negative control). A: tick samples from a farm located in Araguari, state of Minas Gerais (1: larvae; 2: male; 3: female < 4 mm; 4: engorged female); B: tick samples from a farm located in Ribeirão Preto, state of São Paulo. Primers: actin, 317 [nonstructural (NS)3], 401 and 2,743 (NS5). Genomic DNA (gDNA) and RNA [represented by complementary DNA (cDNA)] from ticks were analysed. [1: pool of tick 1; 2: pool of tick 2; 3: pool of tick 4; 4: pool of tick 8 (all from females); 5: pool from larvae].

Fig. S2. detection of Mogiana tick virus in mammalian and arthropod cell cultures. Two percent agarose gel electrophoresis demonstrating the products generated by nonstructural 5 amplification (primer set 401-5-65 and 401-3-349) in baby hamster kidney (BHK), Vero (monkey), Boophilus microplus cattle tick (BME) 26 and C6/36 (mosquito) cell lines. Lane L: 100 bp DNA ladder; C-: reverse-transcription-polymerase chain reaction (RT-PCR) negative control; C+: RT-PCR positive control; 1: isolates from pool 1; 2: isolates from pool 2; 3: isolates from pool 4; 4: isolates from pool 8; 5: isolates from larval pool; 6: mock infected cells.

Fig. S3. molecular detection of purified Mogiana tick virus after ultracentrifugation of culture supernatant from second passage in Vero cells. Two percent agarose gel electrophoresis demonstrating the amplicons generated by nonstructural 5 amplification (primer set 401-5-65 and 401-3-349) in virus purified from Vero cells. Lane L: 100 bp DNA ladder; C-: reverse-transcription-polymerase chain reaction (RT-PCR) negative control; 1: purified pool 4 isolate; 2: purified pool 4 isolate treated with RNAse and DNAse (which was used in deep sequencing); 3: pool 4 isolate in culture supernatant before ultracentrifugation; 4: purified pool 1 isolate treated with RNAse and DNAse; 5: pool 1 isolate from culture supernatant before ultracentrifugation.

Fig. S4. multiple sequence alignment of nonstructural 3 from Mogiana tick virus and representative of tick-borne encephalitis virus (TBEV), mosquito-borne [dengue virus type 2 (DENV-2) and yellow fever virus (YFV)], insect-only [cell fusing agent virus (CFAV)] and not known vector [Apoi virus (APOIV) and Tamana bat virus (TABV)] flavivirus groups. Conserved residues and motifs are indicated by narrows and curly brackets, respectively.

Fig. S5. multiple sequence alignment of nonstructural 5 from Mogiana tick virus and representative of tick-borne encephalitis virus (TBEV), mosquito-borne [dengue virus type 2 (DENV-2) and yellow fever virus (YFV)], insect-only [cell fusing agent virus (CFAV)] and not known vector [Apoi virus (APOIV) and Tamana bat virus (TABV)] flavivirus groups. Conserved motifs are indicated by curly brackets.

Fig. S7. supplemental results for codon usage session.