Signature patterns in region V4 of small ruminant lentivirus surface protein in sheep and goats

Journal Pre-proof Signature patterns in region V4 of small ruminant lentivirus surface protein in sheep and goats ´ ´ ´ Tellez, ´ Ana Silvia Gonzalez Mendez, Fernando Ceron Jorge Luis ´ ´ Tortora Perez, Humberto Alejandro Mart´ınez Rodr´ıguez, Mar´ıa ´ Martha Garc´ıa Flores, Hugo Ram´ırez Alvarez

PII:

S0168-1702(19)30688-4

DOI:

https://doi.org/10.1016/j.virusres.2020.197900

Reference:

VIRUS 197900

To appear in:

Virus Research

Received Date:

24 September 2019

Revised Date:

25 January 2020

Accepted Date:

14 February 2020

´ ´ ´ Please cite this article as: Mendez ASG, Tellez FC, Perez JLT, Rodr´ıguez HAM, Flores MMG, ´ Alvarez HR, Signature patterns in region V4 of small ruminant lentivirus surface protein in sheep and goats, Virus Research (2020), doi: https://doi.org/10.1016/j.virusres.2020.197900

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

SIGNATURE PATTERNS IN REGION V4 OF SMALL RUMINANT LENTIVIRUS SURFACE PROTEIN IN SHEEP AND GOATS Ana Silvia González Méndeza, Fernando Cerón Télleza, Jorge Luis Tórtora Péreza, Humberto Alejandro Martínez Rodrígueza, María Martha García Floresb, Hugo Ramírez Álvareza*.

Affiliations a

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Virology, Genetics and Molecular Biology Laboratory. Faculty of Higher Education,

Cuautitlan, Veterinary Medicine, Campus 4. National Autonomous University of Mexico.

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Km. 2.5 Carretera Cuautitlán-Teoloyucan San Sebastián Xhala. Cuautitlan Izcalli Estado de

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México. C.P. 54714. Mexico.

Laboratory of Immunovirology, Medical Research in Immunology Unit, Pediatric Hospital,

Corresponding author: telephone: 00 +52 55 56 23 19 20

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*

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National Medical Center XXI Century, Mexican Institute of Social Security

E-mail address: [email protected] (Hugo Ramírez Álvarez).

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[email protected] (Ana Silvia González Méndez), [email protected] (Fernando Cerón Téllez), [email protected] (Jorge Luis Tórtora Pérez), [email protected] (Humberto Alejandro Martínez Rodríguez), [email protected] (María

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Martha García Flores)

Highlights  We identified “signature patterns” in the V4 region of the SRLV surface protein in sheep and goats infected with genotypes A and B.



The signature pattern identified in residue 54 was associated with different clinical status (arthritis or asymptomatic) in sheep and goats.



First report of the identification of genotype A of SRLV infecting sheep and goats from Mexico.

Abstract The env gene in Small Ruminant Lentiviruses (SRLV) encodes the surface glycoprotein (SU) that divides into conserved (C1-C4) and variable regions (V1-V5). SRLV region V4 has been found to be homologous to the V3 region of human lentivirus (HIV). HIV V3 is responsible for tropism and the development of nervous clinical patterns when there is a tendency to

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conserve amino acids in specific “signature pattern” positions. The goal of this study was to identify signature patterns in the V4 region of the SU, which is encoded by the SRLV env

clinical status in naturally infected sheep and goats.

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gene. Secondarily, to understand how these signature patterns are associated with different

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Starting with 244 samples from seropositive animals from nine Mexican states, we amplified the V4 region using nested PCR and obtained 49 SRLV sequences from peripheral blood leukocytes. Based on phylogenetic analysis results, we identified three groups: asymptomatic

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genotypes A (Ssx GA) and B (Ssx GB), as well as animals with arthritic presentation, genotype B (A GB). Similarity levels between group sequences ranged from 67.9% to 86.7%,

negative selection.

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with a genetic diversity ranging from 12.7% to 29.5% and a dN / dS ratio that indicated

Analyses using Vespa and Entropy programs identified four residues at positions 54, 78, 79

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and 82 in SU region V4 as possible signature patterns, although with variable statistical significance. However, position 54 residues “N” (p = 0.017), "T" (p = 0.001) and "G" (p = 0.024) in groups A GB, Ssx GA and Ssx GB respectively, best characterized the signature patterns. The results obtained identified a signature pattern related to different genotypes and

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clinical status by SRLV in sheep and goats.

Keywords: SRLV, env gen, V4 region, Arthritis, Asymptomatic, Mexico

1 Introduction Small ruminant lentiviruses (SRLV) which infect sheep and goats (Greenlandb et al., 2004) belong to the order Ortervirales, family Retroviridae, subfamily Orthoretrovirinae and the genus Lentivirus (King et al., 2017). Phylogenetic studies have grouped them into five

different genotypes (A-E) and into several subtypes (Workman et al., 2017). The SRLV genome is flanked by the LTR region, and contains regulatory genes (vpr-like, vif and rev) as well as structural genes (gag, pol, env). The env gene codes for transmembrane (TM) and surface (SU) proteins; the SU protein contains four conserved regions (C1-C4) and five variable regions (V1-V5; Valas et al., 2000). It has been suggested that the V4 variable region of SRLVs may have a function analogous to that of V3 in Human Immunodeficiency Virus1 (HIV-1; Pisoni et al., 2007), for which the epitopes responsible for cell infection have been identified, in addition to being related to the production of neutralizing antibodies (Skraban

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et al., 1999).

SRLV tropism has been linked both to host genetics and to the heterogeneity of the viral

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genome (Ramírez et al., 2013). Both elements may or may not be associated with the development of nervous, respiratory, mammary and arthritic clinical presentations. Arthritis

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is the most frequent presentation in goats in Mexico, and there are no reports of clinical development in infected sheep.

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Previous studies on other lentivirus indicate a tendency for amino acids to be conserved at specific positions in certain viral proteins and form hairpins or loops; these are referred to as

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“signature patterns” (Korber and Myers, 1992; Korber et al., 1993). These patterns, in turn, are associated with viral genetic patterns and specific clinical presentations, such as studies of signature patterns in the V3 region of surface proteins of HIV in brain tissue linked to

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monocytotropic strains isolated in vitro (Korber et al., 1994). In Feline Immunodeficiency Lentivirus (FIV), changes described in V3 and V4 regions of surface proteins, are associated with neurotropism and neurovirulence (Pinghuang et al., 2006), while signature patterns found in the V1, V2 and V3 region of simian immunodeficiency virus (SIV) have been associated with disease progression (Dehghani et al., 2003).

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The presence of signature patterns and their relationship to viral genotypes and clinical and/or asymptomatic presentations in sheep and goats has not yet been identified. Thus, the objective of our study is to find whether there are signature patterns in the V4 region of SRLV surface proteins and whether these are linked to various clinical status in naturally infected sheep and goats. We identified signature patterns in both asymptomatic animals and those presenting arthritis signs, and detected genotypes A and B infecting both sheep and goats from different herds in Mexico.

2 Materials and methods 2.1 Study animals We collected 244 blood samples (139 and 105 from goats and sheep respectively) through non-probabilistic sampling in cooperative herd animals. Animals in the study group were above two years of age, and were chosen regardless of breed or sex. We obtained samples from nine states throughout Mexico: Baja California Sur (BCS) n = 12, Coahuila (Coah) n = 41, Estado de México (EdoMex) n = 75, Hidalgo (Hgo) n = 9, Guanajuato (Gto) n = 34,

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Querétaro (Qro) n = 15, Sinaloa (Sin) n = 5, Sonora (Son) n = 33 and Veracruz (Ver) n = 20. The study received a permit (code MC-2017 / 1-6-UNAM) from the Internal Committee on

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Animal Use and Experimentation of the National Autonomous University of Mexico,

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through the Graduate Program in animal production and health sciences.

2.2 Obtaining PBLs and plasma

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We collected blood samples through jugular vein puncture into tubes with anticoagulant (BD Vacutainer® with Lithium Heparin, México). Samples were centrifuged at 350g for 15

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minutes for phase separation, plasma and peripheral blood leukocytes (PBLs). Plasma was collected in microtubes and we then processed the white layer using lysis solutions (Mendiola

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et al., 2019) to obtain PBLs. Samples were stored at -70° C until use.

2.3 Detection of SRLV infection

To determine serological condition in study animals, we tested for the presence of SRLV antibodies in plasma using commercial Caprine Arthritis Encephalitis Virus Antibody test

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kits, cELISA (VMRD, Pullman, WA, USA), following the manufacturer's instructions.

2.4 DNA extraction We extracted DNA from PBLs using the FavorPrepTM Tissue Genomic DNA Extraction Mini Kit™ (Favorgen, Bioech Corp., Pingtung, Taiwan), and following the manufacturer's instructions. We then quantified DNA in a nanodrop at 260-280 nm absorbance (Thermo fisher Scientific, USA), and stored at -70° C until use.

2.5 Positive control of PCRs We obtained cDNA from RNA in the supernatant of fetal goat synovial membrane cells infected with the SRLV strain FESC-752 (Ramírez et al., 2011). RNA extraction was carried out with the RNeasy Mini Kit (Qiagen, USA) and we obtained cDNA using the SuperScript ™ II Reverse Transcriptase kit (Invitrogen, USA), following the manufacturer's instructions.

2.6 SRLV V4 PCR Two pairs of primers were designed to amplify the V4 region of the SRLV env gene: FW3a-

5'-CAGCCACTATTGCCATGAT-3',

AATTGGGATGGATGTAATTGC-3'

FW4b-V4

RV3b-V4

(position

(position

7,332)

7,730)

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7,911)

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env (position 6,951) 5'-AGAGTATGGGTAAGAWTGGCAAA-3', RV3a-env (position 5'-

5'-

TTGGTGCAGCATTCCATCTA-3' using the FESC-752 virus (HM210570) as a reference

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sequence. The V4-PCR amplification mixture was: 10x Buffer with 1.5 MgCl2 (KAPA Taq PCR Kit, USA), 230 μM DNTP´s (Kapa, USA), 500 nM of each primer (IDT, USA), 5 U of

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Taq DNA polymerase (KAPA Taq PCR Kit, USA), 500 ng of DNA in a final volume of 30 μl. For the second PCR reaction, we used the same reagent concentrations and added 5 μl of

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a 1:10 diluted product obtained from the first amplification, with FW4b-V4 and RV3b-V4 primers. We used a thermocycler (MRC Laboratory Equipment Ltd. Holon, Israel) for the PCR reaction, setting the following amplification conditions: initial denaturation at 95° C for

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5 min, followed by 45 cycles of 95° C / 50s and alignment at 58° C / 50s and 52° C / 40s for the first and second round respectively. Then, extension at 72° C / 60s and a final extension of 72° C / 10 min. The expected amplification product from the first round was 946 bp and the second was 398 bp. Amplicons were separated through electrophoresis on a 1.5% agarose gel stained with 5 µg/ml ethidium bromide. A commercial base pair marker (Cleaver

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Scientific, UK) was used to identify the specific amplicons, by visualizing them under UV light in a transilluminator (UVP®, USA). 2.7 Endogenous gene In order to corroborate samples DNA integrity, we designed primers to amplify a fragment of

the

endogenous

succinate

dehydrogenase

(SDHA)

gene:

Fw-SDHA

5’-

CATGGAGGAGGACAACTG-3’ and Rv-SDHA 5´-TGGTAGATCTTCCCATCTTC-3’. The PCR reaction mixture contained: buffer (10X) with 1.5mM MgCl2 (Amplificasa®

Biotecmol, México), 230 µM dNTP (Kapa Biosystems, USA), 5U Taq DNA Polymerase (Amplificasa Biotecmol, México), 500 nM of each primer, and 300 ng of DNA in a 25 μL reaction mixture. The PCR amplification conditions were: initial denaturation at 95° C for 5 min, followed by 30 cycles of denaturation at 95° C/ 40 s, alignment of 53° C 40 s and extension at 72° C 30 s, with a final extension 72° C 10 min.

2.8 Sequencing Positive amplicons were purified using a commercial kit (Favorgen, Bioech Corp., Pingtung,

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Taiwan) and sent for bidirectional sequencing using Sanger´s method at the Biotechnology

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and Prototype Unit of FES-Iztacala, UNAM.

2.9 Genotyping

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We built a phylogenetic tree including both nucleotide sequences obtained in this study and reference sequences available from GenBank: Gansu (AY900630), Shanxi (GU120138), 496

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(FJ195346), 697 (HQ848062), EV1 (S51392), KV1514 (M60610), LV1 and KV1772 (L06906), Roccaverano (EU293537), Seui (GQ381130), Fonni (JF502416), Volterra

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(JF502417), FESC ‐ 752 (HM210570), 1GA (AF322109), P1OLV (AF479638), SAOMVV (M34193), SRLV ‐ A4 (AY445885), CAEV ‐ CO (M33677), 85/34 (U64439) and USMARC (KY358787). The tree was built with the GENEIOUS, USA program, using the

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MrBayes algorithm which is based on the Markov Monte Carlo (MCMC) model (Huelsenbeck and Ronquist, 2001). FigTree v1.4.3. was used to edit the tree.

2.10 Signature pattern and mutation detection Sequence alignments were made with ClustalW and identity matrices with GENEIOUS.

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To determine the presence of signature patterns, we used the VESPA (Viral Epidemiology Signature

Pattern

Analysis)

program

(https://www.hiv.lanl.gov/content/sequence/VESPA/vespa.html; Korber and Myers, 1992), which identifies differences between two groups of sequences based on position. To assess population diversity in a transversal sense, we determined Shannon's entropy, using online ENTROPY-TWO (https://www.hiv.lanl.gov/content/sequence/ENTROPY/entropy.html). We determined the non-synonymous and synonymous substitution rate using SNAP

(Synonymous No-synonymous Analysis Program) v2.1.1 (Kryazhimskiy and Plotkin, 2008); https://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html).

2.11 Statistical evaluation of signature patterns The results obtained in the amino acid sequences were analyzed with non-parametric Kruskal-Wallis Tests applied to independent samples, using the statistical program SYSTAT 13.

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3 Results 3.1 Detection of SRLV infection

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We identified the presence of antibodies against SRLV in 212 of 244 evaluated plasma samples. Out of 139 goats and 105 sheep sampled, we found 80 asymptomatic goats, 28 with

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clinical signs of arthritis and 104 asymptomatic sheep. V4-PCR detected 69 (59.16%) positive goats, from which 38 sequences were obtained, and 27 sheep (26.92%), from which

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we obtained 11 sequences. Details of the samples are in table 1. Sequences derived from this study were deposited in GenBank and are available with access numbers MG675073 to

MG675150.

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MG675077, MG675080 to MG675119, MG675132, MG675133, MG675149 and

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3.2 Genotyping and variability

We built a phylogenetic tree to determine the association between the 49 SRLV proviral sequences obtained from PBLs. The tree shows a clear grouping of 11 (21.56%) sequences with genotype A (8 sheep and 3 goats; Figure 1). This is the first identification of infection by this genotype in sheep and goats of Mexico, in addition to 38 sequences (78.43%)

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associated with genotype B (35 goats and 3 sheep; Figure 1). The phylogenetic tree distribution shows a clear grouping by region rather than species, or the presence or absence of clinical signs. Based on our results, study sequences were classified into three groups: genotype A asymptomatic animals (Ssx GA), genotype B asymptomatic animals (Ssx GB) and genotype B animals with arthritis (A GB). We found high genetic distance between groups, ranging from 0.127 to 0.295. The Ssx GA group had the least distance within, whereas samples in the Ssx GB group showed the greatest distance. Additionally, we

examined synonymous and non-synonymous substitution ratios, and found negative selection among the study sequences (Table 2).

3.4 Signature patterns The V4 region was 83 amino acids long, and its tertiary structure was made up of three loops (data not shown). VESPA and ENTROPY identified signature patterns in the last loop (position 45 through 83), specifically in residues 54, 78, 79 and 82 (Figure 2). The amino acids deduced in the last loop and their frequency in the three study groups are

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shown in Figure 3. In position 54 for group A GB the residue “N” (p = 0.017) was identified most frequently, as were residues T (p = 0.001) and G (p = 0.024) respectively for groups

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Ssx GA and GB. Residue “G” (p=0.000) was identified as the most frequent at position 78 for the Ssx GA group. Residue “R” was most common for the Ssx GB group and showed a

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tendency toward statistical significance (p=0.056). Similarly, at position 79, residue “D” was identified in group A GB and was not statistically significant (p = 0.095) although it was

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most frequent. The same occurred with residue "N" (p = 0.064) in group Ssx GB and residue "R" (p = 0.000) in group Ssx GA. Finally, at position 82, residues "T" (p = 0.000) and "K"

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were identified as more frequent for the Ssx GA and A GB groups respectively, although neither were statistically significant (Table 3).

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4 Discussion

In this paper, as a first approach, we used the Vespa and Entropy programs to identify a possible signature pattern in the last loop of the V4 region of surface proteins encoded by the env gene of SRLV. Specifically, we found these possible signature patterns at positions 54, 78, 79 and 80, in sequences derived from goats infected with arthritis, as well as from

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asymptomatic sheep and goats. The most important statistical significance was found at position 54 in residues T, N and G, in all three of the groups analyzed. Although the threonine residue at position 54 of the SRLV surface protein is very frequent in the available sequences of the genetic subtypes A1, A3, and A4, obtained mainly from infected sheep, it is also possible to identify the variability of the residue T in this position in sheep infected with SRLV strains of subtype A2, with and without the presence of a clinical manifestations.

These findings suggest an association between the signature pattern from residue 54, clinical status, and SRLV genotype in sheep and goats. While residue "R" from position 78, and residues "N" and "D" from position 79 may be relevant to signature patterns, corroborating this hypothesis requires the analysis of a greater number of sequences. Similar studies in other retroviruses have identified signature patterns at specific positions of the lentivirus surface protein, and these in turn have been associated with tropism towards the central nervous system which generates different neurological pathologies (Korber et al., 1994), dementia (Holman and Gabuzda, 2012) and neurocognitive deficit (Pillai et al., 2006).

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Viruses that generate patterns such as HIV have been found in compartments that function as virus reservoir organs (lymphoid organs and brain; Korber et al., 1994).

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Most studies related to signature patterns in lentiviruses such as HIV, SIV and FIV have identified viral strains with a marked tropism towards the central nervous system (Korber et

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al., 1994). These strains are also characterized by having macrophages as target cells for infection, modifying their natural tropism towards infecting lymphocytes (Pépin et al., 1998).

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Conversely, SRLVs naturally only infect cells from the monocyte/macrophage and dendritic lines, however nervous signs related to SRLV infection are not so frequent and have only

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been reported in certain regions of the world with specific genotypes. The most recent report was described in Spanish sheep related to an A3 genotype (Glaria et al., 2012). Studies in SRLV infected sheep and goats that developed nervous clinical signs and mastitis did not

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identify signature patterns, although they found clear virus compartmentalization in some organs. This indicated microevolution following initial infection, which, in turn, favored nervous or mammary disease generated by the quasi-species (Pisoni et al., 2007; Ramírez et al., 2012). To date, there have been no reports on the presence of nervous clinical signs related to SRLV infection in Mexico. However, the presence of arthritic signs in goats is frequent

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(Mendiola et al., 2019), as is the asymptomatic infection of a large number of sheep and goats (Días et al., 2005). In the present study, we analyzed DNA samples from sheep and goats infected with SRLV. This analysis allowed us to identify a specific signature pattern in both species infected with genotype A and B, both in goats with arthritis as well as in asymptomatic sheep and goats. This highlights the importance of conducting more studies in asymptomatic animals to understand what factors promote disease development, not forgetting that they are an

important source of virus and can spread infection to healthy animals. The signature pattern we identified at position 54 in arthritic goats was also identified in 11 sequences derived from tissues such as spleen, mammary gland and synovial membrane in three goats with signs of arthritis (data not shown), which reinforces the finding of the signature pattern identified in goats with arthritis.

SRLV region V4 forms three loops and has an approximate length of 80-84 amino acids. This differs from what has been described for HIV where the V3 region forms a single loop

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33 to 35 amino acids long (Hötzel et al., 2002; Korber et al., 1993). Additionally, the V3 region has been described as consisting of a stem-loop structure (Holman and Gabuzda,

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2012) that has not been studied in the SRLV region V4. The region where the signature pattern was identified, and which is part of the third loop of the SU protein region V4,

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contains two linear epitopes that correlate with the evasion of neutralizing antibodies (Skraban et al., 1999). Our alignments identified five conserved cysteines in the amino acid

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sequences of SRLV V4, and this conservation was independent of both clinical phase and viral genotype, which is consistent with previous studies (Skraban et al., 1999; Valas et al.,

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2000). It is worth mentioning that we obtained some atypical viral sequences in this study, which presented deletions that have been previously reported by other studies (Bertolotti et

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al., 2011; Gjerset et al., 2006).

On the other hand, the phylogenetic tree we built showed a grouping of 11 sequences (eight from sheep and three from goats) with reference sequences from genotype A. This was the first time this genotype was identified in asymptomatic sheep and goats in Mexico. Ramírez et al., (2011) reported the presence of genotype B1 in sheep and goats in the Estado de

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México, which is consistent with the findings in the same state and both species from the present study. Additionally, we identified the presence of genotype B in sheep and goats in seven states throughout the country, which indicates that SRLV genotype B1 is widely distributed nationally in herds of sheep and goats, as previously described (Mendiola et al., 2019).

We found that the sequences from asymptomatic animals infected with genotype B, were more divergent than sequences derived from asymptomatic animals of genotype A. This divergence within asymptomatic B genotype animals was also greater than that observed in sequences from arthritic goats with the same genotype. In the sequence analysis, we found mutations tending towards negative selection, although we found genetic distances as great as 29.6%, mainly in the Ssx GB group- these mutations were more frequently synonymous. Shah et al., (2004) proposed the current SRLV classification where 12% to 29% of divergence is used to identify subtypes. With the results we obtained regarding genetic

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distance, it is possible to suggest that among the sequences identified as genotype B, some may be a new genetic subtype that would extend the previously described B1-B3. However,

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it is necessary to amplify the gag and pol to support a new genetic subtype.

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SRLV related clinical signs were only found in goats (arthritis), more infections were detected with the PCR-V4 in goats than sheep, and all infected sheep were asymptomatic. It

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is known that SRLV pathogenesis is the result of complex multifactorial interactions including animal genetics, virus genetics and immune pressure (Minguijón et al., 2015).

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These factors could be causing low infection levels, which in turn could explain our difficulty in detecting positive sheep through PCR, as well as the lack of development of clinical

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conditions in sheep. However, other analyses are required to support this hypothesis.

5. Conclusion

As a first approach, we identified a possible “signature pattern” of 4 discontinuous residues at positions 54, 78, 79 and 82 of the V4 region of the SRLV surface protein., although only residue 54 was significant in all groups studied. The results we obtained identified a signature

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pattern related to different SRLV genotypes and clinical status in sheep and goats. We identified genotype A which infects goats and sheep in Mexico for the first time.

Compliance with ethical standards Conflict of interest: The authors declare that they have no conflicts of interest.

Ethical approval; All applicable international, national, and institutional guidelines for the care and use of animals were followed.

Authors and contributors Ramírez H., Tórtora J. and González A. conceived and designed the experiments; González A and Cerón F. performed the experiments; Tórtora J., Martínez H., García M. and Ramírez H. revised the manuscript; González A. and Ramírez H. analyzed the data; García M.,

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González A. and Ramírez H. wrote the paper.

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CRediT author statement

Ramírez H., Tórtora J. and González A. conceived and designed the

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experiments; González A and Cerón F. performed the experiments; Tórtora J., Martínez H., García M. and Ramírez H. revised the manuscript; González A. and Ramírez H. analyzed the data; García M., González A. and Ramírez H.

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wrote the paper.

Acknowledgements

We thank the DMV responsible for the herds, the cattlemen who kindly provided the samples,

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and the staff of the Virology, genetics, and molecular biology lab at FES-Cuautitlan. UNAM.

This study was funded by the CONACyT project 221285 "Genotyping the env gene of retroviruses that impact the health of domestic ruminants", by the PAPIIT code IT201217 “ELISAs based on using recombinant proteins and synthetic peptides for the serological

detection of lentiviruses in goats”, and by PIAPI1610. FESC. UNAM “Use of molecular and

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bioinformatics tools for the identification of infectious agents”.

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Minguijón, E., Reina, R., Pérez, M., Polledo, L., Villoria, M., Ramírez, H., Leginagoikoa, I., Badiola, J.J., García-marín, J.F., Andrés, D. De, Luján, L., Amorena, B., 2015. Small lentivirus

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Figure captions

Figure 1. Phylogenetic tree constructed using the Bayesian method and 1000 bootstraps. A1A4, B1, B2-3, E show reference sequences grouping by genotype (⚫) and sequences obtained in this study. Identification of sequences in the phylogenetic tree, from the center out: accession number, identification of the animal or letter to the state (BCS: Baja California Sur, COA: Coahuila, GTO: Guanajuato, HGO: Hidalgo, EDO: Estado de México, QRO:

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Querétaro, SON: Sonora, SIN: Sinaloa and VER: Veracruz). Clinical phase: ◼ with arthritis;

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/ Ov: samples of sheep, (C +) positive control sequences of strain FESC-752.

Figure 2: Schematic representation of the main loop by infectious phase, formed between positions 45 to 83 and generated from the consensus sequences obtained in the present study. Ssx GA: sequences obtained from asymptomatic animals genotype A; Ssx GB: sequences obtained from asymptomatic animals genotype B; A GB: sequences obtained from animals with arthritis of genotype B; +: amino acids with positive charges; -: amino acids with

negative charges. Dotted box: discontinuous signature patterns (modified from Skraban et

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al., 1999).

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Figure 3: Alignment of SRLV amino acid sequences derived from goat and sheep PBL (Ssx GA: asymptomatic of genotype A; Ssx GB: asymptomatic of genotype B; A GB: with arthritis of genotype B), black boxes represent positions 54, 78, 79 and 82.

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Table 1: Sample distribution of sheep and goats by clinical status, ELISA, PCR and sequences obtained. PCR-V4

Sequences Goats Sheep STATE (+) (+) A Ssx A Ssx Ssx B.C.S. 2/0 1 Coahuila 10/30 1 Estado de México 11/1 0 11 4 Guanajuato 9/5 7/13 6 7 Hidalgo 9/0 7/2 4 Querétaro 2/0 13/0 1/1 10/3 1 4 Sinaloa 5/0 5/0 3 Sonora 19/0 14/0 2/17 2/12 1 2 Veracruz 11/1 8/0 12/0 1/8 3 1 Total 28/0 80/31 104/1 21/7 48/63 27/79 18 20 11 cELISA: competitive ELISA; (+/-): positive/negative; A: arthritis; Ssx: animals without clinical signs; B.C.S.: Sheep (+/-) Ssx 9/1 1/0 63/0

Goats (+/-)

Sheep (+/-) Ssx 0/10 0/1 17/46

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Baja California Sur.

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cELISA Goats (+/-) A Ssx 2/0 10/30 12/0 0 14/0 20/0

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Table 2: Variability of SRLV sequences by group and genotype Group

Nº of sequences

dN/dS

Genetic distances

Mean

Standard error

Mean

Standard error

Ssx GA

11

0.13

0.029

0.127

0.017

Ssx GB

20

0.26

0.001

0.295

0.008

A GB

18

0.27

0.029

0.208

0.005

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Ssx GA: Nucleotide sequences from asymptomatic animals genotype A; Ssx GB: Nucleotide sequences from asymptomatic animals genotype B; A GB: Nucleotide sequences from animals with arthritis genotype B; dN/dS: non-synonymous/synonymous ratios.

Table 3: Nonparametric statistical analysis (Kruskal-Wallis Test) results. Comparisons between groups of SRLV infected sheep and goats, analyzed by position and V4 protein region amino acids. Position

A.A.

p-Value (n) Media

A GB SD

SE

Ssx GB (n) Media SD

SE

Ssx GA (n) Media SD

SE

0.001 0.017 0.024 0.535

(2)0.111 (10)0.556 (4)0.222 (2)0.111

0.323 0.511 0.428 0.323

0.076 0.120 0.100 0.076

(6)0.300 (3)0.150 (9)0.450 (2)0.100

0.470 0.366 0.510 0.308

0.105 0.082 0.114 0.069

(9)0.818 (2)0.182

0.405 0.405

0.12 0.122

0.000 0.151 0.056 0.102 0.542 0.423 0.484 0.484

(1)0.056 (3)0.167 (6)0.286 (6)0.381 (1)0.056 (1)0.056

0.236 0.383 0.463 0.498 0.236 0.236

0.055 0.090 0.109 0.117 0.055 0.055

(2)0.100

0.308

0.068

(9)0.881 (2)0.182

0.405 0.405

0.122 0.122

78

G N R K Q S A E

(8)0.400 (6)0.300 (2)0.100

0.503 0.470 0.308

0.112 0.105 0.068

(1)0.050 (1)0.050

0.224 0.224

R G E N D K T Q H P S

*0.000 0.878 0.787 0.064 0.095 0.301 0.484 0.172 0.423 0.484 0.484

(9)0.818 (1)0.091 (1)0.091

0.405 0.302 0.302

0.122 0.091 0.091

T K A R S

0.000 0.221 0.566 0.165 0.741

(9)0.818 (1)0.091 (1)0.091

0.405 0.302 0.302

0.122 0.091 0.091

0.323 0.218

0.076 0.052

(1)0.056 (6)0.333 (4)0.222 (5)0.278 (1)0.056

0.236 0.485 0.428 0.461 0.236

0.055 0.114 0.100 0.108 0055.

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(2)0.111 (1)0.048

0.050 0.050

(3)0.150 (1)0.050 (7)0.350 (2)0.100 (4)0.200 (1)0.050

0.366 0.224 0.489 0.308 0.401 0.224

0.082 0.050 0.109 0.069 0.092 0.050

(1)0.050 (1)0.050

0.224 0.224

0.050 0.050

(6)0.300 (3)0.150 (5)0.250 (5)0.250 (1)0.050

0.470 0.366 0.444 0.444 0.224

0.105 0.082 0.099 0.099 0.050

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0.076 0.076 0.090 0.108 0.090

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0.323 0.323 0.383 0.461 0.383

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79

(2)0.111 (2)0.111 (3)0.167 (5)0.278 (3)0.167

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54

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Ssx GA: nucleotide sequences from asymptomatic animals genotype A. Ssx GB: nucleotide sequences from asymptomatic animals genotype B. A GB: nucleotide sequences from animals with arthritis genotype B. Values in bold: statistically representative; *: value statistically representative with a dominance for genotype but not for clinical status. Values underlined: Values with trend to be statistically significant, requiring larger sample sizes to confirm; A.A: amino acids.