Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014–2018)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethical Considerations
2.2. Specimens
2.3. Viral RNA Extraction, Detection, and Sanger Sequencing
2.4. RNA Replication for MIDV and SINV Positive Follow-Up Specimens
2.5. Virus Isolation and Sequence-Independent Single-Primer Amplification (SISPA) with Rapid Amplification of cDNA Ends (RACE) for Full Genome Sequencing
2.6. Illumina Full Genome Sequencing
2.7. Phylogenetic Analysis
2.8. Statistical Analysis
2.9. Recombination Analysis
3. Results
3.1. MIDV and SINV Detection and Clinical Disease Description
3.2. Seasonality of Alphaviruses in South Africa
3.3. Geographic Distribution
3.4. Detection and Phylogenetic Analysis of Partial Sequences
3.5. Virus Isolations for Full Genome Sequence Analysis
3.6. Recombination Event Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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PCR | Primer Name (Orientation/Channel Dye) | Sequence 5′–3′ | Position (MIDV/SINV) | Region and Amplicon Size | Reference |
---|---|---|---|---|---|
Alpha first round | Alpha1+ (sense) | GAYGCITAYYTIGAYATGGTIGAIGG | 5888–6368/ 6162–6642 | nsP4 480 bp | Sánchez-Seco et al., 2001 [15] |
Alpha1− (antisense) | KYTCYTCIGTRTGYTTIGTICCIGG | ||||
Alpha nested | Alpha2+ (sense) | GIAAYTGYAAYGTIACICARATG | 6066–6264/ 6340–6538 | nsP4 198 bp | |
Alpha2− (antisense) | GCRAAIARIGCIGCIGCYYTIGGICC | ||||
MIDV probe | GCTTTAAGAAGTACGCATGCAACA | 6132–6155 | nsP4 N/A | van Niekerk et al., 2015 [13] | |
SINV probe | ATGACGAGTATTGGGAGGAGTTTG | 6427–6450 | |||
Nested specific MIDV | MNF (sense | GCAGCCTTTTGTCCGTCYAA | 5936–6283 | nsP4 347 bp | Steyn et al., 2020 [14] |
MNR (antisense) | GGCTTCAAGTCRTAGGTTT | ||||
Nested specific SINV | SNF (sense) | GCAACCTTYTGCCCCGCYAA | 6209–6556 | nsP4 347 bp | |
SNR (antisense) | GGGACCAAATTATRCGTCT | ||||
MIDV E first round | MIDV EF (sense) | TTGTCAACGGAGAGAGCAC | 10,231–11,048 | E1 817 bp | This study |
MIDV ER (antisense) | CTATGGGCGGAGCTACTGTG | ||||
MIDV E nested | MIDV EN 9F (sense) | ACCGGGTAGATTTGGGGACT | 10,379–10,930 | E1 551 bp | This study |
MIDV 10,911 EN (antisense) | CACTTTGCTGTGCAAGTGGT | van Niekerk et al., 2015 [13] |
MIDV and SINV Positive Fatal Cases in Horses January 2014–December 2018 | |||||||
---|---|---|---|---|---|---|---|
Year | 2014 | 2015 | 2016 | 2017 | 2018 | Total | |
Virus | No. samples received | 195 | 197 | 129 | 427 | 136 | 1084 |
MIDV | % Positive | 4.61% (9/195) | 5.07% (10/197) | 077% (1/129) | 9.84% (42/427) | 6.48% (7/108) | 6.36% (69/1084) |
% Fatal | 22.22% (2/9) | 10.00% (1/10) | 0.00% (0/1) | 7.14% (3/42) | 0.00% (0/7) | 8.69% (6/69) | |
% Fatal co-infections | 100% (2 WNV/2) | 0.00% (0/1) | N/A | 66.66% (2 WNV/3) | N/A | 66.66% (4/6) | |
SINV | % Positive | 1.02% (2/195) | 4.06% (8/197) | 0.00% (0/0) (0/129) | 0.00% (0/427) | 0.92% (1/108) | 1.01% (11/1084) |
% Fatal | 100% (2/2) | 0.00% (0/8) | 0.00% (0/0) | 0.00% (0/0) | 0.00% (0/1) | 18.18% (2/11) | |
% Fatal co-infections | 50.00% (1 AHSV/EEV/2) | 0.00% (0/0) | 0.00% (0/0) | 0.00% (0/0) | 0.00% (0/0) | 50.00% (1/2) |
Middelburg | Sign | MIDV Positive n = 69 (%) | MIDV Negative n = 1015 (%) | p-Value | Odds Ratio (95% CI) |
---|---|---|---|---|---|
Died or euthanized | 6 (8.70) | 235 (23.15) | 0.004 | 0.31 (0.13–0.73) | |
Neurological signs | Any neurological signs | 51 (73.91) | 667 (65.71) | 0.188 | 1.48 (0.85–2.59) |
Ataxia | 34 (49.23) | 388 (38.23) | 0.074 | 1.56 (0.96–2.55) | |
Paresis | 11 (15.94) | 122 (12.02) | 0.342 | 1.38 (0.70–2.71) | |
Hind leg paralysis | 6 (8.70) | 47 (4.63) | 0.142 | 1.96 (0.80–4.76) | |
Recumbency | 6 (8.70) | 159 (15.67) | 0.163 | 0.51 (0.21–1.20) | |
Paralysis | 3 (4.35) | 97 (9.56) | 0.195 | 0.43 (0.13–1.39) | |
Tremors/fasciculations | 3 (4.35) | 28 (2.76) | 0.441 | 1.6 (0.47–5.40) | |
Seizures | 1 (1.45) | 49 (4.83) | 0.365 | 0.28 (0.03–2.13) | |
Stiffness | 6 (8.70) | 7 (0.69) | 0.0001 | 13.71 (4.47–42.02) | |
Fever | Fever | 52 (75.36) | 529 (52.12) | 0.00025 | 2.81 (1.60–4.92) |
Fever only | 13 (18.84) | 168 (16.55) | 1.000 | 1.015 (0.543–1.895) | |
Fever and neurological signs | 38 (55.07) | 188 (18.52) | 2 × 10−10 | 5.25 (3.18–8.66) | |
Fever, neurological and respiratory signs | 1 (1.45) | 3 (0.29) | 0.235 | 4.85 (0.49–47.38) | |
Other | Icterus | 20 (28.99) | 118 (11.63) | 0.00021 | 3.1 (1.78–5.40) |
Anorexia/inappetence | 20 (28.99) | 189 (18.62) | 0.039 | 1.78 (1.03–3.07) | |
Pallor | 10 (14.49) | 69 (5.91) | 0.010 | 2.69 (1.31–5.53) | |
Sindbis | Sign | SINV Positive n = 11 (%) | SINV Negative n = 1073 (%) | p-Value | Odds Ratio (95% CI) |
Died or euthanized | 2 (18.18) | 239 (22.27) | 1 | 0.77 (0.16–3.61) | |
Neurological signs | Any neurological signs | 6 (54.54) | 713 (66.45) | 0.522 | 0.42 (0.12–1.38) |
Ataxia | 3 (27.27) | 419 (39.05) | 0.543 | 0.58 (0.15–2.21) | |
Recumbency | 2 (18.18) | 163 (15.19) | 0.678 | 1.24 (0.26–5.79) | |
Circling/paddling | 1 (9.09) | 17 (1.58) | 0.168 | 6.21 (0.75–51.27) | |
Facial nerve paralysis | 1 (9.09) | 3 (0.28) | 0.040 | 35.67 (3.41–372.98) | |
Seizures | 1 (9.19) | 49 (4.57) | 0.406 | 2.08 (0.26–16.65) | |
Tongue paralysis | 1 (9.09) | 7 (0.65) | 0.078 | 15.22 (1.71–135.51) | |
Tremors/fasciculations | 1 (9.09) | 30 (2.80) | 0.274 | 3.47 (0.43–28.03) | |
Fever | Fever | 8 (72.73) | 573 (53.40) | 0.237 | 2.33 (0.61–8.81) |
Fever only | 3 (27.27) | 179 (16.68) | 0.406 | 1.88 (0.49–7.17) | |
Fever and neurological signs | 4 (36.36) | 220 (20.50) | 0.260 | 2.13 (0.61–7.36) | |
Fever, neurological and respiratory signs | 0 (0.00) | 4 (0.37) | 1.000 | Undefined(undefined) | |
Other | Anorexia/inappetence | 4 (36.36) | 205 (19.11) | 0.237 | 2.41 (0.71–8.34) |
Flaccid tail | 1 (9.09) | 7 (0.65) | 0.078 | 15.22 (1.71–135.51) | |
Icterus | 1 (9.09) | 137 (12.77) | 1 | 0.68 (0.08–5.37) | |
Nasal discharge | 2 (18.18) | 19 (1.78) | 0.021 | 12.26 (2.48–60.64) |
Sample Group. | Horse 1 | Horse 2 | Horse 3 | Horse 4 |
---|---|---|---|---|
Sample ID’s | ZRU074/17 ZRU141/17/6 | ZRU060/17 ZRU141/17/1 | ZRU034/18 ZRU031/18 ZRU038/18 | ZRU194/17/3 ZRU209/17 |
Date sick | 2017/03/07 | 2017/03/03 | 2018/02/03 | 2017/04/03 |
Date first sampled | 2017/03/07 | 2017/03/03 | 2018/02/05 | 2017/04/03 |
Days from onset for first sample (PCR positive) | 0 days | 0 days | 2 days | 0 days |
Initial signs | Fever & Icterus | Ataxia, Fever & Icterus | Fever | Anorexia/inappetence, Fever, Icterus, Pallor |
Virus result on first submission (test method) | MIDV/EEV (PCR) | MIDV (PCR) | MIDV (PCR) | MIDV (PCR) |
Date follow-up sampled | 2017/03/24 | 2017/03/24 | 2018/02/07 2018/02/17 | 2017/04/10 |
Days from onset for follow-up sample (PCR positive) | 17 days | 21 days | 4 and 14 days | 7 days |
Signs at follow-up | Healthy | Healthy | Fever, Healthy | Icterus |
Virus result for follow-up specimen (test method) | MIDV/EEV (PCR) | MIDV (PCR) | MIDV (PCR) | MIDV/WNV (MIDV PCR/WNV IgM ELISA) |
Location | Benoni | Benoni | Bapsfontein | Onderstepoort |
ZRU Number | Year | Signs | Location (Province, City) | Co-Infection | Clinical Outcome |
---|---|---|---|---|---|
ZRU080/14 | 2014 | Stiffness | North West, Vryburg | None detected | Alive |
ZRU089/14 | 2014 | Ataxia, icterus, paresis, fever | Free State, Ladybrand | AHSV | Alive |
ZRU044/17 | 2017 | Ataxia, icterus, fever | Gauteng, Pretoria | None detected | Alive |
ZRU059/17/1 | 2017 | Ataxia, icterus, fever | Gauteng, Pretoria | None detected | Alive |
ZRU075/17 | 2017 | Ataxia, fever | Gauteng, Benoni | None detected | Alive |
ZRU103/17 | 2017 | Fever | Gauteng, Benoni | None detected | Alive |
Possible Event | Parental Sequence | RDP4 Recombination Detection Model and Corresponding p-Values | |||||
---|---|---|---|---|---|---|---|
SP nt position | Major | Minor | RDP | MaxChi | Chimaera | SciScan | 3Seq |
2582–3093 (509 nt) | SFV | Unknown | 1.833 × 10−2 | 3.921 × 10−4 | 8.034 × 10−4 | 5.921 × 10−11 | 1.242 × 10−9 |
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Fourie, I.; Snyman, J.; Williams, J.; Ismail, A.; Jansen van Vuren, P.; Venter, M. Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014–2018). Viruses 2022, 14, 2013. https://doi.org/10.3390/v14092013
Fourie I, Snyman J, Williams J, Ismail A, Jansen van Vuren P, Venter M. Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014–2018). Viruses. 2022; 14(9):2013. https://doi.org/10.3390/v14092013
Chicago/Turabian StyleFourie, Isabel, Jumari Snyman, June Williams, Arshad Ismail, Petrus Jansen van Vuren, and Marietjie Venter. 2022. "Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014–2018)" Viruses 14, no. 9: 2013. https://doi.org/10.3390/v14092013
APA StyleFourie, I., Snyman, J., Williams, J., Ismail, A., Jansen van Vuren, P., & Venter, M. (2022). Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014–2018). Viruses, 14(9), 2013. https://doi.org/10.3390/v14092013