Foot-and-mouth disease virus persistence and evolution. Bryan Charleston, Pirbright Institute

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1 Foot-and-mouth disease virus persistence and evolution Bryan Charleston, Pirbright Institute

2 FMD: a priority impediment to wellbeing Animal health and welfare impacts on human health

3 Conjectured Status of FMD KEY Endemic Intermediate, sporadic Free with vaccination Free. Virus present in game parks Free

4 One-to-one challenge experiment

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6 FMDV 2001 transmission during early phases Farm 1 22/02/01 Farm 2 23/02/01 Farm 3 24/02/01 Farm 4 25/02/01 26/02/01 27/02/01 28/02/01 Farm 5 01/03/01 time

7 Inter-animal transmission + full-genome sequencing Short infectious period (mean 1.7 days) + highly contagious (R o 4.5) + rapid host immune response = Limited time to accumulate substitutions (vary antigenic sites) before transmit/infect subsequent host?? Controlled inter-animal transmission and sequencing (O1 BFS) 1 to 4 substitutions (consensus level) Interestingly: similar for inter-farm transmission UK (21 IPs, 7 months) (15 IPs, 3 months) UK to 5 (11 IPs, 2 months) Does antigenic variation and recombination occur predominantly during long term carriage? Malirat 1994 reported higher rate of fixation of mutations during persistence than during acute disease

8 MLN 38DPCI LZ DZ FMDV D46 DAPI 80 µm FMDV D46

9 Kruger National Park, South Africa

10 Study design 3 KNP buffalo isolates SAT1/KNP/196/91 SAT2/KNP/19/89 SAT3/KNP/1/08/3 PK cells OVI cattle titration TCID 50 6 month old Nguni cattle (n = 4) SAT-3 SAT-2 SAT-1 Skukuza buffalo challenge study TCID 50 Necropsy N = 4 buffalo 35DPC Necropsy N = 4 buffalo Necropsy N = 4 buffalo Necropsy N = 4 buffalo 95DPC 156DPC 163DPC 185DPC 299DPC 400DPC 1.25IU/kg 1.25IU/kg N = 16 FMD free buffalo (7 9 ) months old Imfolozi Game Reserve, KZN N = 4 ACTH stimulation (Synacthen ) N = 4 buffalo Day 302 Progesterone IUD Estrumate

11 Acute FMD: naïve buffalo 1DPC 2DPC

12 FMDV RNA copies/ml plasma Acute FMD: naïve buffalo Viraemia qrt-pcr X X2 X X Day 0 Day 2 Day 4 Day 6 Days post challenge Day 8 Day 11 Transient pyrexia; 5 animals, day 2 and 4 ( C) All buffalo +ve for antibodies to SAT-1, 2 and 3 at 14DPC (liquid-phase blocking ELISA) All buffalo PCR +ve for FMDV on oesophageal/pharyngeal scrapings between 29 and 35DPC

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14 Acute FMD: naïve buffalo Conclusion SAT co-infection in cattle severe clinical FMD 10 dose in naïve buffalo: mild clinical FMD integrin αvβ6 expression in tissues typically associated with FMD lesions viraemic for at least 5 to 7 days No leucopenia High levels type 1 IFN seroconverted to all three serotypes by day 14 PCR +ve oesophageal/pharyngeal scrapings between 29 and 35DPC

15 Chronic FMD: buffalo 95DPC 100µm 100µm 10µm Anti-3B (D5) IB11 Merge 10µm

16 Chronic FMD: buffalo Casteleyn et al., Budras, Bovine Anatomy Follicles with central crypt

17 Wellcome Trust Fellowship African buffalo are naturally co-infected with all three SAT serotypes Experiment: buffalo co-infected SAT 1, and monitored for 400 days Virus monitored in probang samples and palatine tonsil swabs SAT specific quantitative real-time RT-PCR Virus isolation and quantitative real-time RT-PCR Probang Tonsil swab Palatine tonsil swab useful tool especially for isolating live virus Co-infection dynamics Viral titres decrease over time SAT 1 dominant (detectable at 400dpi), no SAT 2 after 172dpi, no SAT3 after 214dpi Similar co-infection dynamics seen invitro during passage in epithelium cell line and novel buffalo kidney cell line

18 Competition assay ZZR21 S A T 1 (M O I 2 ) S A T 1 (M O I 1 ) S A T 1 (M O I 0.5 ) S A T 2 (M O I 2 ) S A T 2 (M O I 2 ) S A T 2 (M O I 2 ) S A T 3 (M O I 2 ) S A T 3 (M O I 2 ) S A T 3 (M O I 2 ) Single infections (MOI 2)

19 Competition assay BK cells S A T 1 (M O I 2 ) S A T 1 (M O I 1 ) S A T 1 (M O I 0.5 ) S A T 2 (M O I 2 ) S A T 2 (M O I 2 ) S A T 2 (M O I 2 ) S A T 3 (M O I 2 ) S A T 3 (M O I 2 ) S A T 3 (M O I 2 ) Single infections (MOI 2)

20 Virus dilution Virus dilution PFUs Killing assay SAT1 SAT2 SAT SAT1 SAT2 SAT Hours post-infection

21 Nucleotide sequence (window size 50) Similarity LPro 1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D Amino acid (window size 10) Similarity

22 FMD: buffalo Despite close contact, +ve virus isolation and ACTH stim no transmission/ seroconversion in cattle Consistent with previous reports: no transmission/ transmission only after months of contact Rx buffalo dexamethasone (Gainaru et al., 1986) Rx cattle dexamethasone for 3 weeks (Ilott et al., 1997) inhibited shedding of FMDV Co-infection cattle rinderpest/ bovine herpes 1 viruses (McVicar et al., 1977) no increase virus recovery/ transmission Pattern of transmission from captive buffalo during acute infection is also variable same pen: buffalo to buffalo / buffalo to cattle (Gainaru et al., 1986)/ buffalo to cattle (Dawe et al., 1994) adjoining pen: buffalo to impala (Gainaru et al., 1986)

23 Acknowledgments Nick Juleff Lin-Mari de Klerk Lorist Roy Bengis Louis van Schalkwyk At Dekker Scalk van Dyk Dave Cooper Francois Maree Berry Mutowembwa Brenda Botha Livio Heath Katherine Scott Pamela Opperman Belinda Blignaut Mark Woolhouse Ivan Morrison Dan Haydon Ferran JORI, Cirad Mammal Research Institute University of Pretoria Markus Hofmeyr Peter Buss Jenny Joubert