Diagnostic capacity and research activities AVIAN INFLUENZA: Diagnostic capacity and research activities at Onderstepoort Veterinary Institute (ARC-OVI)

AVIAN INFLUENZA IN THE SPOTLIGHT Human pandemics are cyclic, next one due- H3? Avian H5N1, H9 or H7 are also candidates Asian H5N1 jumped directly from infected birds to humans (previously, pandemic strains arose by reassortment of human and avian virus genes-intermediary hosts required) Asian HPAI H5N1 seems to be becoming more virulent for poultry (as indicated by molecular markers and pathogenicity tests) 174 human cases; 94 deaths (54% mortality) Asian HPAI H5N1 is spreading rapidly: AsiaEurope  Africa

THE CHALLENGES ENCOUNTERED WITH AIV Multiple lineages and pathotypes 16x H-types; 9xN-types 16 x 9= 144 AIV strains, theoretically Segmented genome- 8 segments- reassortment; Multiple lineages of internal genes, some of which are virulence determinants (e.g. NS1 and PB2) Zoonosis: H5 and H7 strains :handling requirements minimum Bio-safety level (BSL)2+ RNA virus- requires reverse transcription to cDNA for analysis (RNA degradation) H0 cleavage site is the major virulence determinant- requires sequencing LPAI vs HPAI LPAI vs HPAI low pathogenicity AI high pathogenicity AI Sequencing of the H0 cleavage site for pathotype prediction: Insertion of multiple basic amino acids (R and K) at the underlined H0 cleavage site results in the conversion of LPAI viruses to HPAI viruses

OVI AND AVIAN INFLUENZA Pre-1998: capacity for virus isolation and serology - 1998: Ostrich research group surveyed wild birds for AIV in Oudtshoorn and isolated H10N9 2001: AIV M PCR implemented in Biotechnology division 2002: H6N2 outbreak: capacity extended to type H5, H6, H7, H9, H10 by PCR; sequencing; phylogenetic analysis commences 2003: capacity for full-length characterization of all 10 genes - 2004: real-time RT-PCR (rRT-PCR) (LightCycler 2); NASBA; implemented for H5 - 2006: LightCycler480 rRT-PCR

OVI CAPACITY BIOTECHNOLOGY VIROLOGY division division Collaborating laboratories (APVL, SPVL, UP-PRL, VLA-Weybridge [UK]) BIOTECHNOLOGY division VIROLOGY division

OVI CAPACITY Virology Division Tests: Serology (HA/HI and ELISA) Virus isolation Access-controlled Biosafely level 2+ (BSL 2+) AIV diagnostic staff: Veterinarian/Division head (G Gerdes) 5 technicians: ELISA (H Harris; R Rudoph) Virus isolation (H Kekana; P Combrink) HI (K Roos; M Molefe) Access control to Virology’s BSL2+ facility

OVI CAPACITY Biotechnology division Tests Nucleic acid extractions RT-PCR and rRT-PCR NASBA Sequencing Molecular characterization Phylogenetic analysis Access controlled PCR labs AI diagnostic & research staff: Veterinarian/PCR laboratory head (M Romito) Molecular biologist (C Abolnik) 2 PCR technicians (A Doboro, R Maluleka) Quality Manager/ Safety officer (L Taljaard) Access control to SANAS-accredited PCR laboratories

Five Dedicated PCR laboratories OVI FACILITIES AND EQUIPMENT Five Dedicated PCR laboratories Lab 2.2 The MagNaPure- automated RNA extraction- 96 samples Ms Rachel Maluleke setting up an RT-PCR mix reaction in Lab 1 Lab 2.1 for sample processing nucleic acid extractions LightCycler480 GelDoc system Gel tanks thermocyclers Lab2.3 the nested PCR room Lab 4: Agarose gel electrophoresis and gel documentation Lab 3: Thermocycling room

Facilities and Equipment… NanoDrop spectrophotometer A backup LightCycler and a NanoDrop Cuvette-less spectrophotometer for 1ul quantities; shortens result turnaround time; saves on precious RNA, eliminates repeat sequencing (cost-saving)

DNA sequencing 4 capillary system (upgradeable to 16 capillaries) FACILITIES AND EQUIPMENT ABI 3130 Genetic Analyzer DNA sequencing 4 capillary system (upgradeable to 16 capillaries) (OVI-Biochemistry division: 16-capillary 3100 Genetic Analyser; OVI-Exotic Diseases division: 3100 4-capillary)

FACILITIES AND EQUIPMENT The new BSL-3 laboratory Interim BSL-3 facility at Exotic Diseases Division

TEST TURNAROUND TIME CAPACITY 2005: RT-PCR/NASBA: ~10 000 pools from 26 000 ostriches Serology: 22 683 HI tests and 4 941 ELISA tests from 18 652 chickens and 8 972 ostriches

AVIAN INFLUENZA IN AFRICA Prior to recent Asian HPAI H5N1 in Nigeria, Egypt and Niger, only South African and Zimbabwe have reported AI outbreaks/virus isolations Lack of information for the prevalence of AIV in poultry and/or wild birds in rest of Africa Historically, AI is a disease of ostriches in South Africa (and Zimbabwe): LPAI H7N1(‘91), LPAI H5N9(‘94), H9N2(‘95), H6N8(‘98), H10N1(’01) 2001 LPAI H6N2 outbreak in chickens 2004 HPAI H5N2 outbreak in E.Cape ostriches Targeted surveillance of wild ducks: 1998: Wild ducks, Oudtshoorn (H10N9) 2004: Wild ducks, Blesbokspruit (H4N8, H3N8, LPAI H5N1), Oudtshoorn (LPAI H5N2) OVI research activities: Phylogenetic analysis conducted to determine origins of the viruses Sequenced and analyzed by Europeans groups

OVI RESEARCH: AIV COMPONENT Ostriches can act as long term reservoirs and mixing vessels for the reassortment of AI viruses that can spill over to the poultry industry The H6N2 chicken outbreak strain was produced by a reassortment of historic H9N2 (’95) and H6N8 (’98) ostrich viruses. It adapted for spread in chickens. Northern Europe, Russia and East Asia are the sources of SA LPAI viruses that circulate in wild ducks before infecting ostriches Based on very close phylogenetic relationships with viruses from these countries- shared gene pool. The H5N1 virus was not closely-related to Asian HPAI H5N1 The 2004 E Cape HPAI H5N2 ostrich outbreak virus was not closely-related to Asian H5N1 and was probably derived from LPAI virus that circulated in wild ducks and geese (see diagram) Phylogenetic tree: The branching pattern shows the genealogy of the viruses. The closer two organisms are to a branch, the closer their relationship.The length of the line does not indicate a length of time, only the degree of change

BIRD MIGRATION IN AFRICA Charadriiformes Some examples of aquatic bird movements in Africa (ADU)-limited data: Anseriiformes Comb duck Red-billed duck Ruff Common tern Egyptian goose

PROPOSED MODEL - - - - - Reservoirs of AI viruses: Shorebirds (waders, gulls, terns) Geese Ducks - - - - - theoretically possible

SURVEILLANCE FOR AIV IN AFRICAN WILD AQUATIC BIRDS Aluminium and cotton swabs slim-tipped for cloacal swabbing of smaller migratory species g a+b c f Faecal samples to be collected from wild aquatic birds by trained teams To be tested at OVI by rRT-PCR or virus isolation for AIV Early-warning system for HPAI H5N1 Characterization of African strains to describe epidemiology of AI in Africa Detailed protocols for sample collection, storage, transport and import permit are available as hand-outs A typical kit for faecal sample collection could contain: a) Face masks b) Dispenser bottle c) Antibiotics d) Phosphate-buffered saline (PBS) e) Latex gloves f) Swabs g) Sterile containers (organs of dead birds)

CONCLUSIONS LPAI is frequently detected in South Africa- it is most probably prevalent in other African countries but has gone undetected Highly likely that HPAI H5N1 will continue to spread into Africa Early detection will be critical for prevention and control Development of prediction and risk models OVI has the expertise and facilities to play the lead role in diagnosis and AIV research for the nations of the Africa continent