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

2. 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

3. 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

4. OVI AND AVIAN INFLUENZA
Pre-1998: capacity for virus isolation and serology
: 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
: real-time RT-PCR (rRT-PCR) (LightCycler 2); NASBA; implemented for H5
: LightCycler480 rRT-PCR

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

6. 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

7. 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

8. 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

9. 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)

10. 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: capillary)

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

12. TEST TURNAROUND TIME CAPACITY 2005:
RT-PCR/NASBA: ~ pools from ostriches
Serology: HI tests and ELISA tests from
chickens and ostriches

13. 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

14. 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

15. 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

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

17. 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)

18. 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