Abolnik_347-351.indd INTRODUCTION Highly pathogenic avian influenza (HPAI) is a dev- astating disease of poultry that has zoonotic poten- tial for humans and is caused by strains of the H5 and H7 subtypes of influenza A viruses (family Or- tho myxoviridae). The low pathogenicity avian influ- enza (LPAI) virus precursors to HPAI viruses are ubiquitous in wild waterfowl and shorebirds and usually only cause asymptomatic infections in these reser voir hosts. Once transferred to terrestrial poul- try, LPAI viruses can convert into HPAI (Webster, Bean, Gorman, Chambers & Kawaoka 1992). Although the virulence determinants of avian influ- enza viruses are multigenic in nature, an insertion of multiple basic amino acids at the cleavage site of the haemagglutinin precursor protein, H0, targets this protein for cleavage by ubiquitous subtilisin-like endonucleases, resulting in rapid systemic spread of the virus. In contrast, LPAI viruses contain a monobasic composition at H0 which is targeted by trypsin-like proteases that are limited to cells of the intestinal and respiratory tracts (Rott 1979; Stieneke- Gröber, Vey, Angliker, Shaw, Thomas, Roberts, Klenk & Garten 1992; Vey, Adler, Klenk, Rott & Gar- ten 1992; Wood, McCauley, Bashiruddin & Alex an- der 1993). The amino acid sequence at H0 is thus recognized as an important and reliable molecular virulence marker (OIE Terrestrial Manual 2004). South Africa was affected by two separate outbreaks of HPAI H5N2 in intensively-farmed ostriches be- tween 2004 and 2006, and intensive serological and virological surveillance was conducted during both outbreaks. Of thousands of tracheal swabs or organ samples tested, 46 were positive or suspect positive for the presence of AIV by real-time reverse tran- scription-PCR (rRT-PCR) or nucleic acid-based se- quence assay (NASBA) (M. Romito, unpublished laboratory data). H5-type viruses were isolated in only 3 (6.5 %) of these cases, viz. the HPAI H5N2 virus isolated in the Eastern Cape outbreak in 2004 (OSZA04N227), the HPAI H5N2 virus isolated in 347 Onderstepoort Journal of Veterinary Research, 75:347–351 (2008) RESEARCH COMMUNICATION A rapid and sensitive real-time reverse transcription PCR for the pathotyping of South African H5N2 avian influenza viruses C. ABOLNIK ARC-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, 0110 South Africa ABSTRACT ABOLNIK, C. 2008. A rapid and sensitive real-time reverse transcription PCR for the pathotyping of South African H5N2 avian influenza viruses. Onderstepoort Journal of Veterinary Research, 75:347– 351 A Fluorescence resonance energy transfer (FRET) real-time reverse-transcription (rRT-PCR) assay was developed that distinguishes stains of South African and European highly pathogenic (HPAI) from low pathogenicity (LPAI) H5 avian influenza viruses in the absence of virus isolation, irrespective of the length of insertion at the hemagglutinin cleavage site (H0). The assay was used to pathotype H5-type viruses detected by rRT-PCR in ostrich tracheal swabs collected during the 2006 HPAI H5N2 outbreak in the Western Cape Province. Keywords: Avian influenza virus, H5N2, pathotyping, real-time reverse transcription PCR, FRET Accepted for publication 10 June 2008—Editor 348 Transcription PCR for the pathotyping of South African H5N2 avian infl uenza viruses the Western Cape outbreak of 2006 (OSZA06AI1091) and the LPAI H5N2 virus isolated in the same out- break (OSZA06AI1160). The isolate OSZA06AI1091 contained fewer multiple basic amino acids at H0 than did isolate OSZA04N227, but was clearly HPAI according to the OIE definition (Abolnik 2007b). Unfortunately however, the nucleic acids detected by rRT-PCR/ NASBA are present in insufficient quantities to allow further molecular characteriza- tion (Hoffmann, Starick, Depner, Werner & Beer 2007; unpublished laboratory data 2007), yet for control purposes there is an urgent need to be able to differentiate between LPAI and HPAI viruses in ostriches especially where virus isolation has not been successful. At least two other groups have de- scribed sensitive rRT-PCR assays for HPAI H5 pathotyping, hoewever the SYBR Green method described by Payungporn, Chutinimitkul, Chaisingh, Damrongwantanapokin, Nuansrichay, Pinyochon, Amon sin, Donis, Theamboonlers & Poovorawan (2006) may give false positive results (Fernández, Gutierrez, Sorlozano, Romero, Soto & Ruiz-Cabello 2006). In the method of Hoffmann and co-workers (2007), a set of hydrolysis probes was described that detects the Asian group of HPAI H5N1, and the H0 cleavage site sequence of the Qinghai strain in particular. However, insertions/substitutions in the H0 cleavage site abolished signal generation. As demonstrated for isolates from in South Africa (Fig. 1), potential variations in the H0 cleavage site se- quence need to be taken into account. Therefore a rapid and sensitive rRT-PCR assay was developed to distinguish between South African HPAI and LPAI H5N2 viruses in the absence of virus isolation that does not rely on the sequence at H0. MATERIALS AND METHODS Primer and probe design A set of primers (InfA_H5_F and InfA_H5_R) and three probes (InfA_H5_640, InfA_H5_FL and InfA_ H5_705) were designed for the amplification and detection of a fragment spanning the cleavage site sequence of the H5N2 HA gene (Table 1). The LCRed640 fluorophore-labelled InfA_H5_640 probe was designed to hybridize over the consensus LPAI sequence that is conserved within the H5 lineage. The InfA_H5_FL binds a sequence adjacent to this, and is labelled at both the 5’ and 3’ ends with fluores- cein. The LCRed705 fluorophore-labelled InfA_H5_ 705 probe binds at the opposite end of InfA_H5_FL, in a conserved region within the H5 sub -lineage. In the presence of an LPAI sequence, the three probes will bind in tandem and both accep tor probes will be excited by the flourescein donor probe, resulting in TABLE 1 Primers and probes used to differentiate between HPAI and LPAI in this studya Primer/probe Sequence (5’-3’), -fluorophore Nucleotide position (Fig. 1) InfA_H5_F InfA_H5_R InfA_H5_640 InfA_H5_FL InfA_H5_705 GTGCCCCAAATACGTGAARTCA CCATCTATTGCTTKKTGAGTGGACTC 640-TCCTCTTGTTTCTYTTTGAGGGACATT-p F-CCTCCTTCTATAAARCCTGCTATRGCCCCAAATA-F -pCCATACCAACCRTCTACCATKCCTTGCC-705 951–972 1169–1143 1029–1067 1069–1102 1105–1133 a Primers and probes were designed in collaboration with and manufactured by TIB MOLBIOL, Eresburgstr. 22-23, D-12103 Berlin, Germany TABLE 2 Results of the H5 rRT-PCR pathotyping assay Sample F2(640) Cp F3(705) Cp Pathotype dH20 OSZA06AI1091 OSZA06AI1160 AI1133.1 AI1133.11 AI1133.12 AI1133.13 AI1133.15 AI1133.16 AI1133.17 AI1133.23 AI1120.37 AI1149.6 Negative control HPAI positive control LPAI positive control Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab Ostrich tracheal swab – – 20.46 – – 26.34 – – – – – – – – 22.34 20.59 – 29.90 26.21 28.83 31.12 – – – 29.80 – – HPAI LPAI – HPAI LPAI HPAI HPAI – – – HPAI – 349 C. ABOLNIK F IG . 1 M u lti p le n u cl e o tid e s e q u e n ce a lig n m e n t o f H 5 s tr a in s fo r th e r e g io n s p a n n in g t h e H 0 c le a va g e s ite ( n u m b e ri n g u p st re a m o f th e H 0 c le a va g e s ite is b a se d o n G e n b a n k se q u e n ce D Q 8 5 1 5 6 1 [ n o t sh o w n ]) 1 0 2 0 1 0 3 0 1 0 4 0 1 0 5 0 1 0 6 0 1 0 7 0 1 0 8 0 1 0 9 0 1 1 0 0 1 1 1 0 1 1 2 0 1 1 3 0 1 1 4 0 1 1 5 0 . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | O S Z A 0 6 A I 1 1 6 0 ( H 5 N 2 ) G C G A C T G G A C T C A G G A A T G T C C C T C A A A G A - - - - - - - - - - - - G A A A C A A G A G G A T T A T T T G G G G C T A T A G C A G G T T T T A T A G A A G G A G G A T G G C A A G G C A T G G T A G A T G G T T G G T A T G G A T A C C A C C A T A G C A A T G A G C A G G O S Z A 0 6 A I 1 0 9 1 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A G A A - - - - - - - - - . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O S Z A 0 4 N 2 2 7 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G A A A A A A G A A G A A . . . A . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . - - - - - - - - - - - - - - . . . . . . . . . . . . . C . . . . . . . A / m a l l a r d / B a v a r i a / 1 / 2 0 0 5 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A / t e a l / I t a l y / 3 8 1 2 / 2 0 0 5 ( H 5 N 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A / m a l l a r d / I t a l y / 3 4 0 1 / 0 5 ( H 5 N 1 ) . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . G C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A / m a l l a r d / S w e d e n / 2 / 0 2 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . T . . . . . . . . . . . C . . . . . . . A / m a l l a r d / S w e d e n / 3 9 / 0 2 ( H 5 N 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . C . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . A . . . . A / d u c k / D e n m a r k / 6 5 0 4 7 / 0 4 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . A / c h i c k e n / I t a l y / 3 1 2 / 1 9 9 7 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A G A A - - - - - - G A A . . . A . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . A . . . . A / c h i c k e n / I t a l y / 8 / 9 8 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A G A A - - - - - - G A A . . . A . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . A . . . . A / t u r k e y / E n g / 5 0 - 9 2 / 9 1 ( H 5 N 1 ) . . . . . . . . . . C . . . A . . C . . . . . . . . . . . . A A A A - - - - - - G A A . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . C . . . . . G . . . . . . . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . . . A / m a l l a r d / S w e d e n / 3 1 / 0 2 ( H 5 N 2 ) . . . . . G . . G . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . A . . . . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . A d u c k / M o n g o l i a / 5 4 / 0 1 ( H 5 N 1 ) . . A . . A . . G . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . C . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . A . A / d u c k / P r i m o r i e / 2 6 3 3 / 0 1 ( H 5 N 3 ) . . A . . A . . G . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . A . A / m a l l a r d / S w e d e n / 8 0 / 0 2 ( H 5 N 9 ) . . A . . G . . G . . . . . . . . . A . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . A . . . . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . A / C h i c k e n / I t a l y / 9 0 9 7 / 9 7 ( H 5 N 9 ) . . . . . . . . G . C . . . . . . C . . . . . . . . . . A G - - - - - - - - - - - - . . G . . . . . . . . . C . . . . . . . . . . C . . . . . . . . C . . C . . . . . G . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . . . A / d u c k / H o k k a i d o / V a c - 1 / 0 4 ( H 5 N 1 ) . . A . . A . . G . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . C . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C A . . . . A . A / d u c k / M o n g o l i a / 5 0 0 / 0 1 ( H 5 N 3 ) . . A . . A . . G . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . C . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . A . A / d u c k / M o n g o l i a / 5 4 / 0 1 ( H 5 N 2 ) . . A . . A . . G . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . C . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . A . A / d u c k / M o n g o l i a / 5 9 6 / 0 1 ( H 5 N 3 ) . . A . . A . . G . . . . . . . . C . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . T . . . C . . . . . . . . C . . C . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . A . A / d u c k / H o k k a i d o / 4 4 7 / 0 0 ( H 5 N 3 ) . . A . . . . . G . . . . . . . . . . . T . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . A . A / c h i c k e n / F R / 0 3 4 2 6 a / 0 3 ( H 5 N 2 ) . . . . . . . . G . C . . . . . . . . . T . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . G . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . A . . . . A / m a l l a r d / S w e d e n / 2 1 / 0 2 ( H 5 N 2 ) . . . . . . . . G . . . . . . . . . . . . . . . . . . C . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . A . . . A . . . . C . . . . . . . . . . . G . . . . . T . . . . . . . . C . . . . . . . A / d u c k / P o t s d a m / 1 4 0 2 - 6 / 8 6 ( H 5 N 2 ) . . . . . . . . . . . . . . . . . C . . T . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . T C . . . . . . . A . . . . . . . . . . . C . . . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . C . . C . . . . . . . . G . . . . . . . . . . . . . . C . . . . . . . A / d u c k / H o n g K o n g / 3 4 2 / 7 8 ( H 5 N 2 ) . . A . . C . . . . . . . . . . . C . . T . . . . . . . . . - - - - - - - - - - - - . . G . . . . . . . . T C . . . . . . . A . . . . . . . . . . . C . . . . . . . . G . . . . . . . . . . . G . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . A . A / s w a n / H o k k a i d o / 6 7 / 9 6 ( H 5 N 3 ) . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . T . . C . . . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . . . A / P e k i n d u c k / S g p o r e / F 5 9 / 0 4 / 9 8 . . A . . . . . . . . . . . A . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . C . . C . . . . . . . . G . . . . . . . . C . . . . . C . . . . . . . A / d u c k / M l a y s i a / F 1 1 9 - 3 / 9 7 ( H 5 N 3 ) . . A . . . . . . . . . . . A . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . C . . . . . . . . . . . G . . . . . . . . . . . . . . C . . . . . . . 350 Transcription PCR for the pathotyping of South African H5N2 avian infl uenza viruses two signals that are detectable at different wave- lengths (LightCycler® channels F2 and F3). The presence of insertions or substitutions in the H0 cleavage site of HPAI virus will however abolish the binding of InfA_H5_640 and only one signal in chan- nel F3 would thus be visible. RNA extraction and first-strand cDNA synthesis Viral RNA was extracted from tracheal swabs or in- fective allantoic fluid with a Total Nucleic Acid Isola- tion kit (Roche) using a MagNALyser and TRIzol® LS Reagent (Gibco, Invitrogen), respectively. First- strand cDNA synthesis was accomplished by incu- bating 5 μℓ RNA, 2 μℓ random hexamer and 6 μℓ dH20 at 65 °C for 10 min. After snap-cooling in an ice bath for 5 min, 4 μℓ of 5 X RT buffer, 0.5 μℓ of RNAse inhibitor, 2 μℓ 10 mM dNTPs and 0.5 μℓ Transcriptor Reverse Transcriptase® (Roche) were added. The reactions were incubated at 55 °C for 30 min. rRT-PCR A LightCycler® FastStart DNA MasterPLUS HybProbe kit (Roche) and LightCycler® 2.0 device were uti- lized for real-time PCR using 5 μℓ of the cDNA in a total volume of 20 μℓ. The temperature profile was used as follows: 10 min at 94 °C, and 45 cycles of 20 s at 95 °C, 15 s at 55 °C, and 20 s at 72 °C. Fluor- o phore-specific emission data were collected during the annealing step. Crossing point (Cp) values were calculated with LightCycler 3.5 software using the second derivative maximum method. The assay was applied to ten samples from the recent outbreaks in 2006 that were positive or suspect positive for the presence of H5 virus by rRT-PCR during surveil- lance but where virus isolation was unsuccessful (Table 2). RESULTS AND DISCUSSION The positive controls, OSZA061160 (LPAI) and OSZA06AI1091 (HPAI), produced fluorescent sig- nals in both and in one channel, respectively (Ta - ble 2). Unknown samples AI1133.11, AI1133.13, AI1133.15 and AI1120.37 were HPAI positive, where as AI1133.12 was LPAI positive. Five of the samples gave negative results, which may have been due to RNA degradation during prolonged storage. Interestingly, LPAI and HPAI were detect- ed within the same flock (AI1133 samples). OSZA04N227 (HPAI, 2004) did not amplify, due to an unusual sequence deletion detected between nucleotides 1121 and 1134 (Fig. 1) (Abolnik 2007a). (The assay has been adapted for high throughput on the LightCycler480® system by replacing the LCRed710 fluorophore with an LCRed610 fluoro- phore, and use of a one-step RT-PCR kit). In South Africa the reemergence of H5N2 in poultry is a constant threat because the LPAI strain exists in the wild bird reservoir (Abolnik 2007b) but this particular LPAI H5N2 lineage is also circulating in the European wild waterfowl population (Abolnik 2007a) and therefore after further validation the primer and probe set described here could be used in Europe since poultry in that region is also at risk of infection and consequent mutation. I have de- scribed here a rapid and sensitive method to patho- type H5-positive swabs where virus isolation has been unsuccessful. This approach of detecting the consensus LPAI H0 sequence with abolishment of binding as an indicator of the presence of an HPAI strain (provided that adjacent or upstream controls are included) could be adapted for the pathotyping of other lineages of AIV H5 and extended to hydrol- ysis probe chemistries. ACKNOWLEDGEMENTS I thank Hans-Henno Dorries for assisting with primer/ probe design, Rachel Maluleke and Bontsi Marumo- Mochothloane for technical assistance, and Truuske Gerdes and Marco Romito for useful discussions. REFERENCES ABOLNIK, C. 2007a. Molecular epidemiology of Newcastle dis- ease and avian influenza in South Africa. Ph.D. thesis. Uni- versity of Pretoria, South Africa. http://upetd.up.ac.za/UPeTD.htm. ABOLNIK, C. 2007b. Molecular characterization of H5N2 avian influenza viruses isolated from South African ostriches in 2006. Avian Diseases, 51:873–879. FERNANDEZ, GUTIERREZ, F.J., SORLOZANO, A., ROMERO, J.M., SOTO, M.J. & RUIZ-CABELLO, F. 2006. Comparison of the SYBR Green and the hybridization probe format for real-time PCR detection of HHV-6. Microbiology Research, 161:158–163. HOFFMANN, B., HARDER, T., STARICK, E., DEPNER, K., WER NER, O. & BEER, M. 2007. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus using real-time RT-PCR. Journal of Clinical Microbiology, 45:600–603. OIE TERRESTRIAL MANUAL 2004. Highly pathogenic avian in- fluenza. Paris: Office International des Epizooties. PAYUNGPORN, S., CHUTINIMITKUL, S., CHAISINGH, A., DAM- RONGWANTANAPOKIN, S., NUANSRICHAY, B., PINY O- CHON, W., AMONSIN, A., DONIS, R.O., THEAMBOONLERS, A. & POOVORAWAN, Y. 2006. Discrimination between high- ly pathogenic and low pathogenic H5 avian influenza A vi- ruses. Emerging Infectious Diseases, 12:700–701. 351 C. ABOLNIK ROTT, R. 1979. Molecular basis of infectivity and pathogenicity of myxoviruses. Archives of Virology, 59:285–298. STIENEKE-GRÖBER, A., VEY, M., ANGLIKER, H., SHAW, E., THOMAS, G., ROBERTS, C., KLENK, H-D. & GARTEN, W. 1992. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin endoprotease. EMBO Journal, 11:2407–2414. VEY, M., ORLICH, M., ADLER, S., KLENK, H-D., ROTT, R. & GAR TEN, W. 1992. Haemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the recogni- tion motif R-X-R/K-R. Virology, 188:408–413. WEBSTER, R.G., BEAN, W.J., GORMAN, O.T., CHAMBERS, T.M. & KAWAOKA, Y. 1992. Evolution and ecology of influ- enza A viruses. Microbiology Reviews, 56:152–179. WOOD, G.W., MCCAULEY, J.W., BASHIRUDDIN, J.B. & ALEX- ANDER, D.J. 1993. Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtypes. Archives of Virology, 130:209–217. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (Europe ISO Coated FOGRA27) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts false /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /Bicubic /ColorImageResolution 600 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages false /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Bicubic /GrayImageResolution 600 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [595.276 841.890] >> setpagedevice