The methods for the diagnosis of Avian Influenza
Two major methods including virus isolation (VI) and reverse transcriptase – polymerase chain reaction (RT-PCR) were adopted for the diagnosis of avian influenza (AI) in AHRI. They are basing on the tests of chapter 2. 3. 4 of "Manual of Diagnostic Tests and Vaccines for Terrestrial Animals” which was published by World Organization for Animal Health (OIE) (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf).
1. Virus Isolation (VI)
This method was implemented for routine diagnosis of field samples and detecting samples of active surveillance.
(1) The procedure is as following:
The supernatant fluids of faeces or tissue suspensions obtained through clarification by centrifugation at 1000 g are inoculated into the allantoic sac of three to five embryonated SPF chicken eggs of 9–11 days’ incubation. The eggs are incubated at 37°C (range 35–39°C) for 2–7 days. Eggs containing dead or dying embryos as they arise, and all eggs remaining at the end of the incubation period, should first be chilled to 4°C for 4 hours or overnight, and the allantoic fluids should then be recovered and tested with a screening test (such as haemagglutination [HA] test) and influenza A subtype-specific test (such as haemagglutintin inhibition [HI] tests or reverse transcriptase polymerase chain reaction [RT-PCR] test). Fluids that give a negative reaction will be passaged into one further batch of eggs.
(2) Assessment of pathogenicity
A. Chicken lethal test
Eight 4- to 8-week-old susceptible chickens were intravenously inoculated with 0.2 ml of a 1/10 dilution of a bacteria-free, infective allantoic fluid. These chickens were observed for 10 days. When chickens are too sick to eat or drink, they will be sacrificed humanely during the observed period. Any influenza virus that is lethal for six, seven or eight of eight inoculated chicken within 10 days, will be determined as a HPAI virus. Conversely, a further analysis of the sequence of haemagglutinin proteolytic cleavage site will be taken. If H5 and H7 isolates that are not virulent for chickens and do not have an HA0 cleavage site amino acid sequence similar to any of those that have been observed in HPAI viruses are designated as low pathogenicity avian influenza (LPAI).
B. The sequence of haemagglutinin proteolytic cleavage site
The heamagglutinin proteolytic cleavage site of HA0 protein gene for H5 and H7 isolated viruses will be amplified by RT-PCR. Then the amplified products will be performed a further sequencing. Basing on their deduced amino acid sequences, if the sequence of HA0 cleavage site containing more than 4 basic amino acids or the sequence is similar to that observed for other highly pathogenic AI isolates, the isolate being tested will be considered to be highly pathogenic (see the table that lists all the reported heamagglutinin proteolytic cleavage site of HA0 protein for H5 and H7 LPAI and HPAI viruses based on deduced amino acid sequence, which can be found on the OFFLU site at: http://www.offlu.net/index.php?id=123). Conversely, if H5 and H7 isolates that are not virulent for chickens and do not have an HA0 cleavage site amino acid sequence similar to any of those that have been observed in HPAI viruses are designated as low pathogenicity avian influenza (LPAI).
2. Reverse transcriptase – polymerase chain reaction (RT-PCR)
RT-PCR techniques on clinical specimens can, with the correctly defined primers, result in rapid detection and subtype identification (at least of H5 and H7), including a cDNA product that can be used for nucleotide sequencing. Modifications to the straightforward RT-PCR method of detection of viral RNA have been designed to reduce the effect of inhibitory substances in the sample taken, the possibility of contaminating nucleic acids and the time taken to produce a result.
A. Universal primers
To detect avian influenza virus by RT-PCR, two primers based on conserved sequences of the NP gene of viruses of avian, human, swine and equine origins were used. The NP-specific primers are:
NP1200 (forward): 5’-CAG(A/G)TACTGGGC(A/T/C)ATAAG(A/G)AC-3’,
NP1529 (reverse): 5’-GCATTGTCTCCGAAGAAATAAG-3’
B. Subtyping primers
To subtype avian influenza H5 and H7 viruses by RT-PCR, 2 sets of primers, each based on conserved sequences of a single HA subtype were used.
The H5-specific primers are:
H5-155f (forward): 5’-ACACATGCYCARGACATACT-3’
H5-699r (reverse): 5’-CTYTGRTTYAGTGTTGATGT-3’
The H7-specific primers are:
H7-12f (forward): 5’-GGGATACAAAATGAAYACTC-3’
H7-645r (reverse): 5’-CCATABARYYTRGTCTGYTC-3’
RT-PCR was carried out in a reaction mixture (25 μl) containing 2.5μl of 10-times reaction buffer (Promega, Madison, WI), 2.5μl dNTP blend (2.5 mM each of four dNTPs, Promega), 0.2μl AMV reverse transcriptase (9 units/μl, Promega), 0.3μl RNase inhibitor (40 units/μl, Promega), 0.5μl Taq DNA polymerase (9 units/μl, Promega), 1μl of each primer (10 pmol each), 1μl of RNA template (about 1 ng), and 17μl of water. The PCR condition for the amplification of H5 and H7 was 42°C for 45 min (reverse transcription), 95°C for 3 min, 35 cycles of 95°C for 30 s (denaturation), 50°C for 40 s (annealing) and 72°C for 40 s (extension), followed by 72°C for 10 min (final extension). Products of the RT-PCR were analyzed by agarose gel electrophoresis. The predicted length of the RT-PCR amplicon for H5 and H7 was 545 bp and 634 bp respectively.
(3) DNA sequencing
The amplified DNA fragments were purified by using a QIAquick PCR purification kit. Purified DNA fragments were sequenced from both directions (using the same primers that amplify the DNA), by an automatic sequencer.