Molecular Study on The Pathogenicity of Avian Influenza Virus

Highly pathogenic avian influenza virus (HPAI) differ from Low pathogenic avian influenza virus (LPAI) based on multiple basic amino acid motif of the carboxylterminus of HA1, especially arginine and lysine. The propose of this work was toamplify and sequence the cleavage site region of HA gene of avian influenza virusisolated from both cases with characteristic or unspecific lesion, using reversetranscriptase polymerase chain reaction (RT-PCR). Primer desaigned for amplification and sequence was H5-F: 5’ ggagactcagcaatcccatgaaaag 3’ and H5R:5’ccataccaaccgtctaccattcc 3’, and expected product size was 246 bp. The result indicated that all avian influenza virus (AIV)-isolates originated from chicken with both specific and non specific lesion show a multiple basic amino acid motif -PQRERRRKKR//GLFand classified as highly pathogenic avian influenza. Philogenetic study of HA genefragment indicated that each type of characteristic lesion created philo-groups.


Introduction
Highly pathogenic Avian Influenza (HPAI) outbreak has been reported in Indonesia since 2003 and still continuously circulated, due to H5N1 sub-type.The field examination focused on clinical signs of the disease that indicated some changes in macroscopic lesions.Formerly the bird exhibited characteristic symptoms, e.g.: high mortality rate, hemorrhages on shank and hock joint, congestion and cyanosis of comb and wattle, which can be followed by edema projection called Hemagglutinin (HA) and Neuraminidase (NA).Approximately 80% o f t h e s u r f a c e ' s p r o j e c t i o n s a r e Hemagglutinin and the remaining Neuraminidase (McCaulay and Mahy, 1983).Antigenic properties of HA and NA glycoprotein determine the sub-type specifity of AIV.Currently, it has been recognized 16-H and 9-N (Suzuki and Nei, 2002;Fourchier et al., 2005).Hemagglutinin is viral glycoprotein which is res-ponsible for both receptor binding and membrane fusion that in turn will facilitate the infection (Matrosovich et al., 1999;Mitnaul et al., 2000;Suzuki et al., 2000).In addition to its binding and fusion functions it is the primary target for neutralizing antibodies.
Although the pathogenicity of AIV is p o l y g e n i c t r a i t , t h e H A ' s s u r f a c e glycoprotein still play a central role (McCauley and Mahy, 1983;Ito et al., 2000).The process of the infection, HA will be cleaved by certain protease enzyme into HA1 and HA2 sub-unit.The HA1 is receptor binding protein as the major target of immune responses, whereas HA2 is an anchor protein of the envelope and mediates fusion of the envelope and the cellular endosomal membrane (Suzuki and Nei, 2002).Another worker emphasize that the cleavage process on HA region will determine further infection.
Cleavage site region of HPAI viruses differ from those of avirulent influenza a viruses by the virtue of possessing multiple basic amino acid of the carboxyl terminus of HA1, especially arginine and lysine.This feature permits cellular protease such as furine will recognize multiple basic amino acid, to cleave the HA and render the virus infection and able to spread to a variety organs, leading to systemic infection.Whereas most avirulent strain have a single arginine or monobasic cleavage site that will cleaved only by tripsin which secreted from cells in the respiratory and intestinal tract, so the virus only produced localized infection (Harimoto and Kawaoka, 2001;Ito et al., 2001).Specific motif consisting of a basic amino acid series at the cleavage site is RRRKKR//GL- (Dharmayanti et al., 2005) or -RRRKKR//G- (Harimoto and Kawaoka, 1994).Most of the motif has been identified for AIV H5N1 sub-type in Indonesia.The presence of the polybasic amino acid region is important as pathotypic marker of AIV (Bank and Polywright, 2003).
Based on fascinating fact, the aim of this study is to determine the molecular pathogenicity marker of both AIV isolates by amplifying and sequencing the cleavage site region of the hemagglutinin.

Materials and Methods
Ten isolates have been identified using RT-PCR.Four isolates were obtained from birds without specific symptoms.Six isolates were obtained from birds that indicated characteristic lesion of avian influenza virus infection.

Viral RNA extraction
Viral RNA was extracted from allantoic fluid using RNA extraction kit Invitrogen Pure Link™ Micro to Midi 50xRxn Total RNA Purification System (Cat.no: 12183-018) according to the manufacturer's protocol.To purify total RNA from 0.2 ml allantoic fluid, in a 1.5 ml RNAse-free micro centrifuge tube was added 0.2 ml of RNA lysis solution contain 1% (v/v) 2mercaptoethanol.The mixture was vortexed throughly to disrupt lyse blood cells, and centrifuged at 12,000xg for 2 min at room temperature.Supernatant was transferred to clean 1.5 ml RNAse-free microcentrifuge tube and 200 ml of 100% ethanol was added.Any precipitate was dispersed by vortexing or pipetting up and down several times.The sample was transferred to the RNA spin cartridge, centrifuged at 12,000xg for 15 s at room temperature.The flow-through was discarded.This step was repeated once.The spin cartridge was centrifuged at 12,000xg for 1 min at room temperature to dry the membrane, and the cartridge was moved into an RNA recovery tube.Fifty microliters of RNase-free water was added to elute the RNA and incubated for 1 min at room temperature then centrifuged for 2 min at 12,000xg at room temperature.This step was repeated once, after that the cartridge was discarded and elutes was stored at -4°C.

Thermocycling condition
One step Reverse transcriptase-PCR (RT-PCR) was carried out in GeneAmp® PCR System 2400 machine and performed using Invitrogen SuperScript™ III One-Step RT-PCR System.Cycling conditions included a reverse transcription step at 50°C for 30 min.After an initial denaturation step at 94°C for 5 min, amplification was performed during 40 cycles including denaturation (94°C for 30 s), annealing (50°C for 1 min) and extension (68°C for 45 s), followed by final extension at 68°C for 5 min.Primer designed for this amplification and s e q u e n c e w a s H 5 -F : 5 ' ggagactcagcaatcccatgaaaag 3' and H5-R: 5'ccataccaaccgtctaccattcc 3', which has expected product size was 246 bp (Ito et al., 2001).

Electrophoresis of PCR product
PCR product were electrophoresis in 1.5% agarose gel in 1x TBE, using Electrophoresis tank (MSMIDIDUO, Cleaver Scientific Ltd), at 100V for 40 min, and read under UV transillumination at 302 nm wave length to determine the size of PCR product fragment.

Sequencing of PCR product
F r a g m e n t H A s e q u e n c i n g w a s performed with Dye Terminator Cycle Sequencing method using ABI PRISM 377A model version 3.4.1.All process was done at BPPT, Gene Technology Laboratory, Tangerang, Jawa Barat.

Sequence Analyses
Nucleotide sequences were analyzed by using Clustal W version 1.83 and Phyllip version 3.65 to determine their genetic relationship.

Discussion
In order to AIV molecular identification, the important and basic thing in this study w a s t o u n d e r s t a n d v i r u s i s o l a t e s pathogenicity HPAI similarity which taken from different clinical character cases, by identified the amino acid motif on cleavage site region.We found polybasic amino acid region on HPAI that especially being filled by arginine and lysine.In LPAI virus, in that restricted site only have single arginine, which is why this motif could be used as AIV pathotypic marker (Kawaoka et al.,1984;Swayne and Suarez, 2000;Banks and Polywright, 2003).The amplification of cleavage site of HA gene fragment at position 775 to 1021 was based on Ito et al. (2001), and the result can be seen in the Figure 1.
All 10 RT-PCR products were sent to BPPT to have them sequenced.Only 7 out of 10 samples give good results amino acids sequences at cleavage site deducted from  1).
Table 1.Translation of amino acid sequence at cleavage site region of seven AIV isolates.
According to Senne et al., (1996), minimal motif amino acid concept is BXBR//, which B is basic amino acid, X is non basic amino acid and R is arginine that related to restriction area (sign //).Follow in amino acid level conversion, samples number 1, 2, 3 and 9 that are virus isolates from unspecific lesion case have same motif with samples number 4, 5, and 7 from characteristic case of AI disease.Amino acid motif in the restriction area above (Tab-2) fulfill the minimal concept of HPAI virus according to Senne et al., (1996) and it still consistent with few kind of studies that reported by another researcher.
In general amino acid motif at cleavage site of HPAIV-H5 is: -RRRKKR//GL- (Dharmayanti et al., 2005) or -RRKKR//GL- (Ito et al., 2001).Basic amino acid series in this study have same motif with isolates that reported by Zhou et al., (1999) to upstream-10: -PQRERRRKKR//.The origin of this basic amino acid series was from human isolate (A/Hong Kong/156/97), 11 isolates from chicken, i.e.: A/chicken/Hong K o n g / 1 9 9 7 a n d 2 d u c k i s o l a t e s (A/Duck/Hong Kong/1997) that isolated in Hong Kong (1997).All isolates have been confirmed as AIV pathogenic for chicken based on the pathogenic marker of the cleavage site position.This amino acid motif in this study show a bit different with motif that reported by Senne et al. (1996).In the cleavage site, we found 3 different pattern that showed until upstream-10 and downstream+3: -PQ-RKRKKR//GLF or - (Senne et al., 1996).The first motif is very clear because of basic amino acid repetition to upstream-6, whereas the second motif still consistent in minimal concept: BXBR//.In the third motif, amino acid position on upstream-2 filled by Threonine (T) that belonging to alcohol group, replacing Arginine or Lycine.This unusual phenomenon still considered as HPAI because of the basic amino acid repetition in upstream-3 to -6.In his view the second position became unimportant, as far as this motif still has a repetition of basic amino acid in upstream -3 to -6.
Most AIV have Arginine (R) on terminal carboxy HA-1 at upstream-1 position,