Study of Prevalence of Bird flu by using RT PCR at Central Veterinary Laboratory, Nepal, 2007

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1 Study of Prevalence of Bird flu by using RT PCR at Central Veterinary Laboratory, Nepal, 2007 Dipesh Dhakal 1, Pawan Dulal 1, Rewati Man Shrestha 2, Salina Manandhar 2 and Janardan Lamichhane 1 1 Department of Biotechnology, Kathmandu University, Dhulikhel, Kavre, Nepal 2 Central Veterinary Laboratory, Tripureshwor, Kathmandu, Nepal ABSTRACT Avian influenza is a contagious respiratory disease caused by strain A of influenza viruses also known as bird flu viruses. These viruses occur naturally among wild, aquatic birds in their intestines without making them sick. Among 144 subtypes of influenza A virus, H5N1 is highly pathogenic and possess threat of becoming a pandemic disease. Viral strains can be detected and compared to each other on the basis of genes segments coding for proteins: Hemagglutinin(H) and Neuraminidase(N). There are different direct and indirect methods for detection of presence of these gene segments. For the purpose of the research, RT PCR technique was used because of its high sensitivity with additional benefit of less time consumption. 35 samples of different type from different bird species were collected from different regions of Nepal. Extraction of RNA from samples and their RT PCR reaction were performed as par the protocols provided with commercial kits from Quagen and Invitrogen respectively. Final results were depicted by performing agarose gel electrophoresis. Among all the samples, none showed any band equivalent to molecular weight of 150kb corresponding to that of band of H5. Thus, based on study done at Central Veterinary Lab, it was affirmed that there was not prevalence of bird flu at least at the suspected areas of Nepal till date. E mail: medipesh@gmail.com

2 INTRODUCTION Avian influenza is a contagious respiratory disease caused by strain A of avian influenza viruses (also called bird flu viruses). These viruses occur naturally among birds. Wild birds worldwide carry the viruses in their intestines, but usually do not get sick of it. However, once they are transmitted to poultry, they can cause serious economic losses arising from high mortality and trade embargo. There are numerous subtypes of influenza A viruses and many of them have infected different animals, including ducks, pigs, whales, horses, and seals. Among almost 144 subtypes of influenza A, illness in people have been caused mainly by H3N2, H2N2, H1N1, and H1N2. Most of these cases have resulted from following two ways: a) Directly from birds or from avian virus contaminated environments to people b) Through an intermediate host, such as a pig. Influenza viruses are enveloped, polymorphic, zoonotic viruses occurring in lower animals and birds as well as in humans. They belong to Orthomyxovirideae family of RNA viruses. There are four genera of this family i.e. Influenza virus A, Influenza virus B, Influenza virus C and Thogotovirus based on the antigenic differences in two of their gene products, nucleoprotein(np) and matrix(m1). These viruses have segmented negative strand RNA genomes with highly conserved 3 and 5 sequences. This genomic structure results in a high mutation rate from the error prone replication of the single stranded RNA, which is the reason for the observed antigenic drift in the surface antigens. There are two different groups for detection or study of influenza virus depending upon the techniques used and time taken for detection i.e. direct and indirect. The direct method includes: Enzyme immuno assays or Immunochromatography assays, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction (RT PCR) etc. These methods do not need isolation of viruses or their culture but use a direct technique. In indirect method sample is inoculated in a live culture system and the presence of virus infection is then detected in the culture system. Amplification of amount of virus in the culture makes the method more sensitive. This method also includes detection of virus using serological techniques. The indirect technique includes: embroynated egg culture, animal cell culture etc. RT PCR is a process whereby RNA is first converted to complementary DNA (cdna) and a section of the genome is then amplified through the use of primers that bind specifically to this target area. This allows for exponential amplification of small amounts of nucleic acid, through the action of a thermo stable DNA polymerase enzyme, which enables highly sensitive detection of minute amounts of viral genome. Not only does RT PCR have superior sensitivity but it can also be used to differentiate between subtypes and conduct phylogenetic analysis. Specimens should be processed as fast as possible after collection to prevent contamination and RNA degrading substances. In the study the target was amplification of the H5 gene segment (equivalent to molecular weight of 150kb) by using RT PCR technique with the help of specific primer designed for this purpose. MATERIALS AND METHODS Source of Sample and Sampling Method: The research focused on the analysis of samples collected from different places of Nepal like Kailali, Baitadi, Kavre, Kathmandu, Nuwakot and Chitwan comprising samples of different types that included serum, tracheal swab, cloacal swab, allantoic fluid after tracheal swab inoculation or allantoic fluid after cloacal swab inoculation. Altogether 35 different samples (Table 1) were collected from different parts of Nepal and analyzed using Reverse Transcription Polymerase Chain BSN E Bulletin Vol. 1. Oct Page 1

3 Reaction (RT PCR). For analysis of serum, 3 ml of blood was injected out from bird and serum was collected and transported to Central Veterinary Laboratory to use as sample. Similarly, tracheal swabs, cloacal swabs were collected and for increasing the virus titer, swab samples were cultured in chicken embryo for first passage and second passage. After hours of inoculation, the allantoic fluid was harvested. Protocol for virus inoculation and extraction of viral sample was as par WHO manual on Animal Influenza Diagnosis and Surveillance. Table 1. Different Samples Considered for Study Sample No Place Sample Type Identity No. Species 1. Kathmandu Tracheal swab 3.3 T1 Local Poultry 2. Kathmandu Cloacal swab 4.4 C1 Parrot 3. Kathmandu Cloacal swab 5.4 C1 Parrot 4. Taudaha Allantoic fluid from cloacal swabs 6.6 Wild birds 5. Taudaha Allantoic fluid from cloacal swabs 7.8 Wild birds 6. Taudaha Allantoic fluid from cloacal swabs 8.9 Wild birds 7. Nuwakot Allantoic fluid from tissue Poultry 8. Nuwakot Allantoic fluid from tissue Poultry 9. Nuwakot Allantoic fluid from tissue Poultry 10. Nuwakot Second passage of allantoic fluid Poultry 11. Nuwakot Second passage of allantoic fluid Poultry 12. Nuwakot Second passage of allantoic fluid Poultry 13. Nuwakot Second passage of allantoic fluid Poultry 14. Nuwakot Second passage of allantoic fluid Poultry 15. Nuwakot Second passage of allantoic fluid Poultry 16. Nuwakot Second passage of allantoic fluid Poultry 17. Nuwakot Second passage of allantoic fluid Poultry 18. Chitwan Serum K1 Poultry 19. Chitwan Serum K2 Poultry 20. Chitwan Serum K3 Poultry 21. Chitwan Serum K4 Poultry BSN E Bulletin Vol. 1. Oct Page 2

4 22. Chitwan Serum S1 Poultry 23. Chitwan Serum S5 Poultry 24. Chitwan Serum S6 Poultry 25. Chitwan Serum S7 Poultry 26. Baitadi Serum 2C Poultry 27. Baitadi Serum 7C Poultry 28. Baitadi Serum 12C Poultry 29. Baitadi Serum 13C Poultry 30. Kailali Serum 17C Poultry 31. Kailali Serum 20C Poultry 32. Kailali Serum 24C Poultry 33. Kailali Serum 30C Poultry 34. Kavre Serum Lau 1 Poultry 35. Kavre Serum Lau 2 Poultry Extraction of RNA: RNA was extracted of from different samples following protocol as in QIAamp Viral RNA Mini Kit (Qiagen GmbH, Hilden) according to manufacturer s recommendations. Primers Used: The forward and reverse primers as recommended by WHO, were used in the solution which were targeted for sequence of haemagglutinin (H5). The primers used for the reaction had specifications as given in Table 2. Table 2. Specifications of Primers Used ID Oligo Name Primer Sequence(5 3 ) Modification size MW 2 Tm AI H5 fwd GGA ATG CCC CAA ATA TGT GAA ATC D AI H5 rev TCT ACC ATT CCC TGC CAT CC D151 Preparation of Master Mix: PCR reaction was done in three batches. Therefore, master mix was prepared for three times for each batch of 15, 10 and 10 samples. Protocol for preparation of master was according BSN E Bulletin Vol. 1. Oct Page 3

5 to the manual (cdna synthesis and PCR amplification in a single tube, Invitrogen). Two extra reaction volumes were included for positive and negative control. This volume of master mix was mixed with sample and pipetted into the PCR tube which was then inserted into the wells present in the thermocycler Tabel 3. Specification of Master Mix for 1 Reaction Volume S. No. Particulars 1 Reaction Volume 1 Water 9µl 2 2 X Reaction buffer 12.5 µl 3. A1H5 forward 0.25 µl 4. A1H5 reverse 0.25 µl 5. RNA template 2 µl 6. Superscript III/Platinum Taq Mix 1 µl Total volume 25 µl Thermocycling: Thermocycler (Biometra T personal model) was used for production of cdna and its amplification. Each tube containing 25 µl of reaction mixture was subjected to RT PCR. The optimal cycling conditions were as given in Table 4. After running the thermocycler, tubes were stored at 4 o C for the following day when Agarose gel electrophoresis of the amplified product was done. Table 4. Optimized Conditions for PCR Reaction Step Temp/ Time Cycle No. RT reaction 48 o C for 30 min 1 cycle Initial denaturation 94 o C for 2 min 1 cycle Denaturation Annealing Extension 94 o C for 30 sec 50 o C for 40 sec. 68 o C for 40 sec 40 cycles Final Extension 68 o C for 7 min 1 cycle Hold 4 o C Final step BSN E Bulletin Vol. 1. Oct Page 4

6 Agarose Gel Electrophoresis: A total of 10 µl of PCR product was added to loading buffer and run on a 2.5% agarose gel (Biometra) at 95 volts for 60 minutes. After electrophoresis DNA bands were stained with ethidium bromide and visualized with UV transillumination. RESULT The oligonucleotide primer set as specified in Table 2 was used for the amplification of H5 gene corresponding to the molecular weight of 150bp. Sample were loaded in each well as indicated by their number specified on Table 1. Size Marker (100bp DNA Ladder, Promega), Positive control (Serum sample of infected bird from Austria) and negative control (Sigma water) were also run along with the samples. Positive control showed a band at level equivalent to 150bp. None of the other lane showed band corresponding to region between 100bp and 200bp.The band profile for different samples were as shown in Fig 1, Fig 2 and Fig 3. Figure 1. Result of Samples 1 15 Figure 2. Result of Samples BSN E Bulletin Vol. 1. Oct Page 5

7 Marker Figure 3. Result of Samples DISCUSSION In PCR technique, if the desired gene (here H5) is present it is amplified during the PCR and the amplified gene segment can be visualized by the gel electrophoresis. In the study, the result indicates that there is not presence of any band corresponding to molecular weight of 150 kb which is trademark of H5. This is shown clearly by Fig 1, 2 and 3. The study was focused on analysis of only 35 samples from selected regions of Nepal and most of the samples were collected from suspected dead birds. Since, there is not presence of H5 gene segment thus, none of the sample was found to be positive for the H5 strain of virus and it can be clearly depicted that there is not prevalence of H5N1 at least in the areas from where sample was collected for analysis. So, based on the experiment carried out in Central Veterinary Laboratory, it can be confirmed that there is not prevalence of bird flu in those suspected areas of Nepal till date. ACKNOWLEDGEMENT This study was supported by Molecular Biology Lab, Central Veterinary Laboratory, Tripureshwor, Nepal, World Health Organization (WHO) and Department of Biotechnology, Kathmandu University, Nepal and we express our gratitude for their generous support. We are also indebted to entire staff of Central Veterinary Lab; faculties of Department of Biotechnology, Kathmandu University; our colleagues and supporters. REFERENCES 1. Allwinn, R et.al. (2002) Laboratory diagnosis of influenza virology or serology? Med Microbiol Immuno (Berl) 191: BSN E Bulletin Vol. 1. Oct Page 6

8 2. Fouchier, R.A. et al. (2005) Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black headed gulls.journal of Virology 79: Holland, J. et al. (2002) Rapid evolution of RNA genomes.science 215 (1982): Hulse, D.J. et al. (2004) Molecular determinants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens. Journal of Virology 78 : Meijer, A. (2006)Importance of rapid testing to combat the global threat of bird flu. European Influenza Surveillance scheme (EISS) 6: Martin, V. et al, (2006) History and Evolution of HPAI Viruses in Southeast Asia. Annals of the New York Academy of Sciences 1081(1): Nicholson,K.G.(2003) Influenza Lancet 362: Padhi, S. et al. (2004) Avian Influenza A (H5N1): A Preliminary. Indian Journal of Medical Microbiology 22(3): Pattnaik, B.et.al. (2006)Phylogenetic analysis revealed genetic similarity of the H5N1 outbreaks in chickens in Maharashtra, India with those isolated from swan in Italy and Iran in Current Science 31: Payungporn,S.(2004) Single Step Multiplex Reverse Transcription Polymerase Chain Reaction(RT PCR) for Influenza A Virus Subtype H5N1 Detection.Viral Immunology 17(4): Poddar,S.K.(2002) Influenza type and subtype detectionby single step single tube multiplex reverese transcription Polymerase chain reaction(rt PCR) and agarose gel electrophoresis. Journal of Virological Methods. 28: Purification of Viral RNA(Spin Protocol), amp Viral RNA Mini Handbook 2005 ed: Sawayne, D. E. (2006) Principles for Vaccine Protection in Chickens and Domestic Waterfowl against Avian Influenza Emphasis on Asian H5N1 High Pathogenicity Avian Influenza Ann. New York Academy Sciences 1081: Senne, D.E et al. (1996) Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the cleavage site as a marker of pathogenicity potential. Avian Diseases 40: Steininger, C. et al.(2002) Effectiveness of reverse transcription PCR, virus isolation, and enzymelinked immunosorbent assay for diagnosis of influenza A virus infection in different age groups. Journal of Clinical Microbiology 40: Stevens, T.D. et al. ( 1969) Rapid identification of viruses by indirect immunofluorescence: standardization and use of antiserum pool to nine respiratory viruses. Application Microbiology17; Wang, W et al. (2006) Fatal infection with influenza A (H5N1) virus in China.N. Engl. J. Med 354.: BSN E Bulletin Vol. 1. Oct Page 7

9 18. WHO recommendations on the use of rapid testing for influenza diagnosis (2005) World Health Organization. Department of Communicable Disease Surveillance and Response 19. WHO manual on Animal Influenza Diagnosis and Surveillance (2002). World Health Organization Department of Communicable Disease Surveillance and Response BSN E Bulletin Vol. 1. Oct Page 8