Abstract

The RNA disease, Avian pneumovirus affects turkeys, chickens and pheasants all over Europe. The virus spread into the United States in the early eighties, and many diagnostic tests have been tested to determine the differences between the United States strain and the European strain. This study concentrated on a diagnostic test run to determine whether primers worked correctly. The diagnostic test was known as a polymerase chain reaction (PCR). The diagnostic tests were run on DNA, but because APV is an RNA virus, APV was first transformed into DNA, and then the PCR was performed. The PCR determined the number of base pairs in the virus’ DNA genome, showing that the primers worked correctly. Evidence of 280 base pairs was found in the virus strain. Eventually, the entire strain will be sequenced, and a vaccination will be made to prevent future breakouts and the deaths of many animal flocks in the U.S. and Europe.

Introduction

The purpose of this project was to sequence the Avian pneumovirus genome. With the knowledge gained from the gene sequencing, the molecular structure can be determined. The molecular structure and DNA sequence will allow ways to see how Avian pneumovirus has evolved since the time if its discovery and help determine how the changes in the disease will continue to affect the virus. Avian pneumovirus presents an economic problem because it has become epidemic in turkeys, chickens and pheasants. An improved vaccination will greatly help. The sequencing will allow for improved diagnostic tests and nucleotide primer sequencing. The genome information will also help improve vaccinations. The question I faced was whether or not the diagnostic tests I ran would work and be able to sequence the DNA of Avian Pneumovirus.

Background

Avian Pneumovirus is an upper respiratory tract viral disease. The disease was first found in Europe in the late 1960s. The disease was first found in America in 1996, in Colorado. The Avian pneumovirus strain of the disease is different than any other strain found in Europe or any other country. Avian Pneumovirus (APV) belongs to the Paramoyxoviridae family and the Pneumovirinae sub-family. The virus is a single-stranded ribonucleic acid (RNA). Its selective nature makes the virus very hard to control. The virus affects turkeys, chickens and pheasants of all ages. Young (3-10 week-old) turkeys seem to be infected the most. The older birds tend to be more resistant. Pigeons, geese and ducks appear immune to the virus (Clanek, 1997). Between July and December of 1985, nearly every flock of susceptible birds in England and Wales was infected with the virus. The disease is also known as Swollen Head Syndrome and Turkey Rhinotracheitis (Jordan, 1996).

APV has very high mortality rates, which can reach up to 90-100% (Jordan, 1996). The extremely high rate of mortality is due to secondary breakouts, resulting from bacterial infections caused by Bordetella avium and Escherichia coli (Clanek, 1997). The causes of secondary breakouts are also attributed to poor ventilation and general hygiene, overstocking, mixed age groups, poor litter conditions, and wet, damp areas (ibid).

Avian Pneumovirus is spread by mechanical means and contact, and it may transmitted through the air, but that is not certain (Jordan, 1996). Symptoms include: snicking, sneezing, wet coughs, nasal discharge, rales, foamy conjunctivitis, swollen infraorbital sinuses, depression, gasping, and change in voice (Clanek, 1997). Avian Pneumovirus allows lowers the egg production rate significantly (ibid). The rapid onset of the virus makes it hard to control and isolate, making the virus epidemic in some regions of the world (ibid).

Diagnosis of APV can be determined many ways. Serologic tests such as immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and serum neutralization (SN) are commonly used to detect antibodies. Another diagnostic test is polymerase chain reaction (PCR). PCR locates and replicates specific deoxyribonucleic acid (DNA) and RNA sequences (Virida, 1996). PCR will detect nucleic acid sequences particular to a certain virus and amplify the sequence millions of times (Virida, 1996). The DNA or RNA sequences are temperature controlled by enzymes which start molecular reactions. The PCR works by unwinding base pairs of DNA when the system is heated. The primers then begin the sequencing of nucleotides and polymerase (Virida, 1996). The product is then cooled for base pairing. As seen in Figure 1, four nitrogenous bases bind together with the help of correctly sequenced primers. The sequence of the primers helped to determine the 280 order of bases in the Avian pneumovirus genome. The PCR cycle is repeated, doubling the gene sequence, producing many copies of the original DNA or RNA sequence. The gene sequence is synthesized and then can be used to diagnose a specific viral infection (Virida, 1996).

Figure 1

Picture from: The Incredible Machine published by The National Geographic Society.

There is no cure for Avian pneumovirus, but antimicrobial drugs such as neomycin, amoxycillin, quinolones, and trimethopirm-sulphonamide combinations can be used to help suppress secondary breakouts (Jordan, 1996). Another preventative actions are vaccinations. If the birds are infected with a weak strain of the virus, their body will produce antibodies to protect them from being infected with APV. If birds become in contact with the virus, their body will have already produced the necessary protection against the disease. The problems with vaccinations results are due to immunosuppressive organisms which restrict the vaccination, and the virus’ antigenic variation which makes it difficult to create a specific vaccination (Clanek, 1997).

Methods and Materials

Virus Isolation

The chicken embryo fibroblast (CEF) tissue cells were inoculated for virus isolation. The tissues were homogenized in 2% Hank’s fetal bovine serum (FBS) by a stomacher machine to produce an approximately 20% suspension. The tissue/Hank’s suspension was centrifuged at 4200 rotations per minute (rpm) for one hour and then the supernatant was poured off. To inoculate, the media was transferred into two wells of a twenty-four well plate, which already contained a monolayer of cells. The supernatant was added to the cells and was incubated for one hour. The cells were washed three times. A squirt of Hank’s balanced salt solution, containing the solution of Antibiotics, was then added along with 4% FBS. The plates were checked daily for contamination, toxicity, and cytopathic effect (CPE). Once CPE occurred, the isolate was used to extract RNA.

RNA Extraction

The RNA was extracted by performing a purification of the viral RNA. To extract RNA, 140 µL cell-culture supernatant, which was thawed, was pipetted into a 1.5 mL microfuge tube. Then, 560 µL of prepared Buffer AVL, containing carrier RNA, was added to the sample, which was mixed by vortexing. The wells were incubated at room temperature for ten minutes, and 560 µL of ethanol (96-100%) was added to the sample and vortexed. A spin column was placed in a 2 mL collection tube. Then, 630 µL of the serum solution was carefully added to the spin column and centrifuged at 8000 rpm for one minute. The filtrate was poured off, and the step was repeated until the sample was completely used. The sample was then heated to 80°C for ten minutes. The moisture collected in the top of the spin column was wiped off, and the column was carefully opened in order to add 500 µL of washing buffer. The column was centrifuged for one minute and placed into a clean 2 mL collection tube. The filtrate was then discarded. The column was carefully opened, and another 500 µL of washing buffer was added and centrifuged for three minutes at full speed. The column was placed in a clean 1.5 mL microfuge tube, and the collection tube containing the filtrate was discarded. The column was carefully opened and eluted with 50 µL of preheated (80°C) RNAse-free water. Then, the column was centrifuged at 8000 rpm for one minute (QIAGEN).

Reverse Transcription (RT)

To change the RNA in the microfuge tube to DNA a reverse transcription was performed. To, 5 µL of the RNA template, retrieved from the viral extraction, 2.5 µL 10x PCR buffer, 5 µL 25mM solution of MgCl₂, 1 µL random hexamers, 0.1 µL Superscript II reverse transcriptase, 1 µL RNAse inhibitor, 6.4 µL diethyl pyrocarboante (DEPC) water, and 4 µL dNTP were mixed in a 2 mL microfuge tube. After the reverse transcription, a polymerase chain reaction (PCR) was run. The complementary DNA (cDNA), which was produced during the reverse transcription, was used as the RNA template during the polymerase chain reaction.

Polymerase Chain Reaction

In order to prepare the PCR, 2.5 µL of PCR buffer, 3 µL of MgCl₂, 0.5 µL Taq (DNA polymerase isolated from Thermus aquaticus), and 4 µL of dNTP (deoxynucleotide triphosohate) were added to four 2 mL microfuge tubes. In the tube labeled number one, 1 µL of the MF1 forward primer and 1 µL of MR3 reverse primer (primers were either forward (F) of reverse (R)) were added along with 4 µL of cDNA, and 9 µL of DEPC water. In the tube labeled number two, 1 µL of the MF1 forward primer and 1 µL of the MR3 reverse primer were added, along with 0.1 µL of PCR product and 12.9 µL of DEPC water. The tube labeled number three was the positive control, so 1 µL of 2F forward primer and 1 µL of 2R reverse primer were added, along with 4 µL of cDNA and 9 µL of DEPC water. The tube labeled tube number four was the negative control, so 1 µL of 2F forward primer, 1 µL of 2R reverse primer, and 13 µL of DEPC water were added. The four tubes were placed in the PCR machine and the temperatures were set to the following degrees: 94°C for two minutes; 94°C for one minute, 49°C for one minute, 72°C for two minutes, 72°C for ten minutes and 4°C for infinity. The 25 µL product was cycled 35 times in the machine. The products were run on a 1% agarose gel for 30 minutes.

Gel

To prepare the gel, 0.5 g of agarose LE was added to 50 mL of TAE buffer, which was microwaved on high for two minutes. Then 0.5 µL Ethidium bromide was added and the gel was poured and cooled until it solidified. A 100 kilobases pair ladder was added to the first well, along with 4 µL of tetracycline loading buffer which was added to each 5 µL sample collected from the PCR. The gel was run on an electrophoresis machine at 106 amps for 30 minutes.

Sequencing

After the product was located, the gene was cleaned, cloned, and the plasmid was grown. The plasmid was then extracted, and the DNA insert was placed into the cell. Primers were then used to complete the sequencing. This part of the methods was performed by Arshud Dar, my Ph.D. mentor, at the University of Minnesota.

Results

By aligning a base-pair ladder to the gel, it was determined that 280 base pairs of APV were sequenced using the procedure described in this project. As seen in the photograph¹ shown in Figure 2, columns one and two show the positive controls used in the experiment, which used primers 2F and 2R² that previously sequenced 107 base pairs of another genome. As seen in columns three and four identical numbers of base pairs of APV genomes were sequenced.

Figure 2.

Discussion

In designing primers, the sequence of base pairs must be predicted from trial and error. If the sequence is correctly predicted, the base pairs will be seen on the gel. Many factors influence the result of a PCR, but the most important factor is that the sequences of the 2F and 2R primers are correct. The diagnostic PCR test determined that the new primer sequences were complementary to the previously known sequence of bases and could further sequence the base pairs. The number of base pairs sequenced in columns one and two was previously known, so by using the genomes as a positive control, it was determined the primers 2F and 2R worked to sequence APV. In this project the results show that the primers were correctly sequenced and that 280 base pairs were sequenced of APV.

The next step in this research project is to extract the APV genome and place it in a plasmid for additional sequencing. After the sequencing, the knowledge of Avian pneumovirus can be used to create a vaccination to prevent epidemic breakouts.

Bibliography

Clanek, B.W., ed. 1997. Diseases of Poultry. 10th ed. Ames: Iowa State University Press.

“Disease.” Encyclopedia Britannica: Children’s Encyclopedia. 1988 ed.

Foo, Ivy. 1991. “Isolation, Identification and Amplification of DNA from Naturally Mummified Remains.” Thesis. University of Minnesota.

Gould, James & Keeton, William T. Biological Sciences. 6th ed. New York: W.& W. Norton & Company, 1996.

Jordan, F.T.W., ed. 1996. Poultry Diseases. 4th ed. Philadelphia: W.B. Saunders Company Ltd.

Raineri, Deanna, et al. University of Illinois. Gel Electrophoresis. Netscape. Molecular Biology Cyberlab. Internet. 15 Dec. 1998. Directory: life.uiuc.edu/molbio/geldigest/electrc.html

“RNA Isolation and Purification.” QIAGEN. 16 Dec. QIAGEN Online. Webcrawler. 16 Dec. 1998.

The National Geographic Society. The Incredible Machine. Washington D.C.: National Geographic Society, 1986.

QIAamp Viral RNA Handbook. Valencia: QIAGEN, Inc., 1997.

Viridae Clinical Sciences. “Polymerase Chain Reaction.” Viridae Clinical Sciences 12 Jul. 1996: Viridae Clinical Sciences, Inc. Online. Altavista. 27 July 1998.

“Virus.” Encyclopedia Britannica: Children’s Encyclopedia. 1988 ed.

¹ The PCR reaction photograph was taken by Arshud Dar.

² Primers (2F and 2R) were designed by my mentor, Arshud Dar, to detect RNA sequences which were extracted from the virus.