Development of an Improved Avian Pneumovirus Vaccine Differentiable from Wild Virus

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1 Development of an Improved Avian Pneumovirus Vaccine Differentiable from Wild Virus Sagar M. Goyal Professor of Virology Department of Veterinary Population Medicine College of Veterinary Medicine University of Minnesota 1

2 Introduction Avian metapneumovirus (ampv) belongs to the genus metapneumovirus, subfamily pneumovirinae and family Paramyxoviridae (Pringle, 1998). The ampv was first reported in the USA in 1996 in Colorado and subsequently was isolated from infected turkey flocks in Minnesota in 1997 (Goyal et al., 2000). This virus posed serious economic threat to Minnesota turkey growers and induced a disease characterized by upper respiratory tract infection and loss of egg production. The increased cost of turkey production is due to the cost of vaccination and to the use of antibiotics to control secondary bacterial infections. Birds such as pheasants, guinea fowl, Muscovy ducks, and Canada geese can also be infected (Bennet et al., 2002). The ampv has also spread to neighboring states of Minnesota e.g., North Dakota, South Dakota and Iowa (Lauer, 2000). To control ampv infections, a commercial vaccine using a live attenuated strain of ampv is available. This vaccine was developed after 63 passages (p63) of ampv (strain MN 1a) in Vero cells (Patnayak and Goyal, 2004) and is currently being used in Minnesota. A lower passage (p41) of this virus (41 passages in cell cultures) was also found to be safe and effective as a vaccine experimentally (Gulati et al., 2001; Patnayak et al., 2004). This virus (p41) was subsequently adapted to grow at suboptimal temperatures (after 24 additional passages) and this cold adapted (ca) strain was also found to be an effective vaccine under experimental conditions (Patnayak et al., 2003). Although the current vaccine based on p63 is effective in preventing disease, there is no marker associated with this vaccine and hence vaccinated flocks cannot be differentiated from flocks that are infected with wild virus. This information is essential in ultimate control and eradication programs. The development and use of a gene deleted vaccine by molecular manipulation of the ampv genome should prove useful in this regard. Hence, the objective of this study was to sequence and compare the whole genomes of five ampv strains to determine differences in the gene sequences of vaccine and wild ampv strains. The ampv is a negative stranded RNA virus and its genome consists of eight genes (N, P, M, F, M2, SH, G, and L) that are flanked by the leader and trailer regions in the 3 and 5 ends of the genome, respectively (Govindarajan and Samal, 2005). Materials and Methods Viruses: Avian metapneumovirus MN 1a and MN 2a were isolated from an outbreak in turkeys in Minnesota in 1997 and MN1a was adapted in chicken embryo fibroblast cells for seven passages and subsequently in Vero cells for 56 passages to derive the vaccine virus, p63. MN 15a was isolated from a wild Canadian goose (Bennett et al., 2005). The isolation and details of subsequent passages of all six viruses used in this study are given in Table 1. Primers: For amplification of all eight genes, overlapping primer pairs (Table 2) targeting the respective genes or part of the genes were designed using Primer 3 software ( and synthesized. 2

3 Reverse Transcription and Sequencing of N, P, M, F, M2, SH, and L genes: Viral genomic RNA was extracted from clarified cell culture supernatants using viral RNA extraction kit (Qiagen, Valencia, CA). The N, P, M, F, M2, SH, and L genes were reverse transcribed and amplified using OneStep Reverse Transcription-Polymerase Chain Reaction (RT-PCR) kit (Qiagen, Valencia, CA). The RT-PCR products were separated by electrophoresis (1.5 % agarose) and the DNA bands of expected sizes were cut and eluted using QIAquick Gel Extraction kit (Qiagen, Valencia, CA). The products were sequenced with gene specific primers from both directions as well as with internal primers, if needed at Advanced Genomic Analysis Center (AGAC), University of Minnesota. Reverse Transcription and Sequencing of G gene: The attachment glycoprotein (G) of ampv is one of the major surface proteins and based on the gene sequence and antigenic differences, the ampv is classified into four subtypes A, B, C, and D. Owing to the higher G+C content and resulting secondary structures, there has been considerable difficulties in sequencing the G gene from viral genomic RNA. (a) Amplification of G gene of MN 2a virus by cold shock technique: To open the secondary structure formed in GC rich region cold shock technique was used. For this, extracted viral RNA was immersed in liquid nitrogen (LN 2 ) for 5 min and then immediately transferred to water 37 C for 1 min. This was repeated 10 times. Then 5 ul of RNA was subjected for OneStep RT-PCR (Qiagen) using GF5982 and GR7986 primers. PCR products were separated by gel electrophoresis and purified using QIAquick Gel Extraction kit (Qiagen, Valencia, CA). For sequencing, PCR products were sequenced with both GF5982 and GR7986 primers with 5% DMSO. (b) Amplification of G gene of P9, P63, P41 and Ca virus: The G gene of these viruses was amplified with two sets of primers. The first set of primer (GFII and GR7022) targeted the 3 half of the gene, from nt 5837 to nt 7022 (nucleotide numbers were based on the ampv genome subtype C CO; AY590688) and the second set of primer (G513 and GR7986) targeted 5 half of the gene, from nt 6650 to nt This portion contained the highest percent of G+C content of the gene. To amplify these targets viral RNA was incubated in salt solution followed by PCR. To amplify the 3 half of the gene, 20 µl of viral RNA with 20 μl of 5M NaOH (prepared in sterile DEPC treated water) and incubated on ice for 1 hr. After incubation, RNA was concentrated using QIAmp Viral RNA extraction kit (Qiagen, Valencia, CA). The extracted RNA was eluted in 20 µl of elution buffer. A 10 μl of it was used to amplify the 3 half of the gene using the primer GFII and GR7022 in OneStep RT-PCR reaction (Qiagen, Valencia, CA). An amplicon of approximately 1.2 kb (1186 bases) was obtained in addition to many amplicons of smaller sizes. The amplicon of 1.2 kb was cloned in pgemt (Promega) vector and sequenced with M13 forward and M13 reverse primers or sequenced directly with gene specific primers. For G+C rich 5 half of the gene, RNA was treated in a manner similar to that described above. For PCR, cdna was synthesized with G513 primer and PCR was carried out using Takara Kit with GC buffer II (TaKaRa Bio, Japan). A 3 μl of cdna was used to amplify the GC rich region using G513 and GR primers at 55 C annealing. The other 3

4 components were used as per the instructions in the kit. The PCR products were purified and submitted for sequencing. (c) Amplification of G gene of MN 15a: RNA was extracted from different passage levels (p24, 25, 30, 35, 40, 45, and 50) using QIAmp Viral RNA extraction kit (Qiagen, Valencia, CA). For amplification of G-gene PCR was performed using one step RT PCR (Qiagen, Valencia, CA) and the RT-PCR products were separated by electrophoresis on 1.5 % agarose gel and the DNA bands of expected sizes were cut and eluted using QIAquick Gel Extraction kit (Qiagen, Valencia, CA). The products were sequenced with gene specific primers from both directions. Sequencing of leader and trailer regions: The leader region of all viruses was initially determined by using leader consensus forward primer and NR151 reverse primers 5 - GTGGTTGTCCCCACATCTCT-3. The leader sequences of viral RNA were amplified using OneStep RT-PCR (Qiagen, Valencia, CA). The products were gel purified and sequenced from both directions. Similarly, the trailer sequences were also determined using trailer consensus primer 5 - GGAGGACGAGAAAAAAACCGTAT -3 and L4IF TCTTGAATTCCCCAAAAGGA-3 reverse primer using OneStep RT-PCR kit (Qiagen, Valencia, CA). The products were gel purified and sequenced from both directions. Subsequently the complete length of leader of each virus was determined by 3 RACE. The RNA from all viruses was poly-a polymerized in the 3 end with a help of poly-a polymerase enzyme (Ambion, USA). Then cdna were synthesized (Invitrogen) using 3 adapter primer (Invitrogen). These were followed by PCR with 3 adapter primer (Invitrogen) and NR151 reverse primer at an annealing temperature of 52º C. All five PCR products were purified using gel extraction kit after electrophoresis, cloned into TOPO plasmid (Invitrogen) vector and sequenced. Sequencing of N and F genes of recombinant fowlpox virus (rfpv): Recombinant fowlpox virus was provided by Dr. D. Tripathi (University of Illinois, Urbana- Champagne). This virus contains two ampv genes, N and F. To generate a recombinant clone, these two genes were fused in frame with specific FPV late promoters and ATI (acidic type inclusion body protein) as flanking sequences. The transfer vector thus created has lacz gene as a marker to screen for recombinant virus under the control of ATI promoter. The generated transfer vector DNA was transfected into FPV infected QT cells and FPV recombinants were screened and plaque purified based on their ability to hydrolyze the substrate Bluo-gal. Presence of N and F genes in recombinant clone was confirmed by PCR using primers listed in Table 3. Afterwards, PCR products were purified and sequenced in both directions using forward and reverse primers to obtain full length sequences. Results and Discussion The N, P, M, F, M2 (1 ORF), M2 (2 ORF), SH, G and L genes of all five viruses coded for 394, 294, 254, 537, 184, 71, 175, 585, and 2005 amino acids, respectively. The comparative amino acid substitution profile of all genes was given in the Table 3 and 4. The genetic make-up of all viruses was compared using that of MN-1a (p9) as a 4

5 reference. No insertion or deletion mutations were observed in the genetic make up of low passage MN 1a and 2a viruses. However, substitution mutations were observed in the genes of all ampv strains obtained after passaging (Table 2). Overall, when compared to the vaccine progenitor virus MN 1a p9, the vaccine virus p63 and ca virus had more amino acid substitutions than in p41. In the case of G gene, p41 had more substitutions than p63 and ca viruses. This apparent anomaly calls for further studies. Amino acid substitutions were also observed throughout the genome of MN-2a virus and these substitutions were more in the divergent domain of G gene when compared to that of MN-1a (p9) virus. The p9, p41, p63, ca, and 2a viruses had the maximum of 42, 40, 40, 42 and 42 bases respectively as leader sequences. Part of the trailer region (27 bases) was sequenced using L4IF13816 forward and consensus trailer reverse primer Tr-R for all five viruses. Sequence analysis of different passage levels of G-gene of MN 15a showed that there is no change in length of G-gene as a result of passaging in Vero cells, though at some place point mutations were observed (Fig. 1). We amplified N and F genes cloned in rfpv (Fig 2). We recommend that future studies be undertaken in turkey poults to determine the safety and efficacy of this clone against ampv. References Bennett RS, Njenga, MK, Nezworski, J, Velayudhan, BT, Nagaraja, KV, Zeman, DH, Dyer, N, Graham, T, Lauer DC and Halvorson DA (2004) Evidence of avian pneumovirus spread among wild birds and domestic turkeys in central North America. Avian Dis 48: Bennett RS, LaRue R., Shaw D., Yu Q., Nagaraja KV, Halvorson DA and Njenga MK (2005) A wild goose metapneumovirus containing a large attachment glycoprotein is avirulent but immunoprotective in domestic turkeys. J Virology 79: Govindarajan D, Samal S (2005) Analysis of the complete genome sequence of avian metapneumovirus subgroup C indicates that it possesses the longest genome among metapneumoviruses. Virus Genes 30: Goyal SM, Chiang SJ, Dar AM, Nagaraja, KV, Shaw DP, Halvorson DA, Kapur V (2000) Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys. J Vet Diagn Invest 12: Gulati BR, Patnayak DP, Sheikh AM, Poss PE, Goyal SM (2001) Protective efficacy of high-passage avian pneumovirus (APV/MN/turkey/1-a/97) in turkeys. Avian Dis 45: Lauer D (2000) Annual report of Minnesota Poultry Testing Laboratory. Board of Animal Health Report, St. Paul, MN. Patnayak DP, Goyal SM (2004) Duration of immunity produced by a live attenuated vaccine against avian pneumovirus type C. Avian Pathol 33: Patnayak DP, Gulati BR, Sheikh MA, Goyal SM (2003) Cold adapted avian pneumovirus for use as live, attenuated vaccine in turkeys. Vaccine 21:

6 Pringle CR, Randhawa JS, Marriott AC, Easton AJ, Wilson SD, Tolley KP and Cavanagh D (1998) Virus taxonomy San Diego. Arch Virol 143:

7 Table 1: Details of five ampv strains used in this study No. Strain Description and characteristics 1 MN-1a (p9) Isolated from an outbreak of respiratory illness in turkeys in Minnesota and passaged 7 times in CEF (Chicken embryo fibroblast) cells and 2 times in Vero cells. This strain has not lost its pathogenicity. 2 MN-2a (p7) Isolated from an outbreak of respiratory illness in turkeys in Minnesota and passaged 7 times in CEF (Chicken embryo fibroblast) cells. This strain has not lost its pathogenicity. 3 MN-1a (p41) MN-1a passaged 7 times in CEF and 34 times in Vero cells. 4 MN-1a (p63) MN-1a passaged 7 times in CEF and 56 times in Vero cells. 5 MN-1a (ca) MN-1a passaged 7 times in CEF and 34 times in Vero cells followed by cold adaptation at suboptimal temperature (additional 24 passages) 6 MN-15a (p24) Isolated from Canadian goose and passaged in Vero cells. 7

8 Table 2: List of primers used in this study. Gene Primer Position (bp) Product size (bp) N P M F M2 SH G G (MN 15a) L F: CGCATATAAGACAACTTCCAAACA R: CTTTCCCCTCAGGAAAGGAC F: AATGAGGAAGGCCAGAATGA R: ACTCCATTTCCACTTGTCCC F: GAGCAACCCAGATGATGACC R: CAAGACATTTTCACTTGTCCCA F: CAATTTCGTTTTTAACCCTCTCA R: GAGCCTTGCGAGACATCTTC F: TCCCAATGGAAATGAATGGT R: TTCTCAAGTATGATATTAAGCCTTGTC F: TCAAATGTAGGCTAGAGGATATTGAA R: CCTTGGCACTGTCCAGACATA GFII: CTGTGAGGGTGAACTGCACG GR-7022: GTTGGTCTTGGTGGGCCT G513: CACAAGCAATAGCACAACTGACAACACCAACAAC G1640: CATCATAGCAACCAGCCGGC GF: CCGACCCTAATGAGGTCCAC GR: ACACATTCACAACCCCTTCA F: ACA AGT CAA CAT GGA GGT CA R: GGC AAG AYC CTA TTG CAT TG F (1): ACTGCTATCCTGCTGCTGTG R (1): TTTTTCAGGTCATTCTGGTCAA F (2): TCAAGTGAAAAATTGAAGACAAGAA R (2): ATTGCACTGTCGCAGAAAAG F (3): AGCAGCAGTCACAAGTGTCC R (3): TCCTGCATAGCCTACAAGTGA F (4): TGACAACTCGTTCTGGAGGA R (4): TGCTAGTTGTGTGCTTCATATTCA

9 Table 3: List of primers used for full length amplification of F and N genes of recombinant fowlpox virus (rfpv). Gene Primer (5-3 ) F F: ATG TCT TGG AAA GTG GTA CTG R: TTA AGG GAT AAA TCC TTT GTT G N F: ATG TCT CTT CAG GGG ATT CAG R: TTA CTC ATA ATC ATT CTG GCC T 9

10 Table 4: Amino acid substitution profiles of virulent and cell culture adapted strains of ampv using MN-1a (p9) as a reference. Gene MN-1a (p41) MN-1a (p63) MN-1a (ca) MN-2a (p9) N P M F M2-1 ORF M2-2 ORF SH G L

11 Table 5: List of substitution mutations in ampv strains with respect to P9. Gene N P M F M2 (ORF-1) M2 (ORF-2) Amino acid ampv strains residue P9 P41 P63 Ca 2a 192 Asparagine Serine 283 Threonine Lysine Lysine Lysine Lysine 307 Asparagine Serine Serine Serine Serine 88 Glutamic acid Lysine 90 Threonine Methionine 91 Proline Leucine 95 Serine Isoleucine 99 Tyrosine Histidine Histidine Histidine 217 Glycine Valine 240 Arginine Lysine 56 Glutamic acid Lysine 109 Alanine Threonine 163 Asparagine Lysine 179 Arginine Lysine 188 Lysine Asparagine 205 Arginine Isoleucine 270 Isoleucine Methionine 388 Methionine Valine 458 Valine Isoleucine 484 Isoleucine Threonine 504 Alanine Glycine 29 Serine Asparagine 88 Threonine Alanine 94 Threonine Alanine 176 Alanine Proline 183 Serine Phenylalanine 12 Threonine Isoleucine 16 Cysteine Tryptophan 11

12 L SH 20 Glycine Alanine 51 Alanine Valine 54 Leucine Phenylalanine 67 Lysine Isoleucine 69 Phenylalanine Serine 7 Glycine Glutamic Acid 309 Arginine Lysine 580 Lysine Arginine 935 Threonine Alanine Alanine 1104 Methionine Isoleucine 1137 Aspartic acid Glycine 1211 Lysine Isoleucine 1554 Isoleucine Valine 18 Leucine Valine 29 Isoleucine Threonine 33 Leucine Phenylalanine 53 Asparagine Lysine 54 Valine Isoleucine 55 Glutamic acid Lysine 56 Asparagine Lysine 59 Glutamine Leucine 64 Glutamic acid Glycine 67 Valine Glycine 68 Alanine Glycine 87 Arginine Isoleucine 92 Threonine Proline 93 Alanine Proline 104 Threonine Proline 105 Histidine Proline 107 Threonine Proline Proline 108 Asparagine Histidine 110 Serine Arginine 12

13 141 Valine Glycine 143 Arginine Isoleucine 144 Glutamic acid Isoleucine 153 Histidine Proline 154 Threonine Proline 162 Cysteine Glycine 174 Tyrosine Histidine 13

14 Consensus MEVKVENVGKSQELKVKVKNFIKRSDCKKKLFALILGLVSFELTMNIMLSVMYVESNEALSLCRIQGTPAPRDNKTNTENTKKETTFHTT 90 Canada goose 15a/ P P P P P P Consensus TTTRDPEVRETKTTKPKTNEGATSPSRNLTTKGDIHQTTRATTEAELEKQSKQT. EPDTSTKKHTPTRPSS. SPTTTQATAQLTTPTAPK 180 Canada goose 15a/ I E P T K P T K P T K P T K P T K P T K Consensus ASIAPKNRQATTKKTETGTTTTSRAKKTNNPTETATTTLKATTETGKGKEGPTQHTIKEQPETTAGETTTPQSRRTTSRPAPTTKTEEEA 270 Canada goose 15a/ P P P P P P Consensus ETTKTRTTKSTQTSTGPPGPTRSTPSKTATENNKRTTTIKRPNTANTDSRQQTRTTAEQDRQ.QTKAKPTTNGAHAQTTTTPEHNTDTTN 360 Canada goose 15a/ I P T P T P T P T P T P T Consensus STKESSKEDKTTRDPSSKTPTDQEDASKGTTAANPRKNTEANTRTPPTTTPTRHTTESATSTTGDKTKAKTTRWKSTADRQPIRNSTTAE 450 Canada goose 15a/ P P P P P P

15 Consensus TKTAQSKQPTPKQLSNNTTPENTTPPNNKSSSQTDAAPTEEIEIRSSLWRRRYVYGPCRENVLEHPMNPCFKDNTTWIYSDNGRNLPAGY 540 Canada goose 15a/ P P P P P P Consensus YDSKTDKIICYGIYRGNSY. YGRIECTCKNGTGLLSYCCNSYNWS 585 Canada goose 15a/ C P C P C P C P R P R P R Fig. 1. Amino acid sequence comparison of the G protein of the different passage levels in Vero cell line and the original Canada goose isolate (15a/01) (DQ009484). The consensus sequences are shown as dots and the variations are denoted by single-letter amino acid code. 15

16 F gene (1.6 Kb) N gene (1.2 Kb) Fig 1:. PCR amplification of F and N genes of recombinant fowlpox virus (rfpv). 16

17 17

Development of an Improved Avian Pneumovirus Vaccine Differentiable from Wild Virus

Development of an Improved Avian Pneumovirus Vaccine Differentiable from Wild Virus AUTHORS Dr. Sagar Goyal, Principal Investigator Department of Veterinary Population Medicine, 1333 Gortner Ave., Saint