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Applications of the RG

Applications of the RG. Study of molecular biology of RNA viruses Pathogenesis Vaccine development Vectors Other applications…. VPM 700 May’06. Molecular Biology of RNA viruses. Provide insights into the many aspects that control the virus life cycle

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Applications of the RG

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  1. Applications of the RG • Study of molecular biology of RNA viruses • Pathogenesis • Vaccine development • Vectors • Other applications…. VPM 700 May’06

  2. Molecular Biology of RNA viruses • Provide insights into the many aspects that control the virus life cycle - the location and boundaries of the cis-acting elements - the secondary structure of promotor regions - the functional domains of the trans-acting proteins - the gene stop-start signals - AND MANY MORE……

  3. Pathogenesis • Genetic determinant of virulence by H5N1 virus in mice (Hatta et al., Science, 2001) A/HongKong/483/97 (HK483) – Highly Pathogenic A/HongKong/486/97 (HK486) – Mildly Pathogenic Prepared the single-gene reassortant viruses In vivo characterization

  4. PB2 Protein is responsible for the difference in virulence

  5. Amino acid residue at position 627 influence the outcome of infection

  6. H7N2 influenza in the U.S. • H7N2 LPAI virus circulation in NY and NJ LBM’s since 1994 • LBM H7N2 virus served as source of infection for commercial poultry operation - 1997-1998, Pennsylvania - 2001-2002, Pennsylvania - 2002, Virginia, West Virginia, North Carolina - 2003, Connecticut - 2004, Delaware, Maryland • LBM H7N2 virus may serve as source of infection for human - 2003, 1st human case of H7N2 infection in New York

  7. Determinant of pathogenicity • Not totally defined • An optimal constellation of viral genes is required for pathogenicity expression • Hemagglutinin (HA) structure is a primary determinant of pathogenicity in chickens and turkeys “HA0 cleavage into HA1 and HA2” - Necessary for fusion peptide to be exposed - Essential for virus to become infectious All AI viruses : trypsin-like enzyme (respiratory, intestinal) HPAI viruses : ubiquitous protease (systemic)

  8. Key features for HA cleavability • The amino acid sequence at the cleavage site - multiple basic a.a. • The carbohydrate in the vicinity of the cleavage site - no glycosylation at Asn11 Receptor binding site Trimer axis Asn11 Cleavage site

  9. Molecular features of H7N2 virus * Basic amino acids (Lysine(K) and Arginine(R)) are in bold character. Is H7N2 virus progress toward HPAI virus ? What additional changes are required ?

  10. Pathogenic Potential of H7N2 Virus In vitro In vivo Parent PEKP---KKR/G - 0/8 - Mutant 1 PEKR---KKR/G - 0/8 - Mutant 2 PEKK---KKR/G---- inefficient - 0/8 - Mutant 3PEKP--RKKR/G + 0/8 ++ Mutant 4PEKP--KKKR/G---- inefficient - 0/8 - Mutant 5 PEKR--RKKR/G ++ 2/8 +++ Mutant 6 PEKR--KKKR/G---- inefficient - 0/8 - Mutant 7PEKP-KKKKR/G + 0/8 + Mutant 8PEIP-KKKKR/G + 0/8 + Mutant 9PEKPKKRKKR/G +++ 3/8 ++++ Mutant 10PEIPKKRKKR/G +++ 4/8 ++++ Mutant 11PENPKKRKKR/G +++ 5/8 ++++ Mutant 12PETPKKRKKR/G +++ 5/8 ++++ Positive control (CK/Chile/02 (H7N3)) +++ 1/8 +++ Virus sequence replication plaquing mortality virulence in ECE efficiency

  11. Transcription Plasmids (8) Avian – PB2 P T Avian – PB1 P T Expression Plasmids (4) Avian – PA P T PB2 P A Mutated H7 P T PB1 + P A Avian – NP P T PA P A Avian – N1 P T NP P A Avian – M P T Avian – NS P T Rescue of viruses carrying mutated HA genes Co-transfection 293T Amplification 10-day eggs Reassortant H7N1 strains with different H7 gene

  12. Reverse genetics using helper virus 4 Expression Plasmids PB2 P A Transcription Plasmids + PB1 P A H7 HA P T PA P A NP P A Co-transfection Selection of HP reassortant by plaque assay in CEF 293T 24hr incubation HP reassortant 48hr incubation 293T no HP reassortant Helper virus infection (wild type H7 virus)

  13. √ √ √ 4 Expression Plasmids PB2 P A Transcription Plasmids + PB1 P A √ H7 HA M 3 PEKP--RKKR/G M 5 PEKR--RKKR/G M 7 PEKP-KKKKR/G M 8 PEIP-KKKKR/G M 9 PEKPKKRKKR/G M 10 PEIPKKRKKR/G M 11 PENPKKRKKR/G M 12 PETPKKRKKR/G CK/Chile/02-H7 Egret/HK/02-H5 CK/Indonesia/04-H5 P T PA P A NP P A Co-transfection Selection of HP reassortant by plaque assay in CEF 293T 24hr incubation Chile/02, Egret/02, Indonesia/04, H7-M3, M5, M9, M12 reassortants 48hr incubation 293T All other H7 mutants Helper virus infection (wild type H7 virus)

  14. Pathogenicity of reassortants rescued by helper virus rescue system a MDT: Mean death time (average days taken to kill the embryo or chickens b Number of birds that died / number of birds inoculated. c The virus titer is express as the log10 EID50 per milliliter ± standard deviation. d No birds survived

  15. Vaccine Development • Non-structural (NS1) protein, a novel target for the development of influenza vaccines • Functions of the NS1 protein - Inhibit the early production of cellular antiviral mRNA - display host unique PL (PDZ domain ligand) motifs at the C-terminus for PDZ domains involved in cell signaling pathways (Science, 2006)

  16. Genomes RNA segments (bp) Protein (aa) 1. Polymerase (basic) 2 (2341) PB2 (759) 2. Polymerase (basic) 1 (2341) PB1 (757), *PB1-F2 3. Polymerase (acidic) (2233) PA (716) 4. Hemagglutinin (1775) HA (565) 5. Nucleoprotein (1565) NP (498) 6. Neuraminidase (1413) NA (454) 7. Matrix (1027) M1 (252) M2 (97) 8. Nonstructural (890) NS2(NEP)(121) NS1 (230) Virion constituents Infected cells

  17. A/turkey/Oregon/71 (H7N3) • Low virulence AI isolate from a turkey flock with mild respiratory illness • Genetically distinct stocks exist TK/OR/71-SEPRL (Suarez et al., 1999) - Low passage stock of the field isolates - Encode a full length NS1 protein (230aa) TK/OR/71-del (Norton et al., 1987) - Unknown passage history - 10 nt deletion in the NS gene resulting in a truncated NS1 protein TK/OR/71-D-dels (Lee et al., 2005) - Derived from TK/OR/71-del stock by passaging in old ECE - Different size of deletion in the middle of the NS gene

  18. stop * SEPRL 890bp NS genes * del 880bp 380-389 (10nt) * D-del 1 705bp 880 bp 267-451 (185nt) 705 bp * 644 bp D-del 2 644bp 487 bp 218-463 (246nt) * 487bp D-del 3 204-606 (403nt) translation RNA binding Effector domain 1 230 73 SEPRL 230 aa 117 ▼ 124 aa del 80 NS1 ▼ 90 aa D-del 1 160 64 ▼ ▼ 148 aa D-del 2 59 ▼ D-del 3 144 aa

  19. Transcription Plasmids (8) A/WSN/33 – PB2 P T A/WSN/33 – PB1 P T Expression Plasmids (4) A/WSN/33 - PA P T PB2 P A Avian – H5 P T PB1 + P A A/WSN/33 - NP P T PA P A Avian – N1 P T NP P A A/WSN/33 - M P T NS P T Rescue of viruses carrying different NS genes Co-transfection SEPRL (890bp) del (880bp) D-del 1 (705bp) D-del 2 (689bp) D-del 3 (644bp) D-del 4 (487bp) 293T Amplification 10-day eggs Reassortant H5N1 strains with different NS gene

  20. DIVA strategy(Differentiating Infected from Vaccinated Animals) • Use of heterologous strain containing the same hemagglutinin subtype as the field virus but a different neuraminidase in an oil-emulsion vaccine (Beard, 1986; Stone, 1987) • H7N3 subtype vaccine was used to control H7N1 LPAI virus in Italy (2000-2002) • H7N3 subtype vaccine is being used to control H7N2 LPAI virus in Connecticut • Limitations 1) availability of heterologous vaccine (The H7N3 outbreak in Northern Italy in 2002.) 2) degree of homology between the hemagglutinin gene in vaccine and challenge virus

  21. Reverse genetics for rapid generation of influenza vaccine banks WSN-PB2 WSN-PB1 WSN-PA WSN-PB2 WSN-PB1 P P P T T T WSN-NP WSN-M WSN-NS WSN-PA WSN-NP P P P T T T 6 transcription plasmids from WSN/33 (H1N1) 4 expression plasmids + H5: A/CK/PA/13609/93 (H5N2) H7: A/TK/VA/55/02 (H7N2) N1: A/DK/Anyang/01 (H5N1) N8: A/DK/NY/191255-59/02 (H5N8) N1 or N8 H5 or H7 P P T T

  22. Transfection 293T 102.0-4.0EID50 / ml 2 days ECE Amplification 2-3 days rH5N1, rH7N1 rH5N8, rH7N8 108.0EID50 / ml Reassortantvaccine strains

  23. Use of Reverse Genetics for Development of DIVA Vaccines(Lee et al., Vaccine 2004) • Use of plasmid-based reverse genetic system to produce viruses with specific HA and NA subtypes *HI titer is expressed as log2 reciprocal of the endpoint in twofold sera dilution **NI test were performed with NI, N2 and N8 antigen and the number of positive samples were indicated ***Virus titer is expressed as log10 50% egg infective doses per milliliter

  24. Differentiation by indirect IFA assay H5N2 infected H5N2 vaccinated rH5N1 vaccinated H7N2 infected H7N2 vaccinated rH7N8 vaccinated

  25. An alternative DIVA approach on the basis of antibodies to NS1 • The NS1 protein is nonstructural • Killed vaccines for influenza are primarily made with whole virion • The NS1 DIVA approach works well with horses receiving purified vaccines (Ozaki et al., 2001). • Vaccinated chickens produce low levels of antibody to the NS1 protein, but virus-infected chickens will produce higher levels of NS1 antibody (Tumpey et al., 2005).

  26. Application – DIVA vaccine RNA binding Effector domain • DIVA strategy - truncated NS1 recombinant protein produced in E. coli can be used as coating antigen in ELISA format - seroconversion will be observed in infected, but not in vaccinated birds 1 230 73 SEPRL 230 aa D-del 1 90 aa 163 aa D-del 2

  27. Application - Vector Insert foreign sequences • Expression of foreign gene - GFP - HIS tag - Foreign antigenic epitope * D-del 1 705bp 267-451 CRS 338 aa D-del-GFP NS1 GFP

  28. Application – Live Vaccine • For humans, - cold-adapted live vaccine - FluMistTM was approved for use in the U.S. • Potential problems of the use of live influenza vaccines in poultry - bird-to-bird transmission - vaccine-induced respiratory disease - potential for recombination with newly introduced AIV

  29. Application – Live Vaccines • Genetically engineered live attenuating virus - changes in the PB2 gene - exchanging promoter region of the NA gene - generating viruses that have truncated NS1 • TK/OR/71-del virus - attenuation in pathogenicity - decreased ability to replicate in chickens - inefficient bird-to-bird transmission => In ovo vaccination with attenuated live influenza virus that induces strong immune response, but has no infectious virus present in the hatched chicks.

  30. Other Application of RG - Antigenic drift study A 154 158 188 Receptor binding site 194 123 126 160 181 B Trimer axis 218 136 137 175 • Antigenic drift occurs away from the vaccine strain with avian influenza virus (Lee et al., J Virology, 2004) • The genetic changes of the HA1 gene were concentrated in or near the predicted antigenic sites in the protein. • We also found a mutation that created a glycosylation site near the antigenic site => Using a mapping approach, amino acid changes that are important for immunity will be identified 113 71 270 C 40 275 VPM 700 May’06

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