0 9:00 :0 DI :00-9:0 DI- DI- DI- Mx - 0 9:0-9:50 DI- -C(SEC) TNF NO DI-5 -C SEC PMN 0 9:50-0:0 DI-6 (SCE) DI-7 Effect of sugar cane extracts (SC

Transcription

1 0 9:00 :5 D-S :00 -:5 D-S- III D-S- D-S- D-S- D-S-5 Bordetella bronchiseptica III Salmonella III 0:00 :00 D-W- 5 0:00 -:00 D-W- D-W- D-W- O57 D-W- CE D-W-5

2 0 9:00 :0 DI :00-9:0 DI- DI- DI- Mx - 0 9:0-9:50 DI- -C(SEC) TNF NO DI-5 -C SEC PMN 0 9:50-0:0 DI-6 (SCE) DI-7 Effect of sugar cane extracts (SCE) on in vitro immunoglobulin production in dogs 0 0:0-0:0 DI-8 Plasmodium berghei DI-9 NK 0 0:0-0:50 DI-0 IL- DI-

3 0 0:50 -:0 DI- PPD PCR Buza Joram J. Bari Abusaleh M. DI- DI- cdna EST STAFF IL- STAFF 0 :0 -:0 DI-5 Mycoplasma hyopneumoniae IL-8 DI-6 IL8 0 :00 7:0 DI-7 8, DB- 6 0 :00 -:0 DI-7 C DI-8 0 :0 -:0 DB- K- DB- 0 :0 -:00 DB- Brachyspira ibaraki

4 DB- A new point mutation associated with tylosin-resistance of Japanese canine intestinal spirochetes. 0 :00 -:0 DB-5 Leptospira interrogans DB-6 Leptospira interrogans LipL ELISA 0 :0 -:50 DB DB-8 DB a 0 :50-5:0 DB-0 Klebsiella pneumoniae K -980 PFGE - DB- PCR 0 5:0-5:0 DB- Salmonella typhimurium DB- Secreted proteins of the multiresistant Salmonella typhimurium DT0 Ngwai Yakubu

5 DB- Characterization of the 9kDa protein of avian Pasteurella multocida by monoclonal antibody 0 5:0-6:0 DB-5 DB-6 ( ( DB-7 0 6:0-6:0 DB-8 DB-9 PCR JRA DB-0 M-like proteins JRA 0 6:0-7:0 DB- ( ) DB- DB- 0 7:0-7:0 DB- Pasteurella multocida DB-5 Actinobacillus pleuropneumoniae

6 DB-6 SCID Coxiella burnetii 9:00 :00 DV- 8 9:00-9:0 DV- DV- ORF- 9:0-9:50 DV- EHV- ge JRA DV- DV-5 9 gi ge JRA gb -helix 9:50-0:0 DV-6 NK DV-7 DV-8 Meq glycoprotein B 0:0-0:50 DV-9 JST PRESTO DV-0 genotype AsfawF. Yilkal JICA

7 DV- BLV 0:50 -:0 DV- BLV DV- (BLV) :0 -:0 DV- gp5 ( ) DV-5 (BLV) Env MHC II DR :0 -:00 DV-6 DV-7 DV-8 short interfering RNAs sirnas FIV 5:00 8:0 DV-9 8 5:00-5:0 DV-9 cdna DV-0 DV- RC-HL N G

8 5:0-6:00 DV- DV- P DV- Lv reverse genetics 6:00-6:0 DV-5 CDV ACAT DV-6 N DV-7 EGFP-CDV 6:0-6:50 DV-8 DV-9 Real-Time RT-PCR 6:50-7:0 DV-0 Molecular characterization of the nucleocapsid protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid pathotyping method DV- HN

9 7:0-7:0 DV- H9N DV- H9N DV- 7:0-8:00 DV-5 A DV-6 A NS NES 8:00-8:0 DV-7 A M vrna DV-8 9:00 :0 DV :00-9:0 DV-9 00 DV-0 ELISA DV-

10 9:0-9:50 DV- (BVDV) JRA 5 6 DV- 9:50-0:0 DV- DV-5 NS 0:0-0:0 DV-6 DV-7 O/JPN/000 DV-8 0:0 -:0 DV-9 DV-50 (PrP Sc ) DV-5

11 :0 -:0 DV-5 BSE ELISA(OFR ELISA) DV-5 PrP PrP DV-5 :0 -:0 DV-55 P P DV-56 DV-57

12 D-S- III D-S- Bordetella bronchiseptica III (enteropathogenic Escherichia coli, III EPEC ) EPEC III III III EPEC III III Tir (translocated intimin receptor) EPEC III III III B. bronchiseptica III SDS-PAGE EPEC TOF-MS III BopB p7 III BopB p7 III III EspA EPEC BopB p7 III BopB III EPEC p7 p7 D-S- Salmonella III D-S- Salmonella Salmonella Salmonella pathogenicity island SPI- III Salmonella SPI- hpvr;cd55 III Tg Salmonella Salmonella-containing vacuole (SCV) NADPH Tg SPI- SCV F- hpvr Salmonella hpvr Salmonella SPI- Salmonella

13 D-S-5

14 D-W- D-W- 998 VRE [ ] 006 IgA IgA Lactobachillus, Bifidobacterium, Bacillus, Enterococcus, Clostridium [ IgA ] Fuller 989 IgA (B.breve YIT06) B. breve YIT06 ] B.breve IgA B.breve B.breve B.breve B.breve IgA D-W- O57 D-W- CE) E. coli MN57 Lactobacillus Clostridium Bifidobacterium Bacteroides (E. coli MN57 NR Streptococcus bovis LCB6 Lactobacillus gallinarum LCB 0 0 cfu/g E. coli MN57 NR 0 0. cfu 97 0 g MPN cfu 00 g O57 9 VFA -80 Salmonella Enteritidis Bifidobacterium spp. Escherichia coli 78 O57 O57 ( 0 ) 8

15 D-W-5

16 DI- Mx DI- (LF) Mx 80kDa (TF) ss(-)rna Mx Mx (CHV) Mx MDCK Mx cdna CHV Mx Mx Mx LF RT-PCR MDCK Mx poly(i)/(c) LF Mx Mx CHV LF (MDCK) (CPE) CHV Mx Mx Mx LF CHV GTP Leucine zipper (Lz) TF (OTF) LF FLAG LF CHV 0. mg/ml CHV FLAG Mx Mx LF Mx (VSV) Mx CHV TF OTF LF Mx LF CHV CHV Mx VSV LF LF CHV DI- - DI- -C(SEC) TNF NO IFN- Th NK -C(SEC) TNF IFN- TNF P50 (CYP50) NO inhibitor NO IL- T SEC TNF NO cdna IFN- (MG) cdna pcr. T7 - SEC(00 g) T7RNA CMT SCC NO E.coliBL(DE) MG (BoSA) IPTG SEC g/ml CO CYP50 IFN- VSV TNF inos )SEC CMT IPTG SCC NO ) SEC His Western MG BoSA TNF inos blotting 0kDa BoSA NO TNF IFN- His- ) SEC mg/ml VSV MG BoSA CYP U mg/ml SEC TNF IFN- CYP50 MG NO SEC NO Oxidative damage

17 DI-5 DI-6 (SCE) -C SEC PMN SEC (SCE) SEC D- PMN SEC PBMC SEC g/ml 0 SCE PMN BALB/c (6-0 ) (LPS, 5 IL-8 IL-8 g/kg) D- (D-GalN, g/kg) ELISA mrna RT-PCR SCE (500 mg/kg) PMN LPS+D-GalN SEC PBMC PS PMN SEC SCE PBMC PN LPS+D-GalN 8 PS PN IL-8 SEC PBMC GRO mrna 7 P 0.05 PS 0 PMN 90% PS PMA P 0.05 PMN LPS+D-GalN PMA PMN Cell viability SEC SCE LPS+D-GalN PBMC PMN DI-7 Effect of sugar cane extracts (SCE) on in vitro immunoglobulin production in dogs DI-8 Plasmodium berghei SCE are known as a biological response modifier, but there is almost little information concerning its action mechanisms. Immunological effects of SCE were studied to establish a basis for the practical use in small animal practice. Mononuclear cells (MNC) of dogs were separated by a density gradient centrifugation method. Separated cells were adjusted at x 0 6 cells/ml in RPMI 60 with 5% fetal calf serum. MNC were cultured with SCE (0-,000 g/ml) at 7 in a 5 CO incubator. After MNC culture with SCE, cytokine expression and immunoglobulin production were evaluated. IgG and IgM in culture supernatants were measured by ELISA method. mrna expression of cytokines (IL-, IL-6, IL-0 and TNF- ) were evaluated by RT-PCR method. MNC cultured with SCE for hr showed an increase in IgM production but not IgG. The mrna expression of these cytokines were corresponded to IgM production. These results show that SCE have a stimulating effect on immunoglobulin production of MNC. Plasmodium berghei(p. berghei) TLR/MyD88 IL- P. berghei MHC NK T SCID NK NKT DX5 T NKT CD DX5 CDd NKT CDd CDd NKT

18 DI-9 DI-0 IL- NK (Th) 5 (IL)- Th (PBMC) NK IL- IL- PBMC PBMC IL- 6Q D 9Y D 7 (calcein-am) TPA leader sequence pcdna. (K56) IL- % 0.% OVA OVA PBMC NK IL- 0% g IL- 0 7 PBMC NK IL- IL-6 TNF- IFN- IgE PBMC NK IL- IL-6 TNF- IFN- IL- NK DI- DI- PPD PCR Buza Joram J. Bari Abusaleh M. (IL-) (Th Th ELISA IL-(pIL-) Mph thioredoxin(trx) PPD pil- Mph PCR LPS TLR PGN Trx-pIL- BALB/c TLR PU Mph PPD LPS PGN pil- RNA PCR IL- TNF- IL- p5 IgG IgGb IL- p0 inos IL-0 PPD 6 immunoblot IL-8 Trx-pIL- LPS PGN pil- IFN- LPS PPD PGN PCR PPD ELISA Mph TLR Mph IFN- PPD TLR IL- IL-0 pil- IL-

19 DI- DI- cdna EST IL- STAFF STAFF IL- 000 T T B NK IL-cDNA cdna EST ConA PHA PMA CD cdna IL- PCR IL- cdna mrna IL- cdna EST pqe70 JM09 5' CAP BLAST EST Radiation Hybrid FISH PHP IL-cDNA 59bp 5 EST IL- 77.7% 58.% 5,0 cdna EST IL- CD CD6 8 RH FISH cdna IL- 8 (8q5. q),56 IL- EST DI-5 Mycoplasma hyopneumoniae DI-6 IL8 IL-8 Mycoplasma hyopneumoniae(mhp) IL8 IFN- IL8 IL-8 Mhp IL-8 IL8 Mhp E- IL8-Histag SPF (BALF) caspase- 5 5 Mhp 7 BALF IL-8 0 BALF IFN- (PEG) ( 8 6%) PEG plasma IL-8 IL-8R BALF Mhp PEG Total-RNA RT-PCR 8% IL-8 Mhp Th Th Th PEG % plasma PGE IL-8 0ml IL-8 CD8 8 PEG plasma PGE 0mM 00mM Mhp IL-8 IFN- plasma IL8 PGE Th IL8 50 g/ml IFN- IL8

20 DI-7 DI-8 C (EGF) C CRP EGF CRP CRP CRP ELISA EGF cdna EGF 0ppm ppm.5 EGF cdna ppm 0ppm EGF 5 0ppm 00ppm 0 cdna PCR pet CRP 8 9 g/ml EGF 6 0ppm 9 00ppm CRP 8kDa TRX 8 EGF NIHT 0ppm CRP MDCK MTT CRP EGF 00 g/kg/day CRP EGF CRP EGF EGF DB- DB- K- K- Stx ETA Yamaguchi ETA ETA Stx DNA ETA PFGE ETA 0, 0, 75 DNA ETA 898, 897 (MMC) EHEC O57 NaCl 0 PEG CsCl Stx K- MMC LE9 DNA XbaI PFGE 0kb DNA EHEC O57 wrba PFGE DNA XbaI 0kbp DNA CsCl L-idonate L-idonate 5-ketogluconate PFGE ETA PCR ETA ETA DNA PCR Stx DNA ETA DNA ETA

21 DB- DB- A new point mutation associated with tylosin-resistance of Japanese canine Brachyspira ibaraki intestinal spirochetes.. Objectives Canine weakly -hemolytic intestinal spirochetes have been reported as the cause of diarrhea in Australian and Swedish dogs. We also confirmed the high prevalence in Japanese dogs (.6%). Their 6S rdna sequencing was closely related to those of B. hyodysentertiae and B. innocens at the similarity rate of %. The purpose of this study was to investigate the gene mutation concerning drug resistance of canine isolates and to compared with those of reference strains, B. hyodysenteriae Laemuli(970) 6SrDNA ATCC76 and B. pilosicoli ATCC59. Materials and Ochiai(997) DNA Methods Tylosin-resistant mutants were obtained by Ezaki : three-time subcultures of tylosin-susceptible isolates on agar 0.5um containing g/ml of tylosin. Results and Discussion After successive three-time passages of tylosin-susceptible isolates on blood agar containing g/ml of tylosin, the SrDNA B.aalborgi isolates acquired tylosin-resistance. The adenine at the base position 06 of SrDNA has been replaced by cytosine, DNA B.aalborgi while, the base position 058 of S rdna changed from Brachyspira adenine to guanine in B. hyodysenteriae and B. pilosicoli. ibaraki DB-5 Leptospira interrogans DB-6 Leptospira interrogans LipL ELISA L.interrogans Leptospira interrogans L.interrogans LipL ELISA Leptospira interrogans serovar autumnalis L.interrogans serovar australis AkiyamiC Akiyami A RNA RT-PCR LipL 0 ELISA L.interrogans LipL ELISA ELISA 0 L.interrogans 5 LipL ELISA L.interrogans LipL LipL LipL ELISA ELISA LipL ELISA 0 5 /50 l LipL LipL ELISA ELISA

22 DB-7 DB (JARPNII) 0 (Balaenoptera acutorostrata) (Balaenoptera edeni) 5 ) (Physeter macrocephalus) 000/00 Lactococcus garvieaekg950 (JARPA) 0 Todd Hewitt (Balaenoptera bonaerensis) Broth % 9% ) KG950 Lactococcus garvieaekg950 0 (Mysticeti) Ohishi et al. (00) Comp. Immunol. Microbiol. Infect. Dis. 6, 5-6. DB DB-0 Klebsiella pneumoniae a K -980 PFGE (CEM) a Klebsiella pneumoniae a 0.0(K) 98 AF K a 86 K. pneumoniae K AF RAPD AF 50 RFLP DNA PFGE a85 b b5 N5 a K b a b a 6 b b a 77 AF SM KM TC CP ABPC SIX a AF D955 TC CP RAPD RAPD a R K a 65 9 AF 7 9 5kb PFGE RAPD RFLP DNA RFLP K RAPD a PFGE RAPD 8 9 K. pneumoniae

23 DB- DB- PCR Salmonella typhimurium (SA) PCR S. typhimurium PCR SA (ETs) TSST- DNA PCR SA SA 8 PCR ETs S. TSST- typhimurium S. typhimurium SR- aroa, spv, phop, rpoe )SA PCR ) aroa, spv, phop SA 8 90 rpoe 7 aroa SL LacZ pcmv 6.7% etc etg eti tsst- BALB/c 9.5% etg eti X-gal etb etc tsst- 00% % ) etg eti SL707 m.o.i. phop ETC SA in vitro in vivo DB- Secreted proteins of the multiresistant Salmonella typhimurium DT0 DB- Characterization of the 9kDa protein of avian Pasteurella multocida by monoclonal antibody Ngwai Yakubu The frequent isolation, multiple antibiotic resistance and virulence of Salmonella typhimurium DT0 is of particular global concern, particularly with respect to food safety. This study compared the protein expression of a selected few isolates of Salmonella typhimurium DT0 and a non-multi-resistant strain Salmonella typhimurium L8 ST with a view to identifying difference s unique to DT0s. Our results showed no detectable difference in the major cell protein profile. Major urea-extractable proteins:. kda and 9. kda in all isolates, 8. kda but 5.7 kda in one DT0 and non-dt0 were observed. Protein secretion into nutrient-rich growth medium was variable, and all except one of the DT0s the isolates did not secrete the 8.5 kda protein when grown iron-limited medium at 7. The non-dt0, however, produced significantly P 0.05) less cell protein based on protein content assay than the DT0s. Thus, the higher cell protein content of DT0s is possible indication of increased production of protein-like determinants of virulence. These observations will further enhance our understanding of differential protein expression and possible factors contributing to the enhanced virulence of Salmonella typhimurium DT0. Our previous results suggested that the 9kDa capsular protein of avian Pasteurella multocida was an adherence factor to chicken embryo fibroblast (CEF) cells (9th Ann. Mtg. Jpn. Soc. Vet. Sci.). Then the present study was done to clarify the role of the 9kDa protein by using monoclonal antibody (Mab). Mabs were prepared by immunizing BALB/c mice with a crude capsular extract (CCE) of strain P-059 (serovar A:), and characterization of the protein was carried out using the Mabs. Totally 8 Mabs were obtained and all the Mabs recognized 9kDa protein antigen. By applying the Mabs to immune electron microscopy the antigen was observed on the bacterial surface of capsulated strains only. The Mabs inhibited the adherence of encapsulated strains to CEF cells. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-059 and X-7(serovar A:). Thus the 9kDa protein was confirmed as capsular adherence factor of avian Pasteurella multocida and was suggested as cross-protective antigen.

24 DB-5 DB-6 ( ( ( Enterococcus VRE STEC VRE VCM 8 9 STEC 8 88 MIC NCCLS VCM NCCLS MIC 09 STEC PCR DNA CP GM KM DSM OTC EM TS LCM AVM (PFGE) EFM 0 ABPC BCM APM CEZ CL CP CTF CXM DM-A DSM 9 9 VanC VRE ERFX GM KM NA OA OFLX OTC TMP 8 5 MIC 6 mg/l VCM van vana vanb VRE VCM MIC 56,08 mg/l STEC O57 VT VT acc(6")-ie-aph(")-ia 5 aph(")-ic VT STEC DB-7 DB-8 Streptococcus suis (S.suis ) OIE C.jejuni CDCD 9 5 CCDA CEM S.suis S CFU/ml ml MIC DdeI flaa CFU/ml 0 PCR-RFLP CFU/ml 8,76,7 7 OTC(6 ),TS(9 ),NA(8 ) 0 S.suis S.suis C.jejuni 7 flaa PCR-RFLP 8 CDCD S.suis S CFU/ml ml

25 DB-9 PCR M-like proteins DB-0 JRA JRA PCR S. equi) M-like protein (Streptococcus equi subsp. (SeM, SzPSe) SeM nested equi) S. equi subsp. PCR zooepidemicus PCR PCR Newton PCR- SeM (SeM-,,) SzPSe M-like proteins (SeM, SzPSe) SzPSe-, nested PCR PCR PCR SeM -aa Tm PCR-,SzPSe- SzPSe -7aa -step SeM PCR S. SzPSe C equi8 PCR- CF S. 5 zooepidemicus W60 SzP S. zooepidemicus0 CF Hidaka/95/ NCTC968 SeM SzPSe ELISA SeM -aa PCR- 58 SzPSe -7aa, S PCR zooepidemicus SeM PCR aa PCR 5. PCR SzPSe PEPK S. zooepidemicus PCR PEPK DB- DB- ( ) ( ) A 0. A SDS No.( ) 0 7 (SM) Eschelichia coli G5 (K88 ). 0 0 CFU/ CFU/ CFU 5 0 A % A 0.%

26 DB- DB- Pasteurella multocida IFN- Pasteurella multocida Pm IFN- Pm. ELISA IFN- Pm 0 IFN- BP65 A: IFN- PPD (J-PPD). BP65 PPD (B-PPD) Concanavalin A (Con A) CFU 5 ml 0 0 CFU 5mL 0mL IFN- IFN- 0 7 Serotec IFN- 0 8 CFU 0 0 CFU 6 ELISA 0 J-PPD CFU IFN- J-PPD IFN- Con A IFN- J-PPD 0.5g CFU 6 IFN- 0 CFU 9 Pm Pm IFN- J-PPD BP65 IFN- Con A B-PPD.5 0 IFN- Pm DB-5 Actinobacillus pleuropneumoniae DB-6 SCID Coxiella burnetii Q C. burnetii SCID Q Nine Mile (Infect.Immun ) SCID Priscilla Actinobacillus pleuropneumoniae App C.B-7/Icr-scidJcl 0 PCR 60 Nine App Mile Priscilla SCID 8 SPF Nine Mile Priscilla SCID Nine Mile Nine Mile SCID SCID App SCID

27 DV- DV- ORF- 000 EHV- 5089, EHV HH (EHV-) 5089 DNA BamHI puc8 EHV ORF DNA BamHI B EHV-Abp Abp 5089 HH ORF PCR 5089 ORF GFP Abp pt7-orf-gfp gc gc mrna RT-PCR HH Abp-ORF-GFP 5089 gc GFP ORF Abp ORF MDBK ORF5 ICP7 ORF6 ICP ORF MDBK 5089 ORF ORF gc gc gc mrna mrna DV- EHV- DV- 9 gi ge ge JRA JRA EHV- 9 EHV-9 ge ge EHV-9 EHV- ge EHV-9 ge EHV-9 ge PFU 8 gi - lacz PFU 0 5 PFU 0 PFU EHV-9 P0 ge MDBK EHV- FHK EHV-9 gi ge lacz EHV-9 FHK gi gi ge 0 6 PFU ge MDBK gi 0 5 PFU ge gi 0 PFU LD₅₀ 00 gi ge EHV-9 gi ge gi ge 0 PFU ge

28 DV-5 DV-6 gb -helix NK MHC NK -helix HIV gp F NKR (MDV) NKR MDV gb -helix (BHV) NKR MDV (HSV) gb -helix 5 NKR moi=0.0 MDV Md5 000PFU cdna NKR 50 PCR 5 M moi=0 NKR 7 NKR NK HSV gb HSV NK gb -helix NKR cell-to-cell HSV gb in vivo DV-7 Meq DV-8 glycoprotein B MDV (MD) meq L-meq (MDV) meq L-meq bzip MDV 00 bp meq meq-del MDV glycoprotein B (MDVgB) meq MDVgB MSB-O MSB-cl.8 RP D Md5 Meq Meq-del MDVgB Meq MDVgB D H L CDR gb 5 kda MDVgB Meq-del MSB-O (IT-gB) 0 kda MSB-cl.8 87kDa TRX IT-gB MDVgB 5 kda Meq-del MDVgB MSB-O 0 kda Meq MDV RT-PCR Meq-del MDVgB IT-gB in vitro MDV IT-gB

29 DV-9 DV-0 genotype AsfawF. Yilkal JST PRESTO JICA -, BLV - -Gal -Gal genotype BLV provirus BLV genotype -Gal 6 -Gal 50 BLV 800 AGID nested PCR BLV AGID PCR PCR nested PCR -Gal gp5 0bp BLV PERV PERV 5 (PERV-A -B) Imerge genotype BioTherapeutics IBT University College London(UCL) genotype PERV-A AGID PCR 5 AGID Env genotype AGID PCR multiple BLV PCR transmembrane-spanning 5 IBT Erricsson Patience UCL Takeuchi Weiss genotype AGID PCR BLV BLV DV- BLV DV- BLV (BLV) (BLV) B (BLVRcp) BLV ex vivo CD5 B I BLV in vitro (AP)- ex vivo BLV Tax BLVLTR BLVRcp Tax BLV (pwt-if phigh-if) 9T BLVRcp caspase Staurosporine(STS) BLVRcp (boap- ) caspase BLVp boap- p BLV (PBMC) ex vivo STS pblv-if GFP 9T BLV (GFP/ ) boap- STS pwt-if GFP/ BLV AP- phigh-if boap- pwt-if BLVRcp boap- PBMC ex vivo STS BLV phigh-if STS BLV BLVRcp Tax

30 DV- (BLV) DV- gp5 ( (BLV) (BLV) (gp5) gp5 gp5 Balb/c (Mab-,,7,8) BLV gp5 ELISA RPMI60 7 BLV FLK RNA Mab RT-PCR RNA ELISA 80% PKC Mab 7 8 BLV FLK SDS+Me 7 SDS+Me RNA Mab-7 Mab- - Mab-8 PKC H-7 Mab Ca BAPTA/AM BLV FLK RNA PKC Mab Mab MEK BLV CC8 PKC PKC Mab ELISA BLV PKC PKC BLV DV-5 (BLV) Env MHC II DR DV-6 MHC(BoLA)-DRB (CAE (BLV) (CAEV) BLV Env gp (Mab) BoLA-DRB 5 CAE DNA BLV gp5 ELISA PCR (AGID) PCR BLV provirus BLV DNA 0 PCR-Restriction Fragment Length Polymorphism 9 CAEV BoLA-DRB PCR-Sequence Based Typing Fisher BLV CAEV -gp5mab ELISA ( ) BoLA-DRB DNA BoLA-DR 7/96 / 9/90 DR 66 DR X DR 66 MHC T CDR CDR CAE DR 66 gp5

31 DV-7 short interfering RNAs sirnas DV-8 FIV short interfering RNAs sirnas HIV- env 0 (SHIV) RNA sirnas RNase mrna mrna FIV sirnas FIV SHIV89.6P FIV Petaluma gag sirnas sigag sigag sins FIV Petaluma CRFK/FIV sirnas 8 FIV gag RNA real-time sequence detection system 0kbp 7 ORF FIV 6 gag-p6/pol-leader pol-rt pol-in env-gp p RT-assay sigag sigag FIV gag RNA env-gp FIV p gag sins pol sirnas FIV RNA FIV DV-9 DV-0 RC-HL N G cdna RC-HL RC-HL cdna J. Virol., 00 T7RNA RC-HL G Open reading frame(orf) R(G) G (J. Virol., 00) T7RNA G 0RF RC-HL Ni(G) ptk-hyg Clontech BHK RC-HL G T7RNA ( ) T7 N N P N ORF L N ORF T7RNA RC-HL Ni(N) BHK/T7-9 N G ORF RC-HL Ni(NG) 9 ddy N P L 0. Ni(N) g 0. g 0. g 0 5 BHK/T7-9 LD50 0.8FFU RC-HL 0.7FFU Ni(NG) 5 CPE.x0 7 FFU/ml RC-HL N G cdna

32 DV- DV- MV RC-HL CDV (G) CDV RC-HL 9 0 MV ( ) C HCV 0 MV RC-HL MV HCV E E RC-HL R(G /55/68) E 55 rmv-e rmv-e rmv-e R(G /55) 55 HCV R(G 55) ddy rmv-e, MV / / HCV 0 LD50 R(G /55/68) HCV LD50.8FFU R(G ER /55) R(G 55) rmv-e, RC-HL HCV G 55 MV rmv-e rmv-e rmv-e 68 E 68 E DV- DV- Lv reverse P genetics RPV MV (RPV) RBOK reverse CDV genetics phosphoprotein P RPV-L RPV-Lv reverse genetics P C RPV-Lv P RPV L RNA RT-PCR cdna direct P sequence RPV-Lv Sf9 full genome plasmid supporting GST P plasmids BacV-GST-P P C -P FA -P Western Blotting WB GST-tag GST P RPV-Lv B95a GST P P rrpv-lv rrpv-lv (V-) WB P 0TCID50/ 0kDa Sf9 P RPV-Lv reverse genetics P P RPV V P

33 DV-5 CDV ACAT DV-6 N (CDV) ACAT(acyl-CoA:cholesterol acyltransferase) N (CE) CE N (CDV) CE ACAT CDV-KG CDV-N CE ACAT ( ) CDV-N CDV-Bjal CDV cdna CDV ACAT Cos-7 [ ]6 BALB/c CDV-Bjal EGFP 5 7 -galactosidase -Gal CE PCR ACAT Cos-7 EGFP (MGC ) CDV-Bjal moi=0. ACAT CDV-N [ ] CDV-Bjal 5' 5 Ala CDV CE ACAT MGC CDV EGFP- -Gal SV0 large T ACAT 5 N CDV ACAT CE ACAT CDV-N DV-7 EGFP-CDV DV-8 (CDV) (CD) RNA CD CDV(Yanaka ) CDV EGFP (IC) CDV(EGFP-CDV) CDV CDV A IC CDV Millicell CM(Millipore ) 5 EGFP-CDV EGFP 5 6 (Mapabc) (GFAP) 500 RT-PCR (CNP) CDV EGFP (DFE-HC Fromm Onderstepoort Snyder Hill) EGFP-CDV cell-to-cell KDK- EGFP-CDV,, CDV EGFP-CDV EGFP 8 RT-PCR EGFP-CDV 8 7 cell-to-cell CDV CDV CDV

34 DV-9 DV-0 Molecular characterization of the nucleocapsid Real-Time RT-PCR protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid pathotyping method PCR Nucleocapsid (NP) protein of Newcastle disease virus (NDV) is essential for virus replication through its interaction with the template for viral RNA synthesis, association with the P-L polymerase during transcription and replication, and most likely interaction with the M protein during virus assembly. In order to characterize the gene encoding the NP protein of some NDV strains isolated between 90 and 00 cross-contamination Real-Time in Japan, and to develop a rapid method for pathotyping of RT-PCR rrt-pcr CDV the NDV strains, the nucleotide and deduced amino acid CV RT-PCR sequences of the NP genes were determined. By the nd PCR comparison of deduced amino acid sequences of the NP genes, CDV H no difference was shown among velogenic strains. RNA Asia/H Phylogenetic analysis revealed that these old and new Asia/H CDV 9 isolates were divided into two major groups. One group CV J. contains velogenic strains, and the other group contains Virol. Methods, 77:7, 999 mesogenic and lentogenic strains. Based on the results of CV 86 6 that sequence analysis of the NP genes, one restriction CDV rrt-pcr RT-PCR enzyme, PstI, was selected for the pathotyping of those NDV strains by the combination of PCR and the restriction nd PCR 0 00 fragment length polymorphism (PCR-RFLP). When the 6 reliability of this PCR-RFLP was examined, no digestion by RT-PCR RT-PCR 5 PstI was detected in the case of the NP gene products of nd PCR 8 rrt-pcr CV rrt-pcr velogenic strains whereas PstI cleaved the NP gene products of mesogenic and lentogenic strains at site. The results of nd PCR this study suggest that, using one restriction endonuclease, PstI, NDV isolates can be rapidly and simply pathotyped though biological significance of this observation remains to be elucidated in the future. DV- DV- HN H9N NDV Goose/Alaska/5/9 5 HPAI 95 00% 997 9a5b F Virology, 00, 0: 06- NDV HN NDV HN RT-PCR.% AIV 5 9a5b (.%) HN F HN F log0EID50 0.0g NDV HAd NA MDT ICPI IVPI F RRQKR-F HN 5 9a5b HN F VII 8 P H 95 E K 57 E AIV H9N HA PARSSR-G 5 HN HAd NA 9a5b HN HAd NA AIV HA HN F 5 Y80 HN 9a5b HN 9a5b F NDV H9N AIV HPAI 5 HN H9 H5 H7 HAd NA H5

35 DV- DV- H9N 00 H9N H9N Staphylococcus aureus H9N A/duck/Hong Kong/86/80(HK86) A/chicken/aq-Y-5/00 (H9N) HK/56/97(H5N) HA A/chicken/aq-Y-55/00 (H9N) HK56(dk/HK) HA HA SPF HK/ EID S.aureus HK/9-- H9N HK56(dk/HK) 70 S.aureus 0 DV-5 A DV-6 A NS NES A 8 RNA RNA (vrna) mrna AUG RNP NS A NS G (Kozak ) CRM RNP A/WSN/(HN) NS nuclear export PB NA AUG signal (NES) CRM A PB NA RNP Neumann U et al. EMBO, 000 NES NP AUG M6A,M9A,LA RNP PB NA AUG NS NES U A MDCK A/WSN/(HN) NS NES -ILMRMSKMQL- MDCK MOI= PB NA nnnnnn vrna LD50 NS PB NA (AUG) NES NES

36 DV-7 DV-8 A M vrna [ ] RNA RNA RNA vrna RNA vrna RNA RNA NA HA NS vrna GFP vrna RNA RNA M vrna A [ ] MDCK M vrna GFP M vrna VLP [ ] vrna vrna RNPs RNA NP M vrna 5 M MvRNA NA HA NSvRNA RNPs vrna DV-9 DV-0 00 ELISA 985/86 BT OIE 997 A) BT (AGID) 00 AGID 7 OIE BT ELISA ( 8 C-ELISA) C-ELISA HCH syndrome ()BT AGID ()BT (8AB.6 ) OIE C-ELISA %Inhibition ()BT BT C-ELISA AGID 5 () 6 9 AGID 7% BT C-ELISA 0 BT C-ELISA palyam D'Aguilar

37 DV- DV- (BVDV) JRA (BVDV) 5 (5 NCR) BVDV 5 (5 NCR N pro E NS NS5B~ NCR) BVDV8 5 GenBank Kunjin St. Louis encephalitis(sle) Murray Valley encephalitis(mve) BVDV BVDV BVDV BVDV a b HmLu- Vero 5 NCR 5 Kunjin NCR BVDV a 5 NCR BVDV a 00 0 E Kunjin So CP/75 Kunjin MVE 90CP ILLC ILLNC ( ) BVDV DV- DV- BVDV (BVDV)KS86-cp BVDV 000 (ncp)bvdv KS86-ncp BVDV 00 (cp)bvdv Nose BVDV BVDV RNA RT-PCR 5' 5'-UTR ncpbvdv(799ncp 89ncp 90ncp ) cpbvdv(ks86-cp T-0 ) cpbvdv(799cp 89cp 90cp ) BVDV RT-PCR 5'-UTR BVDV BVDV- cpbvdv BVDV-a BVDV-b BVDV-c BVDV ncpbvdv cpbvdv BVDV- 799cp N pro 89cp N pro 90cp NSB BVDV BVDV- 99 BVDV- cpbvdv (Jiv ) BVDV- 799cp 89cp 90cp KS86-cp T-0 BVDV- NS- BVDV- 990 cpbvdv BVDV cpbvdv NS-

38 DV-5 DV-6 NS (BVDV) (CSFV) CPE NS BVDV Nose RNA RT-PCR NS NS cdna NS RNA BVDV targeted RNA recombination CSFV Nose NS pol, abc, M, N BVDV CSFV 7b S C BVDV CSFV NS CPE 0kd NS- CPE FCWF 80kd NS NS BVDV CSFV S C NS DV-7 DV-8 O/JPN/000 0 Rabbit Haemorrhagic Disease:RHD FMDV O/JPN/000 RHDV 99 5 BALB/c 5 00 PBS 0TCID50 0TCID50 O/JPN/ RT-PCR ELISA HA RT-PCR 0TCID50 0TCID50 5 RHDV LD50 0TCID50 O HA RT-PCR RHDV FMDV RT-PCR RHDV CPE ELISA 6 O/JPN/000 HA RT-PCR RHD RHDV

39 DV-9 DV-50 (PrP Sc ) [ ] (BSE) (PrP Sc ) BSE open reading frame (ORF) PrP Sc (PrP) BSE PrP Sc octapeptide-repeat domain (WB) BSE ( BSE) [ ]BSE 0 ( 7 ) PrP Sc octapeptide-repeat domain [ ] WB PrP Sc PrP Sc PrP7-0( K ) BSE ORF PrP Sc Octapeptide-repeat domain ELISA DNA DAPI DNA BSE PrP Sc PrP7-0 octapeptide-repeat ELISA domain BSE DV-5 DV-5 BSE ELISA(OFR ELISA) (PrP c ) BSE Bio-Rad PLATELIA BSE DETECTION KIT (PrP sc ) (BSE) BSE PrP c PrP sc PrP Sc ELISA (PrP) OFR ELISA BSE PrP(bPrP) HRP PrPmAb capture (ELISA) BSE bprp bprp(rbprp) BSE 500pg/ml rbprp BSE PrP(rBoPrP) 0 bprp BSE BSE PrP BSE PrP c HRP (6 8 g/g) (7 PrP PrP 890ng/g) PrP c -HRP PrP Sc PrP c PrP c rboprp PrP BSE pg/well OFR ELISA PrP sc PrP c PLATELIA (WB) BSE PrP sc OFR ELISA PLATELIA WB ELISA 6 BSE 89 OFR ELISA

40 DV-5 DV-5 PrP PrP (PrP C ) ( ) (PrP Sc ) PrP Sc PrP Sc (rprp) NMR Phage display peptide PrP C PrP Sc library PrP Sc peptide PrPmAb PrP PrP Sc Obihiro aa56-90 PrP rmoprp aa9-7 aa7- aa-9 aa7-5 aa6-69 Phage display peptide library Ph.D.- aa9-9 mab aa89- Ph.D.-C7C(New England Biolabs, Inc) M GdnHCl aa55- PrP Sc panning mab PrP Sc ELISA Phage PrP ELISA 5 NeuroA PrP C panning phage peptide (FACS) PrP C Ph.D.-C7C (IFA) KPHPYTL(Phage#) 9.7% KPHPYSL 9.7% aa56-90 mab Proteinase K(PK) WLWPQHR.% Ph.D.- PrP Sc PK QAPHLNWWSTWL 6.9% ACFPWWESCLEH(Phage#7) PrP Sc PK PrP Sc GdnHCl 9.0% Phage# PrP Sc FACS proteinase K(PK) PrP Sc PK PrP aa56-90 aa-9 mab Sc GdnHCl PrP C Phage# PrP FACS Sc Phage# S-S mab IFA PrP C Phage#7 PK PrP aa7- Sc PK PrP mab (ER) aa7-5, aa9-9 Sc GdnHCl Phage#7 PrP mab ER Phage#7 PrP PrP 55- Peptide PrP C phage display PrP Sc PrP C DV-55 DV-56 P P BSE BSE P P S P P PrP P P S DNA P P S P P P P

41 DV-57 PrP Sc ICR G Obihiro I/I5 G PrP Sc PrP Sc PrP Sc Obihiro I/I5 ICR 60 G 0 G 60ml/ 0ml/ G PrP Sc Obihiro I/I5 G PrP Sc Obihiro / G Obihiro PrP Sc I/I5 PrP Sc Obihiro I/I5 G G PrP Sc Obihiro I/I5 G PrP Sc G