International standards for rabies diagnosis. Dong-Kun Yang/ OIE expert for rabies & JE in Japan at Aug 2014

Transcription

1 International standards for rabies diagnosis Dong-Kun Yang/ OIE expert for rabies & JE in Japan at Aug 2014

2 Contents Collection of brain samples Detection of rabies antigen Detection of antibody against rabies

3 Reservoir species of rabies in Asia Bat Wolf Fox Fox Wolf Raccoon dogs Dogs? Dogs Dogs Ferrat badger Dogs Various reservoir species make us use different type of strategy to control rabies.

4 Brain Salivary gland Spinal cord Transport Along nerve axons Bite infection Viral replication in muscle Virus travels within axons in peripheral nerves via retrograde fast axonal transport. Replication in motor neurons of the spinal cord and local dorsal root ganglia and rapid ascent to brain. Infection of brain neurons with neuronal dysfunction. Centrifugal spread along nerves to salivary glands, skin, cornea.

5 Clinical course and transmission of Host A rabies virus By biting with rabid animals Occasional, albeit rare, transmission by inhalation of infected aerosol has been described Host B Infection Symptoms Death Dog: 2 weeks 3 month Cattle: 4 weeks 5 month Lower than 4 days Incubation period No viremia No detection No symptom Secretion of RABV in saliva Host C

6 Receptors of rabies virus The nicotinic acetylcholine receptor (nachr) is located at the postsynaptic muscle membrane. Neural cell adhesion molecule (NCAM) is cell adhesion glycoprotein from the immunoglobulin superfamily and is involved in the mobilization and cycling of synaptic vesicles. The p75 neurotrophin receptor (p75ntr) is a type I transmembrane protein from the tumor Necrosis factor receptor.

7 Collection of brain samples As rabies virus is rapidly inactivated, refrigerated diagnostic specimen should be sent to the lab. The brain is collected following the opening of the skull in a necropsy room. A receipt number 12D101 (Dogs) 12Q116 (Dogs) Detection of antigen with tissue Detection of antigen with Molecular work FAT, H&E, IHC Real time RT-PCR VI impossible to test because of autolysis

8 Preparing sample from brain Samples are collected from cortex, hippocampus, cerebellum and medulla oblongata. The hippocampus is located in the medial temporal lobe of the brain.

9 Shipment of samples Shipment conditions must be considered to be part of the rabies diagnosis chain. Brain is sectioned using a sagittal cut, half is submitted fresh, the other half is submitted in 10 % buffered formalin. After collection, the samples should be forwarded to the laboratory by the fastest method available. If shipment cannot be made within 48 h, the samples should be frozen. The shipper should ensure that there is no leakage during shipment.

10 2 Detection of rabies antigen FAT, VI, RT-PCR, Negri body, Rapid kit, MIT, RT-LAMP

11 Identification test of Rabies virus Fluorescent antibody test (FAT)- golden standard Virus isolation with NG108-15, Neuro-2a, BHK-21 cell Rapid immunodiagnostic test (RIDT) Reverse transcription Polymerase Chain Reaction (RT-PCR) : real time, LAMP Histological test: Negri body in neuron Mouse inoculation test (MIT) is not recommended

12 Commonly used OIE tests for rabies Tests Use Cost Expected time Sensitivity (%) Specificity (%) FAT Virus Isolation Mouse Inoculation Confirmatory test Confirmatory test Confirmatory test Cheap 2-4 h Medium High Moderate 5 days High Medium Expensive 28 days High Medium RT-PCR Screening Cheap 4-6 h High High Rapid kit Supplementary Cheap < 30 min Medium High

13 Preparation of frozen section for FAT Freeze the brain sample at -70. Turn on the Frozen sectioning machine, Paste the frozen brain sample on disc and make frozen section with cutter of machine. Put the section on slide. Proceed to FAT.

14 The underline principle of FAT Rabies virus Antibodies FITC conjugate

15 Fluorescent Antibody Test (FAT) Gold-standard test to confirm the presence of rabies antigen. Frozen section of brain tissue (hippocampus and cerebellum) are fixed in 100% cold acetone for 20 min. Stained with antibody and fluorescent conjugate. FAT slide are examined for specific fluorescence using fluorescent microscope. hippocampus cerebellum

16 Positive sample of FAT1 Brain sample from Korean cattle confirmed as rabies is showing Strong fluorescent in purkinje cells using monoclonal antibody (3E6)

17 Positive sample of FAT2 Brain sample from Korean cattle confirmed as rabies is showing Strong fluorescent in purkinje cells using monoclonal antibody (commercial kit)

18 Negative samples of FAT Cerebrum-1 Cerebrum-2 Cerebellum-1 Cerebellum-2

19 Slide smear technique with suspected brain Drop of brain sample slide 1. Fix the slide with 100% chilled acetone 2. Stain the slide with antibody against RABV

20 Example of impression method of slide

21 Virus isolation using cell culture-1 NG cells (ATCC HB-12317) were cultured in α-mem. About 10% of the brain homogenate materials were centrifuged at 8,000 x g for 5 min. The supernatant was filtered through a 0.45µm membrane (Millipore, USA) and 100 L of filtrate was used to inoculate a monolayer of cells The cells were then incubated at 37 C for 1 h. After 1 h the inoculation medium was removed and replaced with fresh a- MEM. The cells were observed for cytopathic effects (CPE) for 5 days. Normal NG108 cell NG108 cells inoculated with vaccine strain

22 Virus isolation using cell culture-2 After removing the supernatant the cells were fixed with 80% chilled acetone for 15 min. For the staining, the cells were incubated with a monoclonal antibody specific for RABV for 45 min stained with FITC-conjugated goat anti-mouse IgA, IgG and IgM (KPL, USA). After washing in PBS, the cells were examined by fluorescent microscopy (Nikon, Japan). Check the fluorescent in cytoplasm of cells KRVB1004 KRVB1002 Normal NG cells

23 Primer set and condition for RT-PCR Target site of cording Sequences Size(bp) Primers N protein GCA GAT AGG ATA GAG CAR A (Forward) AAA GTG AAT GAG ATT GAA C (Reverse) 467 1cycle Inactivation of reverse transcriptase min 1cycle Initial denaturation min Condition 35cycles Denaturation sec Annealing sec Extension sec 1cycle Final Extension min 1&8 Marker, 2&3 Ammon's horn, 4&5 Cerebellum, 6&7 Positive

24 Sensitivity of RT-PCR against RABV M: 1kb ladder, lane 1-7: 10-1 to 10-7 of CVS 11 and ERA strain respectively. Sensitivity of RT-PCR with the primer sets comes to about 10 TCID 50 /ml. Oligonucleotide of primer sets should be selected in the part of the N gene, because the N gene is considered a very conserved region.

25 Diagnosis of rabies virus with RT-PCR A B M: 1kb ladder, lane 1-4: normal dog brain, lane 5-10: normal swine brain Lane 11: normal bovine brain, P: positive, N: negative. M: 1kb ladder, P: positive, N: negative. The RT-PCR is playing an increasingly important role in many countries but, is not recommended currently for routine post-mortem diagnosis of rabies if brain tissue is available when the FAT should be used.

26 Negri body in the purkinje cells In the early 1900s, Adelchi Negri undertook studies of rabies virus infected animal brains and described the inclusions that now bear his name. Negri body has been the hallmark of the pathology of rabies for 100 years. Negri bodies are eosinophilic, sharply outlined, pathognomonic inclusion bodies (2 10 µm in diameter) found in the cytoplasm of certain nerve cells containing the virus of rabies, especially in Ammon's horn of the hippocampus. These bodies are found in the purkinje cells of the brain in cases of rabies, street rabies virus strain but not observed with fixed virus strain. No longer recommended if other OIE tests are available.

27 Principle of rapid Immunochromatography Captured Ag-labelled Abcomplex Captured labelled Ab Labelled AB-AGcomplex captured by bound AB of control band Sample pad Conjugate pad membrane Absorbent pad GOLD Ab conjugate: pretreated at conjugate pad Labeled AB-AGcomplex captured by bound AB of test band Antigen If antigen is present, some labelled antibody will be trapped on the test line. Excess-labelled antibody is trapped on the control line No specialized equipment or infrastructure required Potential for ready to use field test

28 Intra structure of immunochromatography kit and application of wild samples to the kit Control line Capture region Nitrocellulose membrane Gold conjugate pad Absorbing pad Sample pad The is used for rapid detection of RABV in clinical samples. The sensitivity of the kit is also shown to be equivalent to that of the FAT. But, this kit is used as supplementary diagnostic tools for detecting RABV in both laboratory and field.

29 Pathogenic Determinant of Rabies Virus G protein Strain Position 333 Pathogenicity Street Arg + CVS, SAD Arg + HEP, SAG2 Glu & Others - Current live vaccine for wild animals ERA Arg + Others mean Isoleucine, Glutamin and, Glycine. The length of Arginine is longer than that of Glu or others

30 RABV containing Arginine at amino acid position 333 in the G protein shows pathogenicity in animals Isolates KRVR0801 PNNLVVEDEGCTNLSGF...GFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLR KRVC D KRVR KRVR KRVR KRVB H KRVB KRVB KRVB KRVR KRVB Numbers represent amino acid positions of the ectodomain of glycoprotein. -The Korean isolates had two putative N-glycosylation sites(aspar- X -Ser/Thre) at 37 and 319 aa within the ectodomain.

31 Start to count numbering position of G protein R G 333

32 Nucleotide sequence alignment of pseudogene of 18 available RABV strains SKRDG9902GY A G. SKRRD9901PJ A G. SKRRD9902PJ A G. CVS T..C...A G T..GA D C A A.C.-...GA ERA 46...GGTTCTT.----.T AAAA.-.-AAC...G. F A.A A C.-...GA FJ C AC A.C.-...GA Flury-LEP 46 T..C G TG.GA NeiMeng1025C G. Ni-CE 46...CTAA C---.G Nishigahara 46...CTAA C---.G NNV-RAB-H A...G. PV 46 GGTTCTT.----.T AAAA.-.-AAC...G...A... RRVON CCTCT A----..CAT AA..-.-.A..CTGC RV TAA T---.CTTTATTGG C.G. SAD B GGTTC.T---T TT.A.-.-AAC...G. Serotype A...G. Korean isolates shows high similarity with Chinese strain, All Korean isolates have the same length (489 bp), but other rabies strains showed variable length ranging from 489 to 496 nucleotides. 32

33 Mouse inoculation test (MIT) observed daily for 28 days Carry out FAT Three to ten mice, 3-week-old (12-14 g) are inoculated intracerebrally. The inoculum is the clarified supernatant of a 10-20% homogengate of brain materials in PBS or medium. The mice are observed daily for 28 days and every dead mouse is examined for rabies using FAT. Any death occurring during the first 4 days are regarded as non-specific. This test can cause animal welfare problem, so it is not recommendable. Only recommended where tissue culture in not available.

34 RT-Loop-mediated isothermal amplification (RT-LAMP) Requires a precision instrument for heating and cooling Select appropriate primer set for rabies virus

35 Detection of antibody against rabies 3 VN, FAVN, RFFIT, ELISA, Rapid kit

36 Serological tests for rabies The main application of serology for rabies is to determine response to vaccination. Domestic animals prior to international travel or wildlife populations following oral vaccination. FAVN (fluoresent antibody virus neurtalisation) test (OIE), RFFIT (rapid fluorescent focus inhibition test) (WHO), ELISA. Poor quality sera can cause cytotoxicity in VN test (false-positive results) use of an indirect ELISA. The European Union Reference Laboratory (EURL) for rabies, Nancy laboratory, approved laboratory for rabies neutralizing antibody test. Internationally approved laboratory (4) in Korea : QIA (Seoul Regional Office), Komipharm, KBNP, Choongang Vaccine Lab.

37 Serological tests for rabies (OIE) VN (Virus neutralization) test FAVN (fluorescent antibody virus neurtalization) test RFFIT (rapid fluorescent focus inhibition test) ELISA (Enzyme-linked immunosorbent assay)

38 Production of challenge virus Inoculate the CVS11 into BHK-21 C13 cell or NG cell culture. After inoculating the virus into the cell, the CVS11 is harvested 2 days later. The infective titer of the harvested virus should be contained ranging from to TCID 50 /ml. The titration of the CVS11 must be performed by FA Dispense in 1 ml aliquots and store at -70. Determining the working dilution of challenge virus.

39 Street virus vs Fixed virus Street virus: strains isolated from naturally infected animals Fixed virus: attenuated laboratory and vaccine strains (ERA, SAD B19, CVS11, CVSN2C etc). Street virus mainly buds from intracellular membrane. Fixed virus preferentially buds from plasma membrane. Street Fixed Incubation period Long Short Peripheral infectivity Strong Poor Virulence High Low-High Immune induction Low High

40 VN (Virus neutralization) test Materials Standard positive serum & negative serum 96 well plates for Virus back titration & Serum sample test Test serum : 56 C heat inactivated serum Serum : 2 fold dilution Virus back titration : 10 fold dilution Virus dose : 100 TCID 50 /50 μl Equipments CO2 incubator, Microscope

41 VN (Virus neutralization) test Methods Virus back titration 100 TCID 50 /50 μl (50~200 TCID 50 /50 μl) * * * * * * * * * * * * * * * * * * * * * : Fluorescence N 10 fold dilution 1. Dilute virus to 100 TCID 50 /50 μl (Stock virus 10^7.0 TCID 50 /ml = 5X10^5.0 TCID 50 /50 μl 5000 fold dilution 100 TCID 50 /50 μl) 2. Serially 10 fold dilution of diluted virus 3. Add each diluted virus 50 μl in 8 wells and media 50 μl in all wells 4. Add 100 μl cells (2X10^5 cell/100 μl) in all wells (But, in the rotavirus, 5. prepare cell monolayer) 6. Incubate at 37 for 3-7 days 7. Observe the CPE 8. Virus titer : 100 TCID 50 /50 μl (50~200 TCID50/50 μl)

42 VN (Virus neutralization) test Methods Serum sample test 2 fold dilution 2 = = = = = = = = 256 Sample 1 Sample 2 Sample 3 Sample 4 Virus 50 μl RABV (cell suspension) Media 50 μl 1. Add serum 50 μl in the 1st row wells (3 wells/sample) 2. Add media 50 μl in all wells 3. Serially 2 fold dilution of serum 4. Dilute stock virus to 200 TCID 50 /100 μl 5. Add diluted virus 50 μl (2wells) and media 50 μl (1 well) min of incubation at Add 100 μl Vero, BHK21cells (2X10^5 cell/100 μl) in all wells 8. Incubate at 37 for 3 days 9. Stain the plates with mab against RABV 10. Observe the fluorescent

43 Results examples Reading VN titer Sample 1 Sample 2 Sample 3 Sample = 4 fold = 5.6 fold 2 5 = 64 fold 2 3 = 8 fold 2 fold dilution 2 = = = = = = = = 256 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 0.5 IU/mL, 8-16 fold (Rabies virus) * : Fluorescence Virus 50 μl Media 50 μl (Serum+cell)

44 FAVN (fluoresent antibody virus neutalization) Materials OIE gold standard method Cell : BHK-21 C13 cells (ATCC number: CCL-10) Virus : CVS-11 (ATCC VR 959), 100 TCID 50 /50 μl 96 well plate for Virus back titration & Serum sample test Positive standard serum : OIE standard serum (0.5 IU/mL)

45 FAVN: virus back titration Methods Virus back titration * * * * 100 TCID 50 /50 μl * * * * * * * * * * * * * * * * * * * * * * * * : Fluorescence 1/4 1/16 1/64 1/256 1/1024 1/ fold dilution 1. Dilute stock virus CVS-11 to 100 TCID 50 /50 μl (Stock virus 10^7.0 TCID 50 /ml = 5X10^5.0 TCID 50 /50 μl 5000 fold dilution 100 TCID 50 /50 μl) 2. Add 150 μl FBS free DMEM to every well 3. Then add 50 μl of diluted CVS-11 (100 TCID 50 /50 μl) to column 1 4. Transfer 50 μl to next column till columm 6, discard 50 μl 5. Add 50 μl of cell suspension ( cells/ml in 10% DMEM) to every well 6. Incubate plate for 48~60 hr at 37 C 7. The cells were fixed in cold acetone for 20 min. 8. After the plates were rinsed with PBS, the cells were reacted with RABV-specific monoclonal antibody for 1 hr at 37 C 9. Stained with FITC conjugated goat-anti mouse IgG. 10. After washings, fluorescence was examined under fluorescent microscope.

46 FAVN: 3 fold dilution Methods Serum sample test Sample 1 Sample 2 Sample 3 3 fold dilution (Log dilution) Log 3 = 0.48 Log 3 2 = 0.95 Log 3 3 = 1.43 Log 3 4 = 1.91 Log 3 5 = 2.39 Log 3 6 = 2.87 Log 3 7 = 3.35 Log 3 8 = Add 100 μl FBS free DMEM in the all wells 2. Then add 50 μl of serum to first row (1 to 4) 3. Transfer 50 μl to next row till the end. discard last 50 μl 4. Add 50 μl of diluted CVS-11 (100 TCID 50 /50 μl) to every well 5. Incubate plate for 1 hr at 37 C 6. Add 50 μl of cell suspension ( cells/ml in 10% DMEM) to every well 7. Incubate plate for 48~60 hr at 37 C 8. The cells were fixed in cold acetone for 20 min. 9. After the plates were rinsed with PBS, the cells were reacted with RABV-specific monoclonal antibody for 1 hr at 37 C 10. Stained with FITC conjugated goat-anti mouse IgG. 11. After washings, fluorescence was examined under fluorescent microscope. 12. A positive reference serum of WHO was adjusted to 0.5 IU/ml

47 FAVN: Converting the log D 50 to IU/ml titer * Formula to convert the log D 50 value in IU/mL titer Rabies neutralizing antibody titer (IU/mL) 0.5 IU/mL All positive : Log 0.24, 0.06 IU/ml Rabies protective level : 0.5 IU/ml = VN (8-16 fold)

48 FAVN: reading results Results examples Sample 1 Sample 2 Sample 3 Log IU Log IU Log IU 3 fold dilution (Log dilution) Log 3 = 0.48 Log 3 2 = 0.95 Log 3 3 = 1.43 Log 3 4 = 1.91 Log 3 5 = 2.39 Log 3 6 = 2.87 Log 3 7 = 3.35 Log 3 8 = 3.83 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 0.5 IU/mL * : Fluorescence Rabies neutralizing antibody titer (IU/mL)

49 There are many testing components influencing FAVN Serum samples, virus titer Virus strain, cells, incubation time Temperature, equipment issues Interpretation of test results Calculations used to determine the value of the results The choice of an identical RABV strain (CVS-11 strain) and the use of an internationally recognized reference serum standard are critical. Documentation of all quality assurance activities is essential

50 Serological test questions!!!! Question : You did FAVN in 3 serum samples of dogs. If you got the below 96 well plate results. What is the neutralizing antibody titer of each sample? And which samples is over the rabies protection level? Sample 1 Sample 2 Sample 3 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * : Fluorescence

51 Question 1 solution!! Sample 1 Sample 2 Sample 3 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Rabies neutralizing antibody titer (IU/mL) Answer 1. Sample 1 : 0.17 IU/ml Sample 2 : 2.62 IU/ml Sample 3 : 0.39 IU/ml 2. Sample 2 is over the rabies protection level (0.5 IU/ml)

52 RFFIT (rapid fluorescent focus inhibition test) Materials WHO Cell : mouse neuroblastoma cell (Neuro2a cell BHK21 cells) Virus : CVS-11 ( FFID50) 8 well chamber slide Reference serum standard : 2.0 IU Positive serum : 0.5 IU Negative serum : 0.1 IU

53 RFFIT (rapid fluorescent focus inhibition test) Serum sample 1 1/5 1/25 1/125 1/625 5 fold dilution Log5 = Positive serum (0.5 IU/ml) 1/5 1/25 1/125 1/625 1/ / / /3125 Serum sample 2 1/5 1/25 1/125 1/625 1/ / / /3125 Negative serum (0.1 IU/ml) 1/5 1/25 1/125 1/625 1/ / / /3125 Serum sample 3 1/5 1/25 1/125 1/625 1/ / / /3125 Reference serum (2.0 IU/ml) 1/5 1/25 1/125 1/625 1/ / / /3125 1/ / / /3125

54 RFFIT (rapid fluorescent focus inhibition test) No. of filelds cont Serum dilution aining infected cel ls Fields containing infected cells Field containing % of fields contain no infected cell ing infected cells s 1:5 0/ :25 0/ :125 0/ :625 0/ : / : / : / : / % end-point titer of serum Starting point dilution : dilution next below 50% dilution factor= 5 ( log 5 = ) The difference of logarithms (between the logarithm of the starting dilution & 50% end-point dilution) 50% - (infectivity next below 50%) (infectivity next above 50%)-(infectivity next below 50%) Log (reciprocal of 50% end point dilution) X log of dilution factor = Log (reciprocal of the starting point dilution) + difference of log International units (IU) per ml end-point titer of test serum end-point titer of reference serum (2 IU) X 2 IU

55 ELISA (Enzyme-linked immunosorbent assay) BIO-RAD, Platelia TM Rabies II

56 ELISA (Enzyme-linked immunosorbent assay) Methods Remove the adhesive film Perform 5 wash cycles with the diluted wash solution (R2) Add 100 μl of the diluted enzymatic development solution (R8+R9) Incubate in the dark for 30 minutes at room temperature Add 100 μl of stop solution (R10) to each well similarly to R8+R9 solution Read the plate at 450 nm 620 nm* (dual wavelength mode)

57 ELISA (Enzyme-linked immunosorbent assay)

58 J Virol Methods Oct;161(1): Development and evaluation of a rapid neutralizing antibody test for rabies. Shiota S 1, Mannen K, Matsumoto T, et al Prenincubation of serum with inactivated virus Absorbent pad Control line Nitrocellulose membrane Plastic holder Test line Anti-G MAb (#4-12) Sample pad Pad containing Gold conjugated Sample hole Anti-G MAb (#4-12) The RABV combined with gold-conjugated Mab is trapped at the test line where the unconjugated mab is fixed, and unbounded gold labeled mab is trapped at the control line.

59 Sample Absorbent pad Nitrocellulo se membrane Control line Plastic holder Test line Anti-G MAb (#4-12) 10cm Pad containing Gold conjugated Anti-G MAb (#4-12) Sample pad Sample hole Diagram of rapid test strip (G detection kit) for the detection of neutralizing antibody. The sample is applied to the test strip at the sample hole and allowed to travel along the nitrocellulose membrane and from an antigen-antibody complex with gold-conjugated MAb in the presence of RABV G in the sample. As the antigen-antibody complex or unbound antibody move further, they ate trapped in the test or control line by RABV G- specific or goat anti-mouse polyclonal antibody, respectively, to produce a red band.

60 Treatment of animals suspected to be infected with rabies There is no treatment once the clinical signs appear. Post-exposure prophylaxis of animals is usually considered inadvisable because it may increase human exposure. PEP procedures for animals have not been validated and not recommended in the U.S and EU. This is not the case in all parts of the world, commercial vaccines are licensed for this purpose in some countries, India.

61 Disinfection RABV can be inactivated by sodium hypochloride, 45-75% ethanol, iodine preparations, quaternary ammonium compounds, formaldehyde, phenol, ether, trypsin, beta propiolacton A very low ph(below 3) or very high ph (greater than 11) Ultraviolet radiation, sunlight and drying make it inactive It does not survive for long periods in the environment

62 Minimum infrastructure requirement The trained person who is vaccinated with inactivated rabies vaccine is required. Frozen sectioning machine Fluorescence microscope Positive and negative samples