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In Great Britain, Recent pandemic in pigs is continually growing. Rapid and early detection of infected animals is important for containment and control of disease. Clinically, SIV is not easy to diagnose in all cases and must need to perform diagnostic tests in the case of enzootic. Sometimes, Pigs may harbour the virus without showing any obvious symptoms. Moreover, various reassortments occurred globally during different period of time such as the recent 'Pandemic H1N1 2009' and there are more likely to emerge potent novel virus in future. As a result, it creates a question mark on modern virus detection techniques.
SIV is important pathogen of economic, veterinary and public concern. Generally, the Respiratory Disease: Swine influenza is endemic in pigs but it can be epidemic. Beyond this, it can become harmful to other species and human. Moreover like other influenza virus, it's genetically unstable in nature. As a result, there are various reassortments occurs during the different period of time. Experts are believed that pandemics are more likely to arise within the intervals of 10-40 years with unknown pattern. (Ref- Web 12). Thus, it continues to be an important economic factor in swine production and a human health. (28 SI/469).It reduces weight and reduces weight gain that cause economic damage around £7/ pig and that is around £65 000 a year.
Swine Influenza is a most prevalent (EEI/Ian) and contagious viral disease in pig population that can have significant economic impact on an affected herd. It causes high level of illnesses and low death rate in pigs. Also, it is known as significant contributor to the porcine respiratory disease complex (PRDC) (Ref R-12). It is characterised by coughing, sneezing, nasal discharge, fever, weight loss lethargy, difficulty in breathing, depressed appetite and reproductive disorder (in some cases). In the enzootic form of disease, these signs may be less obvious. Incubation period is short and clinical signs occur within 24 hours. It can transmit easily by fomites. It can transmit through direct contact or close proximity with SIV containing secretion such as nasal discharges and aerosols created by coughing or sneezing.
It (SIV) can occurs in human as there are limited number of death have been reported. Also, it can transmit from human (human influenza virus) to pigs. Similarly, Influenza virus can transmit from poultry to pigs or/and pigs to poultry.
Transmission of SIV can be controlled by facility management, Herd management and Vaccination. (Refer presentation to describe more). In addition, current vaccine may give protection to the pigs against different types of SIV if the pigs have sufficiently high concentrations of serum antibodies. (Gramer MR 'Defining Swine influenza virus')
2.1. Biology of SIV
Swine influenza viruses are the member of genus Influenza virus A, family Orthomyxoviridae. They are further classified according to their haemagglutinin (HA) and neuraminidase proteins (NA). ... ... ...
Figure of Virus:
The various viruses are occurring frequently in pigs are reassortments of classical and avian H1N1 (rH1N1), r (H1N2), r (H3N2), AvianH4N6 (avH4N6), av(H3N3), and av(H9N2). Currently, the viruses occurring in swine population are H1N1, H1N2, H3N2 and pandemic (P)-H1N1. In addition, viruses in Europe are genetically and antigenecally varies than virus in North America or Asia.
Pigs have receptors in their respiratory tract that have ability to bind swine, human and avian influenza viruses. So, changes can occur as result of reassortment.
2.2 History of Swine influenza virus
It is believed that SIV appeared in pigs after 1918 Influenza incident in human. It is proved that it was 'classical H1N1'. But In 1998, H3N2 identified from pigs in USA which was occurred due to the either double reassortment (human + avian) strains of influenza or triple reassortment (Avian + Human+ Swine) influenza strains. Then in 1999, H1N2 emerged which was occurred due to the reassortment of (Classical H1N1 + H3N2) and that challenged vaccine as that wasn't inactivating by classical H1N1 vaccine. Another reassortment occurred in late 2002 where in both HA and NA genes of H3N2. As a result, novel H1N1 emerged. Recently pandemic H1N1 is emerged in mid 2009. Till now, we had 12 cases reported in UK. In addition, viruses from UK are genetically different than USA and Asia.
May be include the figure of types of SIV
History: Ian's note somewhere
2.3 Diagnostic techniques
Scientists have made outstanding development in the field of Virology. As a result we have wide range of virus detection techniques are available. The various techniques available are as follows:
Samples: Nasal swabs, Lungs, trachea,
SIV replicate primarily in the epithelial cells of respiratory tract. People used trachea for their experiments. (Ref - 5)
Nasal swabs: shows good result
Lungs: Swine lungs are easy to homogenise
Trachea: It is quite strong to homogenise
Sample processing (SOP: VIR 0167)
Lung/ Trachea Tissue can be processed for virus isolation using various equipments such as mortar and pestle, Stomacher, homogeniser or mincing with a scalpel blade or scissors. Processing of tissue should be done in cell culture medium with antibiotic supplement.
Nasal swabs can be collected in a cell culture medium / phosphate buffered saline, supplemented with antibiotics and bovine serum albumin (5mg/ml). # FBS. Sample should be shipped to laboratory overnight on wet ice. At lab, sample should vigorously agitate by hand or on a vortex mixer.
This processed material (Nasal swabs / Lungs/ Trachea) are centrifuged at 1500-1900 rgm for 15-30 minutes at 4Ëšc.
The collected supernatant should maintain at 4Ëš c until inoculation. If sample needs to be held more than 24 hrs, it should be stored at -70Ëšc. (Ref- 11)
Egg + HA :
Use 3-4 ECE per sample ( eggs should be 10-11 days old)
Inoculate 0.1-0.3 ml of inoculum in to allantoic cavity amniotic sac.
Incubate eggs at 35-37Ëšc for 3-4 days and observe under the candle daily. Eggs within died embryo in 24 hrs should be discarded.
After 24 hrs, eggs with died embryo should be refrigerated.
Harvest fluids from eggs after incubation period and centrifuge that at 1500-1900 rgm for 10-20 minutes at 4 Ëšc. Collect the supernatant in another bottle for further testing.
Fluids are evaluated for the presence of SIV with the haemagglutination (HA) test. If HA comes up negative first time (First passage) than repeat ECE+ HA (called 2nd passage).
Finally, results should be recorded after 2nd or 3rd passage.
Positive samples are typed by HI tests (see below).
Make a solution of 0.5% erythrocyte suspension from male turkey or chicken blood. Washed and 0.5% suspensions of erythrocytes can be stored at 4Ëšc for up to 1 week.
Dispense 50 µl PBS in a row of 8-12 wells on a 96 well V- or U- bottom micro titre plate for unknown samples and add one row for positive control.
Add 50 µl of undiluted isolate to the first well of each corresponding row.
Cell Culture + HA
Virus isolation can be conducted in cell lines and primary cell susceptible to SIV infection. MDCK (Madin- Darby canine kidney) and CACO2 are preferred cell line but primary swine kidney, swine testicle, swine lung are swine tracheal cells can be used.
Prepare a cell culture medium with antibiotics. For the MDCK cell lines, add final concentration of 1 µg/ml â€¦? Of TPCK- treated trypsin. For the CACO2 cell lines, there is no need to add such trypsin asâ€¦..
Wash confluent cell monolayer (48-72 hours post-seeding) three times with saline solution in order to remove residual growth.
Inoculate cell cultures with an appropriate volume of tissue suspension or swab supernatant and add equal volume? Of maintain media (Without FBS).
Incubate cell cultures for 45 minutes at 37Ëšc with 5% Co2. Add remaining media.
Incubate them at 37Ëšc for 5-7 days with periodic examination of cytopathic effect (CPE).
End of the incubation Period, CPE will be recorded and results confirmed by HA tests. Negative results go through 2nd passage + HA .Finally, Results recorded after 2nd passage.
Positive samples are typed by HI tests (see below).
Traditionally it remains 'Golden Standard Method'.
It achieves quite the sensitivity of virus isolation in cell culture.
Theoretically at least, a single viable virion present in specimen can be grown cultured cells.
Thus, it is expending millions of virions.
It is only the technique that can detect unexpected, unidentified, unforeseen virus.
Even, discover entirely new agent.
It is the only technique producing a supply of live viruses for further examination such as drug sensitivity testing, characterisation of new virus, to produce diagnostic antigen and antibody.
Also, cells can be stored for years with liquid nitrogen.
So, cell culture is the best to replace Eggs.
Prepare a lysis solution of 10µl of mercaptoethanol per 1ml of RLT buffer. For each sample, 600 µl lysis solutions is needed.
Take a 200 µl samples in eppendorf tube.
Add 600 µl lysis solution to each sample.
Leave samples to lyse in the dark for approximately 1hour at room temperature. After lysis briefly vortex at high speed.
Sub typing Agents
HI test (Haemagglutination inhibition test):
Dilute reference HA antigens (H1, H3, etc) to a concentration of 8 HA units (HAU) per 50 µl in 0.01MPBS, pH7.standardise unknown influenza A viruses to contain 8HAU in 50 µl
Conduct a back titration for all unknown isolates and the H subtype antigens to assure that the correct HAU are present. The back titration is performed as described in the HA procedures except the six well plates used instead of eleven.
Treat each reference serum with RDE( receprtor desroting enzyme) ; add 50 µl to 200 µl RDE.. Incubate 12-18hrs in a 37.c water bath. Add 150 µl 2.5% sodium citrate solution and heat inactivate at 56.c for 30 minutes. Combine 200 µl treated sample and 25 µl.
Currently, the various virus detection techniques available are embryonated chicken egg test, using different cell lines and RT- PCR. All these techniques) have different sensitivity for virus detection. They have various merits and demerits. One of the recent paper claims that Caco2 cell lines are more sensitive than Egg method. The big question arises for this type of techniques is that "Are they really detecting virus at reasonable level?"
Traditional techniques for virus detection are not reasonable in terms of time, expense, ethical issue and for the various types of viruses. For this reason, Scientists are looking for best method of virus detection and have started to look at culture media and RT-PCR techniques.
In UK, currently free available influenza virus testing to primary customer at VLA funded by government is not assured for long time period.
2.4 Rediscovery and recent research (similar such as tech comparison)
In most of the laboratory people are using egg inoculation technique, cell cultures (using various cell lines, Generally MDCK cell lines), RT- PCR. But due to the improvements, we have a CaCo2 cell line which shows more sensitivity than MDCK whereas RRT - PCR shows better sensitivity than others.
3.0 Aim of experiment
The aim of experiment was to compare of various techniques in the diagnosis of swine influenza virus (particularly in UK). Four different types of techniques were used against three different types of viruses to compare speed, simplicity, sensitivity, specificity, and expense. It was expected that sensitivity of cell culture method is quite good than ECE method and RRT- PCR better than those.
Materials: All the samples were pre collected and stored them safely at VLA for other experimental purpose.
3.2 Cell lines
3.3 RRT-PCR (M gene
Isolation of pandemic H1N1 in UK
The best method is the one who can fulfil prerequisite:
The site from virus was isolated
Isolation of virus from several cases
Knowledge of virus
Various elements needed for virus
http://vet.sagepub.com/content/39/1/10.full - interesting about various techniques.
http://www.google.co.uk/imgres?imgurl=http://4.bp.blogspot.com/_-Sz2IZ6Pp9k/SWYCIgMJLfI/AAAAAAAAAPg/wjVLzQah_Bk/s320/134.bmp&imgrefurl=http://microbiologyb.blogspot.com/&usg=__vn3cTftlSTfHbFWZY8mji11BHcI=&h=272&w=320&sz=34&hl=en&start=1&itbs=1&tbnid=NZT6HAHlOu31gM:&tbnh=100&tbnw=118&prev=/images%3Fq%3Dcytopathic%2Beffect%2B-%2Bpandamic%2Bvirus%2Bon%2BMDCK%2Bcell%2Bline%26hl%3Den%26tbs%3Disch:1 virus and various technique
http://www.flickr.com/ - Pictures/ Imagesâ€¦
http://download.thelancet.com/flatcontentassets/H1N1-flu/diagnosis/diagnosis-6.pdf - techniques
http://www.searo.who.int/LinkFiles/Dengue_Bulletins_c24.pdf comparison on dengue