PRESENTATION BY DR. SAMIR CHACHOUA M.B.B.S. AVIAN INFLUENZA JANUARY 2006

1. PRESENTATION BY DR. SAMIR CHACHOUA M.B.B.S. AVIAN INFLUENZA JANUARY 2006

2. Virulent Mutation • Ebola Virus • Mahrburg Virus • Avian Flu

3. Introduction Rapidly mutating viruses pose particular difficulties for the human organism as well as in the development of coping strategies. Vaccines have met with very limited success in dealing with these diseases. The seasonal fluctuation of the influenza virus, for example, proceeds unhindered despite massive campaign of flu vaccinations

4. AVIAN FLU H5N1 is a subtype of the species called avian influenza virus (bird flu). Avian flu is a disease and avian flu virus is a species. Flu viruses are named for their Hemagglutinin (H) and Neuraminidase(N) genes. Avian flu viral subtype is H5N1. Influenza A virus, the virus that causes Avian flu. Transmission electron micrograph of negatively stained virus particles in late passage. (Source: Dr. Erskine Palmer, Centers for Disease Control and Prevention Public Health Image Library). Colorized transmission electron micrograph of H5N1 (golden) grown in Madin-Darby canine kidney cells (green). (Source: C. Goldsmith, J. Katz and S. Zaki. Centers for Disease Control & Prevention Public Health Image Library. Image #1841.).

5. MAGNITUDE & EXTENT OF THE PROBLEM Outbreak of human cases from birds in Hong Kong in 1997. Current epidemic since 2003. As of January 11, 2006, 155 cases of infections in humans, resulting in 78 deaths, have been confirmed worldwide. Not all cases of H5N1 infection are reported and consequently the exact mortality rate is unknown. Tens of millions of birds died of H5N1 influenza and hundreds of millions of birds were culled (slaughtered and disposed of) to protect humans from H5N1. Thirteen countries across Asia and Europe have been affected. Infected wild birds found in Europe. Bird Flu Spread on October 26th, 2005. Official UN FAO

6. MAGNITUDE & EXTENT OF THE PROBLEM BY COUNTRY

7. PATHOGENESIS OF AVIAN INFLUENZA VIRUS Characterization of Virus Studies of isolates of avian influenza A (H5N1) from patientsin 1997 revealed that virulence factors included the highlycleavable hemagglutinin that can be activated by multiple cellularproteases, a specific substitution in the polymerase basic protein2 (Glu627Lys) that enhances replication,and a substitutionin nonstructural protein 1 (Asp92Glu) that confers increasedresistance to inhibition by interferons and tumor necrosis factor(TNF-) in vitro and prolonged replication in swine, as wellas greater elaboration of cytokines, particularly TNF-, in humanmacrophages exposed to the virus. Since 1997, studies of influenzaA (H5N1)indicate that these viruses continue to evolve,with changes in antigenicity and internal gene constellations;an expanded host range in avian species and the abilityto infect felids; enhanced pathogenicity in experimentallyinfected mice and ferrets, in which they cause systemic infections;and increased environmental stability. Phylogenetic analyses indicate that the Z genotype has becomedominant and that the virus has evolved into two distinctclades, one encompassing isolates from Cambodia, Laos, Malaysia,Thailand, and Vietnam and the other isolates from China, Indonesia,Japan, and South Korea Recently, a separate cluster of isolateshas appeared in northern Vietnam and Thailand, which includesvariable changes near the receptor-binding site and one fewerarginine residue in the polybasic cleavage site of the hemagglutinin.However, the importance of these genetic and biologic changeswith respect to human epidemiology or virulence is uncertain.

8. PATHOGENESIS OF AVIAN INFLUENZA VIRUS Patterns of Viral Replication The virologic course of human influenza A (H5N1) is incompletelycharacterized, but studies of hospitalized patients indicatethat viral replication is prolonged. In 1997, virus could bedetected in nasopharyngeal isolates for a median of 6.5 days(range, 1 to 16), and in Thailand, the interval from the onsetof illness to the first positive culture ranged from 3 to 16days. Nasopharyngeal replication is less than in human influenza,27andstudies of lower respiratory tract replication are needed. Themajority of fecal samples tested have been positive for viralRNA (seven of nine), whereas urine samples were negative. Thehigh frequency of diarrhea among affected patients and the detectionof viral RNA in fecal samples, including infectious virus inone case, suggest that the virus replicates in the gastrointestinaltract. The findings in one autopsy confirmed this observation. Highly pathogenic influenza A (H5N1) viruses possess the polybasicamino acid sequence at the hemagglutinin-cleavage site thatis associated with visceral dissemination in avian species.Invasive infection has been documented in mammals,and in humans, six of six serum specimens were positive forviral RNA four to nine days after the onset of illness. Infectiousvirus and RNA were detected in blood, cerebrospinal fluid, andfeces in one patient.Whether feces or blood serves to transmitinfection under some circumstances is unknown.

9. PATHOGENESIS OF AVIAN INFLUENZA VIRUS Host Immune Responses The relatively low frequencies of influenza A (H5N1) illnessin humans despite widespread exposure to infected poultry indicatethat the species barrier to acquisition of this avian virusis substantial. Clusters of cases in family members may be causedby common exposures, although the genetic factors that may affecta host's susceptibility to disease warrant study. The innate immune responses to influenza A (H5N1) may contributeto disease pathogenesis. In the 1997 outbreaks, elevated bloodlevels of interleukin-6, TNF-, interferon-, and soluble interleukin-2receptor were observed in individual patients, and in thepatients in 2003, elevated levels of the chemokines interferon-inducibleprotein 10, monocyte chemoattractant protein 1, and monokineinduced by interferon- were found three to eight days afterthe onset of illness.27 Recently, plasma levels of inflammatorymediators (interleukin-6, interleukin-8, interleukin-1, andmonocyte chemoattractant protein 1) were found to be higheramong patients who died than among those who survived (SimmonsC: personal communication), and the average levels of plasmainterferon- were about three times as high among patients withavian influenza A who died as among healthy controls. Such responsesmay be responsible in part for the sepsis syndrome, ARDS, andmultiorgan failure observed in many patients. Among survivors, specific humoral immune responses to influenzaA (H5N1) are detectable by microneutralization assay 10 to 14days after the onset of illness. Corticosteroid use may delayor blunt these responses.

10. PATHOGENESIS OF AVIAN INFLUENZA VIRUS Pathological Findings Limited postmortem analyses have documented severe pulmonaryinjury with histopathological changes of diffuse alveolar damage,consistent with findings in other reports of pneumonia due tohuman influenza virus.Changes include filling of the alveolarspaces with fibrinous exudates and red cells, hyaline-membraneformation, vascular congestion, infiltration of lymphocytesinto the interstitial areas, and the proliferation of reactivefibroblasts. Infection of type II pneumocytes occurs. Antemortembiopsy of bone marrow specimens has shown reactive histiocytosiswith hemophagocytosis in several patients, and lymphoid depletionand atypical lymphocytes have been noted in spleen and lymphoidtissues at autopsy.Centrilobular hepatic necrosisand acute tubular necrosis have been noted in several instances.

11. MODE OF INFECTION OF AVIAN INFLUENZA VIRUS H5N1 RK7First sampled in Qinghai, this is the most powerful strain found so far. This can be transmitted from poultry to human and from human to human. However, this strain of virus is unable to transmit from human to poultry. The diagnosis of this virus is extremely complicated. Incubation period is short and causes of deaths are mainly mis-diagnosis.The H5N1 from Qinghai can kill laboratory chickens in 20 hours and lab mice in 3 days.  As noted above and in additional boxun reports, it can passes from human-to-human with a short incubation period and an easily mis-diagnosed clinical presentation.These documents reveal a diverse gene pool of H5N1, which is rapidly evolving via recombination.  Human isolates from China have not been deposited or even acknowledge by China. HUMAN VIRUS AVIAN VIRUS – H5N1 – Can spread between Bird Cells Cannot Spread Between Humans DEADLY REASSORTED VIRUS THAT CAN SPREAD BETWEEN HUMANS. One Such Could Be H5N1 RK7

12. CONTROL MECHANISMS DETECTION/DIAGNOSIS The diagnostic yield of different types of samples and virologicassays is not well defined. In contrast to infections with humaninfluenza virus, throat samples may have better yields thannasal samples. Rapid antigen assays may help provide supportfor a diagnosis of influenza A infection, but they have poornegative predictive value and lack specificity for influenzaA (H5N1). The detection of viral RNA in respiratory samplesappears to offer the greatest sensitivity for early identification,but the sensitivity depends heavily on the primers and assaymethod used. Laboratory confirmation of influenza A (H5N1) requiresone or more of the following: a positive viral culture, a positivePCR assay for influenza A (H5N1) RNA, a positive immunofluorescencetest for antigen with the use of monoclonal antibody againstH5, and at least a fourfold rise in H5-specific antibody titerin paired serum samples.

13. CONTROL MECHANISMS Antiviral Agents Patients with suspected influenza A (H5N1) should promptly receivea neuraminidase inhibitor pending the results of diagnosticlaboratory testing. The optimal dose and duration of treatmentwith neuraminidase inhibitors are uncertain, and currently approvedregimens likely represent the minimum required. These virusesare susceptible in vitro to oseltamivir and zanamivir.Oral osel-tamivir and topical zanamivir are active in animalmodels of influenza A (H5N1). Recent murine studies indicatethat as compared with an influenza A (H5N1) strain from 1997,the strain isolated in 2004 requires higher oseltamivir dosesand more prolonged administration (eight days) to induce similarantiviral effects and survival rates.Inhaled zanamivir hasnot been studied in cases of influenza A (H5N1) in humans.

14. CONTROL MECHANISMS Antiviral Agents (cont’d) Early treatment will provide the greatest clinical benefit,although the use of therapy is reasonable when there is a likelihoodof ongoing viral replication. Placebo-controlled clinical studiesof oral oseltamivir and inhaled zanamivir comparingcurrently approved doses with doses that are twice as high foundthat the two doses had similar tolerability but no consistentdifference in clinical or antiviral benefits in adults withuncomplicated human influenza. Although approved doses of oseltamivir(75 mg twice daily for five days in adults and weight-adjustedtwice-daily doses for five days in children older than one yearof age — twice-daily doses of 30 mg for those weighing15 kg or less, 45 mg for those weighing more than 15 to 23 kg,60 mg for those weighing more than 23 to 40 kg, and 75 mg forthose weighing more than 40 kg) are reasonable for treatingearly, mild cases of influenza A (H5N1), higher doses (150 mgtwice daily in adults) and treatment for 7 to 10 days are considerationsin treating severe infections, but prospective studies are needed. High-level antiviral resistance to oseltamivir results fromthe substitution of a single amino acid in N1 neuraminidase(His274Tyr). Such variants have been detected in up to 16 percentof children with human influenza A (H1N1) who have receivedoseltamivir.Not surprisingly, this resistant variant hasbeen detected recently in several patients with influenza A(H5N1) who were treated with oseltamivir.Although less infectiousin cell culture and in animals than susceptible parental virus, oseltamivir-resistant H1N1 variants are transmissible in ferrets.Such variants retain full susceptibility to zanamivir and partialsusceptibility to the investigational neuraminidase inhibitorperamivir in vitro

15. CONTROL MECHANISMS Antiviral Agents (Cont’d) In contrast to isolates from the 1997 outbreak, recent humaninfluenza A (H5N1) isolates are highly resistant to the M2 inhibitorsamantadine and rimantadine, and consequently, these drugs donot have a therapeutic role. Agents of clinical investigationalinterest for treatment include zanamivir, peramivir, long-actingtopical neuraminidase inhibitors, ribavirin,and possibly,interferon alfa Immunomodulators Corticosteroids have been used frequently in treating patientswith influenza A (H5N1), with uncertain effects. Among fivepatients given corticosteroids in 1997, two treated later intheir course for the fibroproliferative phase of ARDS survived.In a randomized trial in Vietnam, all four patients given dexamethasonedied. Interferon alfa possesses both antiviral and immunomodulatoryactivities, but appropriately controlled trials of immunomodulatoryinterventions are needed before routine use is recommended. Disposal of Birds More than Hundreds of Millions of Birds Disposed.

16. CONTROL MECHANISMS Mechanisms of Oseltamivir Oseltamivir is an antiviral drug, a neuraminidase inhibitor used in the treatment and prophylaxis of both influenza A and influenza B. Oseltamivir was the first orally active neuraminidase inhibitor commercially developed. Oseltamivir is a prodrug (usually administered as phosphate), it is hydrolyzed hepatically to the active metabolite, the free carboxylate of oseltamivir (GS4071). Oseltamivir acts as a transition-state analogue inhibitor of influenza neuraminidase (3R,4R,5S)-4-acetylamino-5-amino-3- (1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester

17. CONTROL MECHANISMS • Contraindications of Oseltamivir • The following information (but not its interpretation) comes from Roche's "Complete Product Information" publication for Tamiflu (intended for the United States). • In the clinical trials performed by Roche (comparing roughly 2,700 individuals given Tamiflu with 2,650 given placebo), nausea and vomiting were the most frequent adverse reactions reported. Other adverse reactions were not reported by Tamiflu-treated patients at a markedly higher rate than those treated with placebo. • According to Roche, in the postmarketing period, voluntary reports have possibly linked oseltamivir to the following other adverse reactions: • General: Rash, swelling of face or tongue, toxic epidermal necrolysis • Digestive: Hepatitis, liver function tests abnormal • Cardiac: Arrhythmia • Neurologic: Seizure, confusion • Metabolic: Aggravation of diabetes • Postmarketing studies are advantageous because the drug is effectively "tested" on a larger population, and previously missed adverse reactions may be discovered. However, given that forms are voluntary, it may be difficult to determine prevalency rates or whether an actual causal relation exists. The number of adverse reaction reports may be a clue, but these number are not reported by Roche in this document.

18. CONTROL MECHANISMS Contraindications of Oseltamivir (Cont’d) Information from Japan: neurological effects and teen deaths In May 2004, the safety division of Japan's health ministry ordered changes to the literature accompanying oseltamivir to add neurological and psychological disorders as possible side effects, including: impaired consciousness, abnormal behavior, and hallucinations. According to Japan's Pharmaceuticals and Medical Devices Agency there were 64 cases of psychological disorders linked to the drug between fiscal years 2000 and 2004. In February 2004, a 17-year-old male jumped in front of a truck and died after taking one capsule of Tamiflu. In February 2005, a 14-year-old male died after falling nine stories from his condominium building. A third teen reportedly attempted to jump from the window of a building. The two deaths were reported to the Japanese health ministry by Chugai Pharmaceutical Co., a corporation half-owned by Roche which distributes Tamiflu in Japan (Japan Times November 13, 2005; Reuters Nov 14, 2005). Roche points out that 32 million doses have been prescribed worldwide, most of them in Japan, and emphasizes the drug's safety. On November 18, 2005, a previously scheduled Advisory Committee to the United States Food and Drug Administration (FDA) met to reconsider the pediatric safety of Tamiflu; they issued a six-page report: Pediatric Safety Update for Tamiflu. The Committee stated that there was insufficient evidence to claim a causal link between oseltamivir use and the deaths of 12 Japanese children (only two from neurological problems). They did recommend adding a warning to prescription information regarding possible rashes. The author(s) of this section have yet to find Japan's actual listing of adverse reactions linked to oseltamivir. However, it is known that one adverse reaction added to the Japanese list was haemorrhagic Colitis (bloody diarrhoea).

19. CHALLENGES FACING CONTROL AND ERADICATION TECHNIQUES • 1. Viral Instability • 2. The true mechanism of successful vaccination • a.      Lag of phase in immune response • as opposed to viral interference and super-infection. • 3. Stability of Conquered Viruses.

20. EVALUATING SAFETY OF NEW VACCINES • HEPATITIS • AIDS • SARS

21. EVALUATING SAFETY OF NEW VACCINES • Years of Development & Safety Testing Needed As opposed to critical nature of current situation. • Forced Coping Mechanism targeted against the modified host

22. Use of Known Agents In Interference • Newcastle Virus, • Polio Virus • Canine Distemper • Feline Panleucopenia Virus • Caprine Arthritis Encephalitis Virus (“CAEV”) • Endogenous Cells Protection Activation (“ERV”) • Measles versus Aids • CAEV versus AVIAN flu • Sense DNA / RNA versus Anti-sense DNA/RNA • Viral Infection

23. The Use of Known Animal Vaccines To Create Interference In Animals and Humans • These can be in raw and/or modified State • Canine Distemper • Feline Panleucopenia Virus • Caprine Arthritis Encephalitis Virus (“CAEV”) • Endogenous Cells Protection Activation (“ERV”) • Smallpox & Bacterial Viruses

24. ANTISENSE RNA VIRUS 1 • The Use of Known Animal Vaccines To Create Interference In Animals and Humans • Characteristics such as positive DNA or RNA. • CAEV versus Avian Virus ANTISENSE RNA VIRUS SENSE RNA 2

25. ANTISENSE RNA CAE VIRUS VIRUS SENSE RNA CAE VIRUS VIRUS BLOCKAGE OF REPLICATION & IMMUNE RESPONSE TARGET CHANGE • The Use of Known Animal Vaccines To Create Interference In Animals and Humans • . • CAEV versus Avian Virus

26. The Use of Known Human Vaccines To Create Interference In Animals and Humans Polio Smallpox & Bacterial Viruses

27. The Use of Phages and Plasmids For Specific and Non-Specific Interference and Immunity 200 Year History

28. The Use of Chemical Agents To Interfere With Viral Growth And Replication (Feed) • BHT • 2MEA • AL721 • Ethoxyquine, • Detergent and pH modifiers

29. Resistant Condition & Biological Membrane For Concentration of Therapeutic Agent Cancer Rheumatoid Arthritis & Autoimmune Diseases Embryo

30. Agent Includes Alpha-1 Antitrypsin, Sense / Anti-sense Nucleic Acid Mono-clonal and Poly-clonal Antibodies Transfer Factor Interferons, Interleukins and Other Modifying Anti-microbial AgentsUniversal Antibiotic Regenerative & Organ Protective Mechanisms

31. Universal Anti-microbial Agent This combines all of the above mechanisms with super-imposed structural analog including Methylthiols in particular Bismuthiol that can occupy and protect extra and intra-cellular sites attacked by the microbial agent.

32. Colorized transmission electron micrograph of H5N1 (golden) grown in Madin-Darby canine kidney cells (green). (Source: C. Goldsmith, J. Katz and S. Zaki. Centers for Disease Control & Prevention Public Health Image Library. Image #1841.).

40. Effective & Immediate Therapeutic Approach 1.           Vaccination With Active Vaccines Based On Safe & Tested Animal, Human and Other Vaccines 2.           Supplementation with Anti-Microbial Agent both in prevention and therapy (feed augmentation) 3.           Long Acting Passive Immunization With Universal Vaccines

41. Advantages of Current Approaches • Safety and Efficacy Tested. • CAEV • Vaccines with Track Record • Supplements of Known Effects and Side-Effects

42. Results From Current Approaches  Results From University of Stockholm

43. Results From Current Approaches  Results As Tested And Reported By Cedars-Sinai Medical Center In Los Angeles, California

44. Results From Current Approaches  Continuation of Results (from prior slide) As Tested And Reported By Cedars-Sinai Medical Center In Los Angeles, California

45. Results From Current Approaches  Results of efficacy as tested at Cedars-Sinai Medical Center in Los Angeles California

46. Results From Current Approaches  Results of Non-Toxicity as Shown and Tested at Cedars-Sinai Medical Center in Los Angeles, California

47. Results From Current Approaches  Correspondence from Cedars-Sinai Medical Center recognizing “Profound Inhibition” of Dr. Chachoua’s Therapy

48. Results From Current Approaches  • 30 year old lady • presented with • Tuberculosis • Septicaemia. • This resolved • completely • within two days • of vaccination

49. Results From Current Approaches  Picture Showing Healthy Cell After Treatment of Septicaemia

50. Successful Treatment Confirmed By The University of Southern California Results From Current Approaches