Lecture 14 - Thursday, October 31, 2024 - Viral & Bacterial Infections PDF

Course HSS 2305 A Molecular Mechanism of Disease Lecture-14 Viral and Bacterial Infections, Pathogens, Background, Intervention options Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of...

Course HSS 2305 A Molecular Mechanism of Disease Lecture-14 Viral and Bacterial Infections, Pathogens, Background, Intervention options Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa, Roger Guindon Building 451 Smyth Road Ottawa, ON K1H 8M5 Tel 613-878-7043 (Cell) E-mail: [email protected] Alternate: [email protected] Affiliate Investigator, Chronic Disease Program, Ottawa Hospital Research Institute Web: https://med.uottawa.ca/pathology/people/basak-ajoy 1 2  Infectious Diseases Viral Infections Bacterial Infections Parasite Infections Role of Proteolysis  Role of Host and Foreign Enzymes Target Identification Intervention Strategies 3  Infectious Diseases (ID)  Caused by Virus, Bacteria or Parasites  These are organisms that are found all around us  They use our body as host for living and/or reproduction /multiplication Organism Micro-organism Macro-organism Commonly called Parasite includes Include viruses & bacteria) which worms, arthropods (insects, mites, ticks replicate within the host and can etc.) & are seen through naked eyes. multiply to produce a very large Here one infectious stage matures into number of progeny, thereby causing one reproducing stage, and the resulting an overwhelming infection progeny leave the host to continue the cycle. The level of infection is therefore determined by the numbers of macro- organisms that enter the body. This distinction between micro- and macro-parasites has important clinical and epidemiologic implications. 4  Infectious Agents 1. Virus (contains DNA or RNA wrapped with protein) [Common cold, Influenza (HN type), Chickenpox, Pneumonia, Gastroenteritis, HIV, SARS corona, Hemorrhagic Fever (Ebola, Lassa, Hanta, Crimean Congo Hemorrhagic), Hepatitis B/C etc] 2. Bacteria (one cell microbe with simple cellular organization) [Anthrax toxin, Escherichia coli, Tuberculosis, Streptococcus, Salmonella] 3. Parasite (Protozoa, Yeasts, or Multi-cellular organisms eg. fungi or worms that live in the host to obtain nourishment) [Malaria, Amoebic dysentery, Intestinal Giardiasis] 5 VIRAL INFECTIONS 6 Electron microscope pictures of some virus particles Ebola virus hSARS corona virus Pneumovirus Influenza A virus hCytomegalo virus hHIV-1 7 Viral & Host Materials/Components in Viral Infections Viral component  Viral enzyme/s  Viral proteins (Surface glycoproteins & Structural proteins)  Viral genetic (DNA or RNA) materials & replication machinery Host component  Host enzyme/s involved in viral surface glycoprotein maturation 8  HISTORY HIV-AIDS AIDS / Acquired Immuno Deficiency Syndrome was first reported in June 5, 1981 U.S. Centers for Disease Control (CDC) & Prevention first recorded a cluster of Pneumocystis carinii pneumonia (opportunistic lung infection) in small group of homosexual men in Los Angeles In 1982, the CDC introduced the term AIDS to describe the newly recognized syndrome. HIV was identified in 1983 with a blood test available in 1985 Nobel prize in 2008 in Physiology & Medicine: Françoise Barré-Sinoussi and Luc Montagnier for their discovery of HIV. Robert Charles Gallo (CDC, USA) made significant contribution in the field HIV. Most recognize both as the discoverer of the HIV virus 9  Acquired Immune Deficiency Syndrome (AIDS)  Genome consists of duplicate copies of positive single-stranded RNA  2 types known HIV-1 and HIV-2  HIV-1 likely originates from cross species transmission of a Simian Immunodeficiency virus (SIV) from a subspecies of Chimpanzee (Pan troglodytes)  Antibody for HIV-1 dates back to 1959 but not known before 1981.  HIV-2 is thought to have originated from cross species transmission of SIV from Sooty Mangabeys (primate monkey)  Spreads via contact with body fluids (Blood / Blood products, Sexual activity associated fluids like seminal and vaginal fluids) but not by physical contact. It is also not transmitted via saliva/tears/sweat unless it contains blood or there is an injury or tear in the tissue. 10 11 12 12  Structure of HIV  Mature HIV virus is a spherical particle (10 nm radius)  Surrounded by a lipid bilayer Surface glycoprotein gp160 (MW 160 KDa) cleaved to form gp120/gp41 complex which adhered to viral membrane  Inside of envelope is matrix protein p17  Within the shell is the conical capsid core made up of protein p24  It is a RNA virus, the core holds two copies of RNA  Possesses three essential enzymes for its infection activities: HIV protease (PR), Reverse Transcriptase (RT) and Integrase (IN) 13  Schematic of Mature HIV-virion Surface glycoprotein gp120 Transmembrane protein gp41 Integrase (IN) Matrix protein p17 Reverse Transcriptase (RT) Capsid protein p24 HIV Protease (PR) Nucleocapsid protein p7 14  HIV virus (More Information)  It is a complex enveloped “Retrovirus”  Its genome encodes Regulatory, Accessory & Structural proteins Key Structural Proteins are “gag”, “pol” and “envelop”, that are common to all the retroviruses.  It is classified as an RNA virus since it has RNA as its genetic material and does not replicate using a DNA intermediate. Its nucleic acid is positive sense single-stranded RNA (ssRNA) but can be double-stranded RNA as well (dsRNA) for others.  This virus invades the central nervous system, ultimately culminating in severe neurological disorders and increased susceptibility to persistent infections due to suppressed immune system.  Examples of ssRNA viruses: SARS, HIV, Influenza, Hepatitis C 15  HIV viral gene LTR: Long Terminal Repeats Identical sequence repeated hundreds to thousands times) - There are 9 genetic domains in HIV 1 (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat, env and rev), encoding 19 proteins. -3 of these genes, 5’-gag, pol, and env-3’ (common to all retroviruses), contain information needed to make the structural proteins for new virus particles. 16 - gag: encodes antigen matrix, capsid and nucleocapsid proteins; -pol: encodes the enzymes: Protease (PR), Reverse transcriptase (RT) and Integrase (IN); -env: encodes the glycoprotein gp160 that after synthesis present on virus surface via its transmembrane domain. -HIV-1 protease (PR) cleaves the gag/pol polyproteins, generating important viral enzymes and structural proteins which are essential for viral replication. - 6 remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease 17  HIV-1 infection mechanism - The various phases of the complex life cycle of HIV-1 which occurs in several interrelated steps is in next slide. - Following infection HIV enters T4 lymphocytes - These small white blood cells are responsible for immune activity. They contain CD4 receptor and functions by inducing the nearby precursor T-cell to differentiate into mature T cells whose number is severely decreased in AIDS patients where the virus particle loses its outer envelope releasing viral RNA and Reverse Transcriptase (RT) enzyme. - This enzyme catalyzes synthesis of a complimentary DNA strand from viral RNA template. The DNA helix then inserts into the host genome where it is known as the provirus and then it is transcribed by the infected lymphocyte, possibly after the cell has been activated by exposure to antigen; new viral RNA and proteins are produced to form new viruses that bud from the cell membrane. - Agents that interrupt any of these phases will have therapeutic potential in HIV infection. One example involves the blockage of a co-receptor “CCR5 (Chemokine Co-Receptor)”. CCR5 binds to and activates CD4 receptor 18 Various stages for life cycle of HIV-1 19 20  The mechanism of initial interaction of HIV with host cell involves (1) Cleavage of gp160 by a host cellular protease (Furin/PCSK3) forming gp120 and transmembrane bound gp41 (2) The binding of outer envelope glycoprotein gp120 to a cell surface receptor CD4 protein in association with co-receptor CCR5. (3) The transmembrane protein gp41, which has fusogenic property, then interacts with a specific fusion receptor (F) on the cell surface. This could alone permit viral entry if the virus is brought in close proximity to it. Both receptors together make viral infection most efficient. (4) Another note is that a co-receptor CCR5 (Chemokine Co-Receptor 5) also called CXCR4 plays a vital role during the viral attachment. 21 Proteolytic maturation of GP160 of HIV by PCSKs. The two hepta repeat domains at the N and C- terminal of gp41 [HR-N (546-579) and HR-C (624-666)] are shown in yellow, while gp120 and gp41 are shown in red and blue respectively. Disulphide bridges are shown by connecting bonds and the positions are indicated within each domain. The N-glycosylation sites (22 within gp120) and (7 in membrane anchored gp41) are indicated by numbers at the bottom. Tm = transmembrane domain (pink). gp 160 SP Tm Gp 160 ER Signal peptidase PTKAKRRVVQREKR511 AVGIG Tm PCSK3/Furin Fusion peptide Golgi domain HR-N HR-C Tm 332 Gp 120 gp 120 Gp4141 gp 22 Following cleavage, gp120 is recognized by T4- lymphocyte cell surface receptor CD4 and co- receptor CCR5 while the other fragment gp41 remains bound with the viral membrane. It contains two helical domains called Hepta/d Repeat (HR) Domains (HR-N and HR-C) which interact with one another and form a complex. During fusion event, HR-N which forms a trimeric helix, associates strongly with HR-C to form a six helical coiled-coil bundle (or “trimer-of-hairpins”) Coiled-coil (homodimer); leading to a channel formation & finally 3 pairs from each HR-N & internalization with host membrane. HR-C form complex Hepta Repeat Sequence: Hd =Hydrophobic amino acid (Leu, Ile, Tyr, Phe, etc) 1 2 3 4 5 6 7 Po = Polar amino acid (Ser, Thr etc) Ch = Charged amino acid (Arg, Lys, His etc) Hd-Po-Po-Hd-Ch-Po-Ch 23  HIV-AIDS - Projecting from viral envelope are glycoprotein molecules with mushroom like structure consisting of 4 molecules each of gp 41 (as stem) and gp 120 (as cap) - Three viral enzymes Reverse Transcriptase (RT) Integrase (IN) Protease (PR) 24  Targets & Strategies for HIV intervention Reverse Transcriptase (RT) blocking - This approach aims at preventing the reverse transcription of viral RNA into viral DNA by keeping the viral RNA and reverse transcriptase (RT) from escaping their protein coat. Thus RT became one of the prime targets for the design of anti-HIV agents. Several dideoxy nucleoside analogs have already been shown to be effective inhibitor of RT. - AZT (also known as Zidovudine, chemical name, 3'-azido 3'-deoxy thymidine) belongs to this class of molecules. These molecules act because of their resemblance to deoxynucleosides, the building block of DNA. AZT, approved by US Food and Drug administration in March, 1987, has been shown to be powerful in vitro inhibitors of replication of HIV-1 in human T cells. Therapeutic ability of AZT is purely temporary and suffers from many serious side effects and complications such as toxicity, bone marrow suppression, anemia, etc. Moreover resistance to AZT may occur on prolonged treatment. 25  Blocking Binding of Virus Particles  This may include therapies with antibodies that bind to viral glycoproteins gp41 or gp120.  In an alternate approach, cellular CD4 protein molecule of host cell could be targeted with monoclonal antibody for binding.  Another approach would be to inject the subject with "soluble CD4" (consisting of a portion of CD4 that normally lies outside the cell membrane) which is expected to bind tightly to gp120, blocking the infection. 26  Arrest of Translation  Another approach relies on arresting the translation by employing Antisense Oligonucleotides which are actually segments of DNA that are complementary to a portion of HIV mRNA.  They bind to the viral mRNA and prevent ribosomes from translating the mRNA into viral proteins.  Oligonucleotides are however rapidly degraded by cellular enzymes. To make them resistant to the enzymes, one could substitute the S-atom for O-atom on the phosphate links between the nucleotides. The resulting compound called “Phosphoro-Thioate”, is resistant to degradation and has been shown to inhibit the expression of HIV in vitro. 27  Suppression of HIV Protease Activity  Another front against AIDS consists of inhibiting the enzymatic activity of retroviral protease, called HIV-1 protease (a 99 amino acid Aspartyl Protease) thereby modifying the important viral proteins. The assembly of a new virion particle which takes place at the cell membrane is made up of envelope protein and two precursor proteins in differing length. One precursor molecule draws two strands of viral RNA into the nascent virion and a protease that cuts itself of along precursor. The protease completes the formation of the virion by cleaving other enzymes such as Integrase, a DNA polymerase, Ribonuclease. In view of the important roles of HIV-1 protease, specific inhibitors are developed in an effort to contain the infection.  Some examples are Ribavircin, Colchicine, 1-Aurothioglucose, Dimyristoyl phosphatidyl derivative of AZT, BCH-189, etc. 28  Antifusion agents for viral entry  Viral entry process is an attractive drug target for blocking infection. Several antiviral agents function by interfering with the formation of HR-N and HR-C assembly.  The concept for small molecules that inhibit the formation of six helical coiled-coil structures is considered as a potential platform for drug development against many viral infections including HIV. In fact several drugs such as “T20”/“Fuzeon”/Enfuvirtide, gp41 (127-162), DP-178) for HIV- 1, GP610 for Ebola virus and HR2 for murine hepatitis virus have been developed as entry inhibitors. These HR domains contain single or multiple glycosylated Asn residues. It is likely that carbohydrate residue in HR domain plays an important role in helix bundle formation.  The fusion or envelope proteins of many other retroviruses eg Respiratory Syncytial Virus (RSV), Simian Immunodeficiency Virus (SIV), Filovirus Ebola Virus, Corona Virus, Hepatitis Virus (HV), human Severe Acute Respiratory Syndrome (hSARS), Paramyxovirus, Measle Virus (MV), and Visna form similar hairpin structures as a prelude to membrane fusion. 29  Blocking dimerization of HIV-1 protease HIV-1 protease contains “Asp-Thr-Gly (D-T-G)” sequence in the catalytic site with a single domain of an Aspartic protease. However it only functions (in active state) in the homodimaric form with a two-fold symmetric active site and a highly flexible b-hairpin "Flap" contained in the amino acid segment [residue 43-58] which interacts with the substrate. It is thus proposed that drugs that interfere with this dimerisation process may be able to inactivate the enzyme via an alternate mechanism. Also attempts have made to develop symmetric dimeric inhibitors. 30  HIV protease (HIV-PR) (99 amino acid), shortest protease, active only in Dimeric form Active site Dimer interface Asp 25 “Fireman” Handshake Asp125 - Aspartic acid protease family –Asp-Thr (Ser)-Gly catalytic triad –HIV-PR is active it is in homodimer form 31  Inactivation of host cellular protease  Another way to block HIV infection would to inhibit the enzymatic activity of host protease responsible for maturation of gp160 leading to gp120 and gp41 which forms a complex in the secretory pathway of mammalian cell and helps in the anchoring of gp120 protein onto the viral envelope.  This host protease has been identified as Furin/PCSK3, a member of PCSK family. The site of proteolysis has been identified as VQREKR512  AV, a consensus motif for PCs. Thus PC-inhibitors are expected to suppress gp160 processing and subsequently the AIDS symptom. Studies already confirmed this hypothesis. This finding led to a significant interest in the development of potent and specific inhibitors of Furin which may find application in HIV intervention. Current notion is that viral pathogenesis of HIV can be effectively suppressed by a Host-Guest Combinatorial target approach where the host protease furin, the viral protease/s and/or viral fusion process are all inhibited at the same time by using combination of drugs. 32  FDA-Approved Anti-retrovirals For HIV Treatment Combivir Viread Hivid Epivir Rescriptor Ziagen Retrovir Videx Zerit Viramune Sustiva Trizivir Emtriva 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 NRTI Invirase Viracept Kaletra Fuzeon Fortovase Agenerase Reyataz nNRTI Norvir PI Crixivan FI  Nucleoside Reverse Transcriptase Inhibitors (NRTI)  Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI)  HIV Protease Inhibitors (PI)  Fusion Inhibitors (FI) Alternate Combination Therapy: Use of 3 separate anti-retroviral agents such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) & protease inhibitors (PIs). 33  Side effects of PI’s Increases in blood sugar and even diabetes in HIV patients and hyperglycemia (high blood sugar levels) in HIV patients using protease inhibitors. Lack of compliance due to HIV-inhibitor pharmacokinetics, toxicity, and tolerance Drug resistance mutants 34 Human SEVERE ACUTE RESPIRATORY SYNDROME CORONA VIRUS (hSARS CoV) 35  hSARS coV-1 Infection with a typical pneumonia Severe Breathing Difficulties Attacks lung (First reported in 2002) Caused by a new type of corona virus, hSARS-coV. Previously two human coV were known hCoV-229E and hCoV-OC43 which cause ~30% common colds but rarely pneumonia Highly infectious, quickly spread to >25 countries in 2003 outbreak Caused respiratory distress & enteric diseases in humans and animals. Animal derived coV cause respiratory, gastrointestinal, neurological, hepatic diseases in hosts 36 Comparison between HIV (original source: Monkey) and hSARS corona virus (original source: Bat) HIV SARSCoV1 1st Case Diagnosed 1981 November 2002 First Sample January 1983 February 2003 Virus Identification May 1983 March 17, 2003 Cloning 1984 April 2003 (1st Week) April 16, 2003 Sequence 1985 (Published Online May 1st, 2003) Size 8,788 Nucleotides 29,727 Nucleotides 4 Years 5 Months 37  Schematic representation of SARS CoV1 virion Single stranded (ss) RNA virus Genome size is 29,725 kB Genome is composed of: A stable region encoding an RNA-dependent RNA polymerase A variable region representing four coding sequences for viral structure proteins (S, E, N, M proteins) 5 putative uncharacterized protein S - Spike glycoprotein: Receptor binding and Membrane fusion activities E - Envelop membrane protein: Plays a role in coronavirus assembly. N - Nucleocapsid phosphoprotein associated with viral RNA inside the virion M - Membrane protein: Required for virus budding 38  Steps in hSARS co V replication that are potential targets for antiviral drugs and vaccination 39  Sequence of the spike glycoprotein of hSARS-CoV1 and its Processing By Host Enzyme Furin/PCSK3 SARS Spike Protein (Urbani-variety) Fusion Inhibitor ? KQIANQFNKAISQIQESL Cleavage by Furin (PCSK3)/PCSK5 S1 S2 TMD SP FP HR-N HR-C CT 1 14 1255 GIAAEQDRNTR761 EVFAQVKQM Peptide from this domain acts as an inhibitor to the entry of virus into host cells  Membrane bound (via Tran Membrane Domain/TMD) SARS Spike Protein (S) is cleaved by host enzyme Furin to produce Two Fragments S1 and S2  S1 upon complexation with S2 is recognized by Cell surface receptor leading to viral fusion via the Hepta Repeat N-terminal and C-terminal domains (HR-N and HR-C) 40  Furin inhibitor used: Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone [CH3-(CH2)8-CO-NH-Arg-Val-Lys-Arg-CH2-CO-Cl] Furin inhibitor inhibits SARS-CoV infectivity and viral spread in Vero cell (kidney epithelial cells of the African Green Monkey) (Right two panels) compared to control (left panel) Control SARS SARS + Furin inhibitor SARS + Furin Inhibitor (lower contrast) Bergeron, Basak, Chretien, Seidah et. al. BBRC 2005, 326(3):554-63. 41 COVID-19 (SARS CoV2) 42  Spike (S) Protein of SARS CoV1 (2003) Cleavage by host proteases (Furin and TTSP) Cleavage by host proteases (Furin and Type 2 Serine Protease (TTSP) SARS CoV1 Spike (S) protein TTSP PTKR797 SFIE S2’ 29 65 73 109 118 119 158 227 269 318 330 357 589 602 691 699 783 1056 1080 1116 1140 1155 1176 SP RBD F I HR-N HR-C TD CT 1 13 318 569 761 762 902 952 1145 1184 1196 1216 1255 323-348 366-419 467-474 Furin S1 GIAAEQDRNTR761 EVFAQVKQM S2 = Glycosylation site (23); SP = Signal peptide; RBD = Receptor Binding Domain = S-S bridge (3 within RBD), F = Fusion peptide; HR-N = Heptad Repeat Domain N-terminal; HR-C = Heptad Repeat Domain C-terminal; TD = Transmembrane Domain 43  Spike (S) Protein of SARS CoV2 (COVID 19) Cleavage by host proteases (Furin and TTSP) TTSP PSKR815 SFIE 61 121 122 165 234 282 331 343 370 603 616 709 717 801 S2’ 1074 1098 1134 1158 1173 1194 SP RBD F I FP HR-N HR-C TD CT 1 17 331 583 685 686 920 970 1163 1202 1214 123 1273 336-361 379-432 480-488 Furin QTQTNSPRRAR685 SVASQSIIA S2 S1 = Glycosylation site (20); SP = Signal peptide; RBD = Receptor Binding Domain = S-S bridge (3 within RBD), F = Fusion peptide; HR-N = Heptad Repeat Domain N-terminal; HR-C = Heptad Repeat Domain C-terminal; TD = Transmembrane Domain 44  Mechanism of SARS CoV infection Binding of SARS CoV2 spike protein derived S1/S2 complex with host receptor ACE2 RBD ACE2 (Receptor) S2 S1 Membrane bound secreted, complexed with S2 (Hexameric S2:S1 complex) CoV cell membrane Host cell membrane 45 SARS CoV2 Spike (S) protein sequence with known disulphide bonds (13 Confirmed from Crystal struct). 14a-C15-115a-C131-4a-C136-29a-C166-124a-C291-9a-C301-34a-C336-24a-C361-17a- C379-11a-C391-40a-C432-47a-C480-7a-C488-36a-C525-12a-C538-51a-C590-26a-C617-31a-C649-12a-C662-8a-C671 -66a-C738-4a-C743-5a-C749-10a-C760-79a-C840-10a-C851-180a-C1032-10a-C1043-38a-C1082-43a-C1126-8a- C1235-C1236-3a-C1240-C1241-1a-C1243-3a-C1247-C1248-1a-C1250-2a-C1253-C1254-18a (1273) SARS CoV1 Spike (S) protein sequence with known disulphide bonds (9 Confirmed from crystal struct)). 18a-C19-118a-C128-4a-C133-25a-C159-118a-C278-9a-C288-34a-C323-24a-C348-17a- C366-11a-C378-40a-C419-46a-C467-10a-C474-32a-C511-12a-C524-51a-C576-26a-C603-31a-C635-12a-C648-8a-C657 -66a-C720-4a-C725-5a-C731-10a-C742-79a-C822-10a-C833-180a-C1014-10a-C1025-38a-C1064-43a-C1108-8a- Basak, Current Proteomics, 2020, 17, 1-8 -C1217-C1218-3a-C1222-C1223-1a-C1225-3a-C1230-C1232-1a-C1235-2a-C1236-18a (1255) Basak, Coronavirus, 2021, 2, 1-10 46  Cleavage of Corona virus S protein by host protease Furin & TTSP TTSP SARS CoV1 S protein PTKR797 SFIE S2’ 29 65 73 109 118 119 158 227 269 318 330 357 589 602 691 699 783 1056 1080 1116 1140 1155 1176 SP RBD F I HR-N HR-C TD CT 1 13 318 569 761 762 902 952 1145 1184 1196 1216 1255 323-348 366-419 467-474 Furin S1 GIAAEQDRNTR761 EVFAQVKQM S2 TTSP SARS CoV2 (COVID) S protein PSKR815 SFIE 61 121 122 165 234 282 331 343 370 603 616 709 717 801 S2’ 1074 1098 1134 1158 1173 1194 SP RBD F I FP HR-N HR-C TD CT 1 17 331 583 685 686 920 970 1163 1202 1214 123 1273 336-361 379-432 480-488 Furin S1 QTQTNSPRRAR685 SVASQSIIA S2 47  Influenza A viruses Influenza A viruses are negative sense single-stranded RNA viruses. The several subtypes are labeled according to an H number (for the type of “Hemagglutinin”, a protein that causes red blood cells to agglutinate or coagulate) and an N number (for the type of “Neuraminadase”, an enzyme that cleaves the glycosidic bonds of the monosaccharide of Neuraminic acid). The O/N-acyl derivative is called Sialic acid. There are 17 different H antigens (H1 to H17) and 9 different N antigens (N1 to N9). 153 different combinations of these proteins are so far known. The newest H antigen type, identified as “H17” by researchers, was isolated from fruit bats in 2012. 48  Influenza Virus Structure Viruses are spherical, containing negative stranded RNA; outer membrane contains Hemagglutinin activity (HA) and Neuraminidase activity (NA). These glycoproteins are anchored to the inner lipid bilayer by M-proteins. Hemagglutinin (H) Attachment to host cell sialic acid receptors, which are found on the surface of red blood cells, and upper respiratory tract cells. HA is needed for absorption of the viral genome. Neuraminidase (N) Cleaves Neuraminic acid of the mucin upper respiratory barrier to expose sialic acid receptors; also cleaves sialic acid receptor to avoid attachment of budding viruses, which will escape to infect new cells. 49  Influenza Virus Infections Avian Influenza virus of Hong Kong or H5N1 type is highly infectious & pathogenic. 87.5-100% mortality in experimentally inoculated white chickens. Avian Influenza A virus (pathogenic to birds) does not replicate or cause disease to humans. However H5N1 gene contains a code for a multibasic amino acid insertion, upstream of the cleavage site. Several forms of genes encoding the insertions of RE, RR, RERR upstream to the processing site were found. 50  Schematic presentation of hemagglutin proteins of H1N1 and H5N1 H1N1 H5N1 Weak PCSK cleavage RPKVREGR243 MN H1N1 (Swine) HA protein HA1 HR-N HA2 HR-C normal type 1 17 243 546 LESS VIRULENT Efficient PCSK cleavage RNTPRRERRKKR330 GL H5N1 HA1 HR-N HA2 HR-C HA protein 1 21 330 552 EXTREMELY VIRULENT Thick arrow ( ) indicates highly efficient cleavage by host enzyme (PCSK3 or PCSK5) 51  Ebola virus - It is extremely pathogenic and virulent. - Its gene encodes 7 structural proteins including the surface glycoprotein (called S protein). - New subtype - Zaire, Ivory coast and Gabon altered from exotic agents to serious pathogens. 52 Proteolytic cleavage of EBOV (Ebola virus) glycoprotein by host enzyme (PCSK3 or Furin) leading to fragments GP1 and GP2 both being crucial for its infection and fusion with host cell 53 Bacterial Infections 54  Anthrax toxin Bacillus Anthracis (BA) is the causative agent of Anthrax, known since late 19th century. It was known as the bovine disease but it strikes all mammals including human. Carnivores may become infected from ingestion of infected carcasses. It is transmitted to humans by contact with infected animals or contaminated animal products. 55  A scanning electron microscopy image of various forms of Bacillus Anthracis (BA) Cutaneous anthrax Bacillus anthracis (BA) BA spore (enlarged) BA spores Sterne strain of BA 56  Bacillus Anthracis BA is gram positive spore-forming bacteria It secretes Anthrax toxin in the form of 3 key proteins which play role in infection - Protective Antigen, PA (82.7 kD, MW) - Edema Factor (EF, 88.8 kD, MW) - Lethal Factor (LF, 90.2 kD, MW) Host enzyme PCSK3 &/or PCSK6 play role in BA infection MW = Molecular Weight 57  Role of Host PCK3 & PCSK6 in Anthrax Toxin Infection Bacillus Anthracis PA83 (PA) binds to cell surface Anthrax Toxin Receptor (ATR) & is cleaved by Furin (PCSK3) or PCSK6 to yield PA63. PA63 forms a heptameric structure in the plasma membrane facilitating EF and LF internalization leading to the spread of infection. 58