Pathogenesis is disease. Normal flora are part of host’s normal microbial community. Primary pathogens infect healthy hosts. Opportunistic pathogens infect compromised hosts. Virulence is a quantifiable measure of pathogenicity. Parasites do not kill the host. Parasite: consume host resources at host expense. A parasite is an organism that lives in or on the living tissue of a host organism at the expense of that host, but without immediately killing the host. The biological interaction between the host and the parasite is called parasitism. Parasitism is a type of symbiosis, by one definition, although another definition of symbiosis excludes parasitism, since certain types of DNA, such as transposable elements and B chromosomes, may also be considered as parasites of the host genome.
Some organisms are parasitic only during a part of their lifecycle. Many cuckoos, for example, are brood parasites: their young are parasitic on the host species, but adult cuckoos fend for themselves.

Salmonella is a gram-negative intracellular pathogen

T breakthru host immunse system and cause diseaseExposure to host, adherence to host titissue & recognize it carbo, lipd etc, invasion host cell invsaion/nucleus/organelles/tissues, growth and disseminationFirulence factors…act directly on host tissues or indirect: produce ppooclined by it
Virulence factors…host cell damage host defenses: physical like pH, skin or immune system innate and adaptive

Parasites do not kill the host.

1) exposure
2) adherence
3) invasion
4) growth and dissemination
5) virulence factors
6) host cell damage
7) evade host defenses

As with bacterial colonies, it is easy to screen for presence of a virus. When overlaid onto a bacterial lawn, a single virion will create a visible clearing in the lawn. These clearings are called viral plaques, and the virion responsible is called a plaque forming unit (pfu). A large percentage of virions produced will not be capable of infection. Therefore, is important to determine particles per pfu. For example, if for every pfu there are 100 ineffective virions, then particles per pfu = 100.

The following experiment shows the growth pattern of a virus. The methodology is as follows:

• Prepare a bacterial culture in exponential growth.
• Add 10⁴/mL virions.
• Incubate, removing 100µL every 2 minutes.
• Dilute each aliquot 10⁶χ
• Overlay each aliquot onto a bacterial lawn.
• Incubate overnight
• Repeat this experiment, except add chloroform (CH₃Cl) to each aliquot before dilution.

The chloroform will dissolve cytoplasmic membranes, lysing the cells, but the virions will remain unharmed. The following graph shows the results. The y-axis represents pfu/mL, and the x-axis represents time. Solid is without chloroform, dotted is with chloroform.

|
|                    ....__.__.__.__.__.__.__
|                   .   /
|                  .   /
|                 .   /
|                .   /
|               .   /
|______________.___/
| .           .
|  .         .
|   .       .
|    .     .
|     .   .
|      ...
|_____________________________________________

Notice there are two differences between pfu/mL with and without chloroform:

• With chloroform, there is a dip. This is because the viable viruses infect cells and degenerate, causing a temporary decrease in pfu.
• With chloroform, the graph peaks sooner. While usually it takes a while for the viruses to lyse the host cell, that phase is eliminated because the lipid bilayer is dissolved by chloroform.

Viruses have three major differences from bacteria: their genome organization, characteristic mode of replication, and effect on host cell (either death, fusion, or transformation). Viruses are not alive in and of themselves. Instead, they are obligate intracellular parasites capable of replication and reproducing within living cells. A complete virus particle or virion may be regarded as a block of genetic material (nucleic acid) surrounded by proteins (capsid) to protect the nucleic acid. In some viruses, the nucleocapsid (nucleic acid and capsid) is surrounded by an envelope (made of lipids, proteins and glycoproteins). In addition, most viruses are much smaller than bacteria. However, the largest viruses are the same size as the smallest bacteria.

Virus multiply in particular host cells. This is defined by the host cell range of the virus. In transfection, entry does not depend on specific receptors. That is why transfection can be performed on a large host cell range. Host cell range is determined by:

• Specificity of attachment (receptor on cell surface)
• Availability of cellular factors required for viral replication

Breakdown of Events in Viral Life Cycle

1. Attachment or adsoprtion: viruses bind to cells via specific receptor on the surface of the cell. htese are generally proteins or glycoprtoeins. cells naturally lacking poliovirus receptors or in which ireceptor for influenza viruss are enzymatically destroyed are resistant to infection from these viruses….for some viruses use both receptor and coreceptor.
2. Penetration…viruses generally penetrate thoruhg one of the following means: direct fusion with cell membrane releaseing content of virus into cell, by being enveloped into a coated pit which is a vesicle whose outer walls are coated with protein clathrin.
3. Uncoated and elcipse…within the cell, the coated pit will fuse wiht lysosomes, and the lower pH inside the lysosome results in: (i) fusion of the viral emmbrane to the mebrane of the lysosome and (ii) partial uncoating of the capsid. the viral nucleocapsid is then released intot he cytoplasm of the cell. it may remain here for some viruses (cytoplasmic viruses) or migrate into the nucleus for tohers (nuclear viruses). Uncoating of the virion results in eclipse or loss of inefectivityy (failure to recover infectious virus from infected cell).
4. synthetic phase…review of gene expression..DNA replication, transcription, splicing and translation. In order to complete their replication, all viruses must accomplish a common series of events…(i) synthesis of mRNA (ii) translation of mRNA to virus-specific proeins (iii) replciation of viral genome….ss and dsDNA viruses replicate their DNA in a manner similar to ellular DNA replication semi-conservative…
5. Release of progeny virion. Capsid protein and nucleic acids are assembled into virion particles. There are 2 ways the virions can be released:
   • Lysis of cells. For some viruses primarily the non-enveloped and POX viruses, infected cells are lysed by a viral proces to liberate viral progeny.
   • Budding: Enveloped viruses are released by a process known as budding through the cell membrane. viral envelope proteins are incorporated into specific regions of host cell membranes. the assembled nucleocapsid ithen buds though these patches containing viral envelope proteins, possibly some cellular proteins and lipids and becomes enveloped by them. thus budding forms viral envelope.

There are 5 common ways to assay viruses:

There are 3 common ways to combat viruses:

Virion coat proteins absorb to specific receptors on host cell surface. This give the virus specificity, meaning that it will only be able to infect a certain host range. Once the viral genome is injected into the cell, then:

• For a DNA phage, bacterial cell’s RNAP begins transcribing RNAs from certain promoters. The first class of viral genes is intermediate-early genes. Following translation of these genes, the early viral genes are transcribed. These genes encode proteins which get virus ready for DNA replication. After DNA replication, usually into hundreds of copies, the late viral RNAs are made and translated into proteins. These act as structural components, either for capsid formation or cleaving and packaging of DNA into capsid, or to form the receptor on the capsid surface.

Nucleocapsid:

• Attachment
• Fusion & entry into the cytoplasm

Viral Envelope:

• Attachment
• Endocytosis
• Endocytic vessicle (low pH)
• Fusion & entry into the cytoplasm

Plasma-Membrane:

• Attachment
• Endocytosis
• Endocytic vessicle (low pH)
• Membrane penetration & entry into the cytoplasm

Composition: Live attenuated virus.
Measles: Schwartz or Moraten substrains of Edmonston B strain.
Mumps: Jeryl Lynn strain.
Rubella: RA/27-3 strain.
Vaccination schedule: at 12-15 months and again at 4-6 years or before middle school.
Efficiency: 95% lifelong immunization with a single dose.

The picornavirus family has these general properties:

• Large groups of viruses with common structure, genome organization, and expression
• Genome is a single strand of + sense RNA enclosed in an icosohedral non-enveloped capsid
• Causes a wide range of diseases including poliomyelities, enteitis, hepatitis, and common colds.

The picornavirus family has these 5 genuses, with a popular species of that genus listed next to it:

• Enterovirus (poliovirus)
• Rhinovirus (human rhinovirus 1A)
• Hepatovirus (hepatitis A virus)
• Cardiovirus (encephalomyocarditis virus)
• Aphtovirus (foot and mouth disease virus)

Poliovirus will be examined more closely as a model organism for the picornavirus family. As the first virus to be grown in culture (by Dr. Enders in 1949), it has been heavily studied. There are 2 vaccines used to combat poliovirus:

The course of infection is as follows:

• Like all enteric viruses, it is secreted in feces.
• Fecally contaminated food and water transmit the virus.
• Once the virus is ingested, it infects the gut.
• The primary viremia (first time the virus enters the bloodstream) leads to fever, drowsiness, headache, nausea, and vomiting, followed by recovery within a few days.
• However, in rare cases, poliovirus can invade the CNS and target neurons. Paralysis ensues due to neuronal damage.

There are many ways for the virus to be transmitted. Although usually it results from sewage leaking into a water supply, it can also happen if somebody does not wipe well and then goes swimming in a pool. Since poliovirus can be such a debilitating disease, vaccination is very important. However, it is not necessary to vaccinate everybody. By a phenomenon called herd immunity, where just enough people are treated to block transmission, the disease can be eradicated. Continued vaccination is crucial, though, because lab stocks are maintained (and can be released) and vaccinated patients shed attenuated virus (which can mutate and become pathogenic).

In the absence of any viral proteins, the poliovirus nucleic acid can still produce all proteins needed to make new virion particles. If it had been minus sense, it would need to be transcribed before use as a template for translation.

The 5′ end of the genome is highly structured. Poliovirus contains a very small protein at the 5′ end called VPg. VPg is attached covalently (phosphodiester bond) to the uridine residues at 5′ end of the RNA chain. VPg is present in packaged virion RNA but not on viral RNA that is being translated by cellular ribosomes. Upon infection, a cellular enzyme cleaves VPg from VPg-containing viral RNA. poly(A) is also unusual because it is encoded in the genome, as opposed to being added after transcription. This means it is genetically coded (copied from poly(U) at 5′ end of minus strand during replication) and not added post-transcriptionally.

The CAP structure is crucial for positioning mRNA on ribosome for translation. How does the poliovirus RNA translate? Instead of the ribosome binding to the 5′ CAP, the ribosome binds internally to the 5′ UTR. It is highly structured, with many hairpin loops. There is the VPG, several loops, then the Internal Ribosome Entry Site (IRES) or Ribosome Landing Pad (RLP). At the 3′ end of the IRES is the initiating AUG. There is only one ORF in polio RNA, and no stop codon between protein coding sequences. This indicates the presence of a single long polyprotein. Internal binding selects 9th AUG in poliovirus RNA (same as mRNA). Mature viral and structural proteins are generated by proteolytic cleavage of a long precursor polypeptide (polyprotein). Cleavages occur fast, so it is difficult to detect this large polyprotein. Strucutural proteins on left translated before non-structural proteins on right. Ineffcient. Non-structural proteins like RNA-dependent RNAP 3D^pol not needed in high oconcetrations.

• Viral 2A proteasse functions while still part of polyprotein precursor. Before termination, it auto-catalyzes cleavage of P1 from the growing polypeptide chain.
• Viral 3C protease is on right end of genome and catalyzes generation of individual proteins
• Viral proteases also function to shut down host cell translation and transcription, thus allocating more resources to viral processes.
  • Eukaryotic translation: unwinding beings at 5′ end by binding of the 5′ cap by cellular protein complex (cap-binding protein, CPB, or eIF-4F) which consists of eIF-4A, 4B, and 4E-p220). Poliovirus lacks a CAP, so it doesn’t depend on p220 for it’s function. 5′ UTR secondary structure recognized by cellular p52 and other proteins. Still recruits eIF4a and 4B to unwind secondary structure. Ribosome binds internally. So, cleavage of p220 by viral 2A inhibits cellular protein synthesis but not poliovirus protein synthesis.
  • eIF2 is involved in recruiting tRNA^met-Met to ribosome and positioning the initiator tRNA^met onto AUG. In this energy-dependent process, Gtp bound to eIF2 is hydrolyzed to GDP. Recycling of eIF2 requires exhange of GFP for GTP to start antoher round of recruitment. GEF (GDP exchange factor) catalyzes this. eIF2 recruits met-tRNA to the 40s ribosome and poliovirus proteins block eIF2 recycling. eIF2 phosphorylation prevents guanine nucleotide exhachange, nblocking recruitment of tRNA to reibosome and formotation of 40s complex.
  • Also, trancription is halted. Viral protease 3C is translocated and cleaves transcription factor TBP (TATA-binding protein) and other factors, leading to cellular transcription shut-off.
  • Poliovirus protease 2A shuts off translation by phosphorylated eIF2 and cleaving p220; poliovirus protease 3C shuts off transcription by TBP and TFIIIC.

The fate of newly synthesized plus sense RNA depends on the time after infection. Newly synthesized plus sense RNA has 3 fates:

• Translation (early)
• Replication Template (requires viral replicase)
• New Viral Genomes (late)

The viral replicase, an RNAP, is unable to initiate synthesis from RNA. This is unusual. Like DNAP, it needs a primer. VPg-uridine (VPg attached to uridine) serves as this primer. The viral replicase copies +RNA to make complementary -RNA. This minus strand is the template for many new plus strands. The replicative intermediate (RI) is one minus strand template hydrogen bonded to many nascent plus strand RNAs and vice-versa.

There are 5 miscellaneous topics which are nonetheless very important:

They are large icosohedral nucleocapsids with an outer envelope consisting of the lipid bilayer membrane from the infected cell. Their genome is dsDNA 124-230kb. They cause lifelong latent infections of the host. Once latency is established, frequency of reactivation depends on multiple factors including virus subfamily and host physiology.

• Clearance (no virus present, no viral nucleic acid)
• Persistence (low level of virus from small percentage of cells)
• Latency (no virus present, viral nucleic acid in some tissues)
  • Virus remains latent
  • Virus is shed asymptomatically
  • Virus is reactivated, causing symptoms

There are 8 herpesviruses which infect humans:

HSV1 and HSV2 are transmitted by skin to skin contact. It does not penetrate intact skin, but rather requires mild abrasion or chapping of skin. HSV1 causes 95% of orofacial herpes (remainder are HSV-2, but seldom recurs there) & causes 10-30% of primary genital herpes. HSV2 causes primary and recurrent genital infections & may cause primary oral herpes, but like HSV-1 it seldom recurs outside its area of tropism.

Diagnosing a Herpesirus Infection

Post and Roizman Experiment

What is the significance of the TAATGARAT motif in immediate-early promoters?

1. Thymidine-kinase gene + Early promoter-TK gene.
2. Add to early promoter + TAATGARAT + early promoter-TK gene.
3. TAATGARAT-TK Virus: synthesizes TK mRNA temporally like an IE gene. Synthesizes TK mRNA in the presence of cycloheximide.
4. When the TAATGARAT-TK is cloned into a plasmid, TK- cells can be transformed into TK+ cells.
5. When these transformed cells are infected by TK- virions, TK RNA synthesis is induced.
6. Perform same experiment after the TK- virion has been exposed to UV to form thymidine dimers on the DNA & block transcription.
7. TK RNA synthesis is still induced.
8. Perform same experiment in presence of cycloheximde, which blocks translation. This means no ICP0 nor ICP4 will be produced.
9. TK RNA synthesis is still induced.
10. Since no viral protein synthesis is required for induction of the TAATGARAT-TK gene, a virion protein must be the inducing agent. That proteinn is called VP16.

Transient Assay
Test to see transcription factors for IE genes

1. Make a plasmid of the ICP4promoter-luciferase.
2. Transform cells with this plasmid (use lipofectin to do so).
3. Incubate for 24-48 hours, harvest and assay for luciferase activity.

Deletion Assay
delete various segments, attach to luciferase, assay for activity to identify for promoters.

Peter O’Hare

HSV-1 naturally infects the trigeminal ganglia of humans. A branch goes to the eyes, one to the nose, and one to the mouth. The virus can also go latent in the TG, and from there can reactivate from the latent state to cause a recurrent infection. IB4 neurons harbor virus that produce a lytic infection with full viral replication. A5 neurons tend to harbor virus that has gone into latency. In the latent state to lytic cycle genes are expressed.

HSV-1 can infect mouse feet, leading to an infection of the dorsal root ganglia. During latency, the only viral RNA expressed is called the latency-associated transcript, aka LAT. Stresses to the nervous system can cause the virus to reactivate from the latent state, leading to viral replication and transport of the newly made viruses back down the axon and out to the surface.

LAT RNA is anti-sense to part of the third exon of the ICP0 mRNA. At first investigators thought that LAT might be an mRNA encoding a reactivation protein, but LAT is actually a stable intron.

Inhibition of viral growth: action of the viral thymdine kinase protein. Thymidine kinase blocks Viral DNAP.

Summary of HSV latency: 1) viral DNA is an episome. 2) IE, early and alte genes shut off. 3) LAT is the only viral RNA 4) LAT is antisense to ICP0 mRNA. 5) Reactivations vary in the amount of virus released and the presence or absence of clinical symptoms.

Airborne spread or skin-to-skin contact. Like EBV, infection is more severe if primary infection of adult. Patients at risk are (1) adults, (2) pregnant women in their 3rd trimester and (3) immunocompromised patients. The mortality rate for varicella pneumonia in leukemic children receiving chemotherapy is 1,000 times higher than in healthy children. But children with isolated agammaglobulinemia are not at risk.

Sequence of events

1. Airborne transmission
2. Viremia-virus in the blood, replication in organs, incubation period.
3. Continued replication results in emergence of virus from capillaries to the skin.

Complications of Varicella

1. Bacterial superinfection of lesions
2. Varicella pneumonia
3. Neonatal varicella
4. Reyes syndrome (from aspirin intake)

Reyes Syndrome is a a neuroencephelopathy. Liver failure results in toxic buildup of bilirubin, damaging brain cells.

Varicella Vaccine

Live, attenuated virus-so limited infection, not as good an immunization as chickenpox disease. Prevents 70-90% of chickenpox, reduces severity in rest. Can still reactivate to cause shingles: Infections are unilateral, painful vesicular eruptions localized to the dermatome, usually in the head or upper trunk; Severe systemic infections are observed in immune suppressed individuals.

Cytomegalovirus is asymptomatic in majority of cases. Causative agent of infectious mononucleosis. Transmission of cytomegalovirus (CMV):

• In utero
• Early childhood (saliva, etc)
• Venereal in young adults
• Blood transfusion (1-5% rate of transmission with a CMV(-) recipient and a CMV(+) donor)
• Organ transplantation

Epstein-Barr Virus

EBV mononucleosis-Viral replication in the oropharynx, production of virus and transmission to others. Late antigen is VCA= viral capsid antigen. Antibodies induced. EBV latency: In B cells in blood. 10 Epstein-Barr nuclear antigens (EBNAs) plus the Latent Membrane Protein (LMP) are only viral antigens. EBNA-1 binds to Origin of replication and partitions circular DNA among B cell chromosomes. Reactivation from latency occurs through the BZLF protein, which activates transcription of viral genes. Eventually virus replicates, allowing transmission of the virus to seronegatives.

EBV and Lymphoproliferative Disorders: a) Burkitt’s lymphoma, b) Nasopharyngeal carcinoma c) persons receiving organ transplants and immunosuppressive therapy d) Non-Hodgkin’s lymphoma in HIV patients e) Clear role for EBV in Hodgkin’s disease-over 50% are EBV DNA positive.

Influenza virus vaccines:

• Formalin-inactivated whole virus
• Chemically disrupted virus (subvirion)
• Purified antigens

Influenza properties:

• Eight RNA segments
• Antigenic shift and drift
• Respiratory transmission
• Frequent childhood and adult disease

It has two binding proteins: a hemagglutinin (H) and a neuramidinase (N). These, combined with the location and strain of the virus, are used in nomenclature:

A/Ann Arbor/6/60 (H2N2)
strain, location, serial #, year, type

HA binds to salicylic acid. These receptors are only in the respiratory tract. There are different types of linkage:

• α2-3 linkage on avian cells
• α2-6 linkage on avian cells
• α2-3 & α2-6 linkage on avian cells

Antigenic Variation in Influenza

Antigenic variation in influenza virus can occur by antigen shift and antigenic drift. In antigenic drift, small changes accumulate in the epitopes of HA and NA due to RNA replication errors (mutations). This causes the annual variations in influenza. In antigenic shift, a human virus and an animal virus co-infect the same cell, resulting in reassortment of viral RNA segments. Antigenic shift can cause a pandemic if a new virus emerges that:

• Has mostly human RNA
• Has human or animal hemagglutinin protein with high affinity for human cells
• Humans have not evolved antibodies against.

Influenza virus is ana vain virus. There are 15 types of HA and 9 types of N. By resassortment, a new HA can come into the human populaiton. BY antigenic drift, the HA protein can addapt to bind better to the human sialic acid receptor α2-6. There are three human A strains (H1N1, H2N2, H3N2) in addition to strain B. They can all adapt easily to bind to the human receptor. The human HA protien is cleaved only in the lung, meaning the humans strains are infectious only in the lung.

H5N1, an influenza, binds well onto to &alpha2-3 sialic acid. It contains RNA segments that help produce disease. For example, one RNA segment turns off a component of the immune system. H5 is cleaved by the protease furin, which is present in all cells. Therefore, H5N1 is a pantropic virus able to infect all tissues of avians. It is so pathogenic because, although most human lung cells express α2-6 sialic acid, there are a few expressing α2-3 sialic acid. The H5N1 virus infects these cells and causes a potentially fatal very strong immune response. It can infect poultry, and may adapt to be transmissble from human-to-human.

Influenza has two modes of transmission: person-to-person and by respiratory droplets. Classic flu-like symptoms include:

• Fever
• Malaise
• Myalgia (muscular pain)
• Sore throat
• Nonproductive cough

Influenza virus uses the caps of eukaryotic mRNAs instead of synthesizing its own. This was concluded by a series of experiments:

• Actinomycin D inhibits DNA-dependent RNA transcription. Influenza virus transcription is inhbited by Actinomycin D. As a result, it was proposed than nucleus plays a role in viral replication.
• α-amantin is an inhibitor of DNA-dependent RNAP II. It was shown that it inhibited transcription of influenza virus in a cell but not in vitro. This inhibition by α-amantin led to experiments regarding the role of cellular RNA transcription in viral RNA synthesis. In vitro transcritpion was performed using:
  • Detergent-disrupted virions, which provide viral RNA templants and polymerases.
  • Labeled nucleotides
• The dinucleotide ApG, which is complementary to the first 2 nucleotides at 3′ end of each viral RNA segment (3′-UpCpG…5′) was shown to stimulate transcription. It was also shown that ApG was incorporated directly into the 5′ end of the newly synthesized transcript. It is unusual for a dinucleotide to stimulate transcription.
• Dr. Krug showed that globin mRNA, even more than ApG, greatly stimulates in vitro transcritption. He went on to show that the globin mRNA m⁷GpppG can structure was transferred to the influenza virus mRNA transcript.

Krug’s Cap-Stealing Experiment was as follows:

• Globin mRNA added to in vitro transcription stimulates that reaction compared to no globin mRNA added.
• Decapped globin mRNA does not stimulate in vitro transcription, compared to normal globin mRNA.
• Decapped globin mRNA is used to put a ³²P-labeled cap back on, which transfers radioactivity to viral mRNA synthesized in vitro, as shown by gel migration.

Summary: viral polymerase is unable to cap the viral mRNAs. Because uncapped mRNAs are unstable, the virus steals caps from cellular mRNAs. Thus, replication of influenza virus is inhibited by drugs that block DNA-dependent RNA synthesis since these drugs remove a source of cap structures for the virus to steal.

Influenza viruses enter cells via receptor-mediated endocytosis, a kind of engulfment. Following internalization, the vesicle is with an endosome. Endosomes are acidic, and this low pH activates the M2 ion channel. This allows ions to enter the virion, leading to a conformational change in the HA protein. The virus is internalized into clathrin-coated, membrane-bound vesicles.

Amantidine blocks influenza virus replication. Viral mutants resistant to amantidine map to the trans membrane domain of the M2 protein.

Summary: Upon entry into the cell, the enveloped virus resides in a low pH endosome. The viral M2 protein, part of the envelope, serves as an ion channel to further reduce the pH. This induces a conformational change in the HA protein, causing it to protrude forward and effecting a fusion of the viral envelope with the membrane of the endosome. This releases the nucleocapsid into the cytoplasm.

Bacteriophage lambda (λ) was discovered by Joshua and Esther Lederberg. While mutagenizing strains E. coli using UV, a strain was found to be a lysogen.

• Genome is 48,502 bp dsDNA with 12 nucleotide ssDNA cohesive termini. Contained in capsid.
• Linear dsDNA circularizes due to annealing between 3′ sticky cos sites.
• The capsid has two parts:
  1. Head, composed of B, C, Nu3, D, and E proteins.
  2. Tail, composed of J and H proteins.
• Viral Receptor is the E. coli maltose receptor (product of lamB) on surface of host cell.
• Temperate phage, meaning that it can undergo either lysogeny or lysis.
• Chooses developmental pathway depending on nutrient availability.
• Bacterophage λ has two different life cycles.
  • Lytic, where new phages are synthesized, which then lyse the host and burst from the cell.
  • Lysogenic, where the phage genome integrates into the host genome. It is replicated along with host genome host cell replication. Such a host is called a lysogen.

Int

Pi

tL

N

OL,PL

cI

PRM

OR3

OR2

OR1

PR

Cro

PRE

cII

O

P

tr2

Q

/ /

——–

———–

——

——

——

//

——-

	protein coding gene required for lytic replication
	promoter active during lytic replication
	protein coding gene required for lysogeny
	promoter required for lysogeny
PR	The rightward promoter, transcribed to make Cro & extended by N to make lytic cycle genes O & P. OR3 overlaps PRM.
PL	The leftward promoter, transcribed by Pol to make N. Later extended by N to make the integrase gene for lysogeny.
Hfl	A cellular protease cleaving cII. Function at high [glucose], promoting lysis.
N	N is a viral protein that extends transcription from PR into O and P and from PLinto integrase gene. DNA binding protein and RNApol cofactor, binds DNA (at Nut sites) and transfers onto any oncoming RNApol. Alters the recognition of stop codons, so normal stop codons are ignored and special N stop codons are effective instead.
cII	Made from PR promoter, cII activates PRE to make cI and activates Pi to make integrase. In this way, cII promotes lysogeny. cII outcompetes Cro, unless Hfl protease degrades cII (this only happens when there is high [glucose]. cII is unstable due to succeptability to cellular proteases (especially in healthy cells and cells undergoing the SOS response), slightly stabilised by binding to cIII.
Cro	Made from PR promoter, Cro represses transcription from PRM by binding to OR3. At high concentrations it binds to OR2 & OR1, thereby blocking Pol from binding to PR. Cro is a repressor just like cII, but it binds the operators (like OR3) with the opposite affinity from cI. When Cro binds OR3 it represses transcription of cI gene from PRM. RNAP is already trancribing CRO from PR as well as O and P genes with help from N. At higher levels, CRO also autoregulates itself by binding to OR2 and OR1 and repressing transcription from PR. Transcription inhibitor, binds OR3, OR2 and OR1 (affinity OR3 > OR2 > OR1, ie. prefferentially binds OR3). At low concentrations blocks the R promoter (preventing cI production). At high concentrations downregulates its own production through OR2 and OR1 binding.
cI	Made by activation of PRE promoter through action of cII on PRE, cI is an activator of PRM, recruiting RNAP to OR3 by binding cooperatively to OR1 and OR2. Simultaneously represses PR, occupying RNAP site near OR1. At high [cI], binds to OR3 to block polymerase’s ability to transcribe cI gene, shutting itself off. Transcription inhibitor, binds OR1, OR2 and OR3 (affinity OR1 > OR2 > OR3, ie. prefferentially binds OR1). At low concentrations blocks the RM promoter (preventing cro production). At high concentrations downregulates its own production through OR2 and OR3 binding. Also inhibits transcription from the L promoter. Succeptable to cleavage by RecA* in cells undergoing the SOS response.
cIII	cII binding protein, protects cII from degradation by cellular proteases.
PR & PL	Strong promoters. The other promoters must be activated. PRE is the promoter for repressor establishment.
Q	DNA binding protein and RNApol cofactor, binds DNA (at Qut sites) and transfers onto any oncoming RNApol. Alters the recognition of stop codons, so normal stop codons are ignored and special Q stop codons are effective instead.
xis	excisionase and integrase regulator, manages excision and insertion of phage genome into the host’s genome.
int	integrase, manages insertion of phage genome into the host’s genome. In Conditions of low int concentration there is no effect. If xis is low in concentration and int high the n this leads to the insertion of the phage genome. If xis and int have high (and approximately equal) concentrations this leads to the excision of phage genomes from the host’s genome.
A-F	code for phage head genes.
Z-J	Z-J code for phage tail genes. The order shown here is as found on the genome, reading in a clockwise direction]; structural proteins, self assemble with the phage genome into daughter phage particles.
S, R	Lysis promoters, cause the host cell to undergo lysis at high enough concentrations.
OP	[Shown on diagram as O replication P]; DNA replication promoter, promotes the specific replication of only the phage genome.
SIB	Not a protein, but a vital conserved DNA sequence]; Forms a stable hairpin loop structure in transcribed mRNA. Attracts degradation of mRNA by RNAaseIII.
attp	attP not a protein, but a vital conserved DNA sequence]; point of action of int and xis in insertion and excision of the phage genome into the host’s genome. Corresponding attb found in the host’s genome at the point of insertion.

Lifecycle

The ‘late early’ transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome.
The lambda genome is replicated in preparation for daughter phage production.
Q binds to Qut sites.
Replication from the R’ promoter can now extend to produce mRNA for the lysis and the structural proteins.
Structural proteins and phage genomes self assemble into new phage particles.
Lytic proteins build sufficiently far in concentration to cause cell lysis, and the mature phage particles escape.

[xis and int regulation of insertion and excision]
xis and int are found on the same piece of mRNA so approximately equal concentrations of xis and int proteins are produced. This results (initially) in the excision of any inserted genomes from the host genome.
The mRNA from the L promoter forms a stable secondary structure with a hairpin loop in the sib section of the mRNA. This targets the 5′ end of the mRNA for RNAaseIII degradation, so a lower effective concentration of xis mRNA than int mRNA is found, so higher concentrations of xis than int.
Higher concentrations of xis than int result in no insertion or excision of phage genomes, the evolutionarily favoured action – leaving any pre-insterted phage genomes inserted (so reducing competition) and preventing the insertion of the phage genome into the genome of a doomed host.

The lambda repressor is a dimer also known as the cI protein. It regulates the transcription of the cI protein and the Cro protein. cI and Cro proteins regulate λ life cycle. If cI predominates, the lysogenic cycle will ensure. If Cro proteins predominate, the lytic cycle will ensue. cI dimer binds to OR1, OR2, and OR3 in the order OR1 > OR2 > OR3. Binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2, an effect called cooperativity. Thus, OR1 and OR2 are almost always simultaneously occupied by cI. However, this does not increase the affinity between cI and OR3, which will be occupied only when the cI concentration is high.

• In the absence of cI proteins, the cro gene may be transcribed.
• In the presence of cI proteins, only the cI gene may be transcribed.
• At high concentration of cI, transcriptions of both genes are repressed.

How does lambda replicate and package viral DNA? Nu1/A (terminase) binds cos sites.

• dsDNA 40kb genome.
• Complex icosohedral head & short tail.
• More completely dominates cell than M13.
• Replication cycle starts; within 6 minutes host cell protein synthesis shut off.
• Viral proteins completely degrade host cell DNA.
• Host nucleotides used for virion genome replication
• Per cell, virion makes 200 copies of viral DNA within 15-20 post-infection.

T7 good model roganism for gene rexpression….dsDNA….simply 55 gene 40kb genome….infect many viral DNAs per cell…isoalte viral mutants

Pulse-Labeling of viral proteins in E. coli

• Add T7 (MOI of 5pfu/cell) at t=0
• Remove 5mL aliquots every 2min
  • Add ³⁵S-methionine for 30 sec (this makes newly synthesized fluoresce)
  • Lyse cells in SDS
  • Subject to polyacrylimide gel electrophoresis
  • Prepare autoradiogram of gel

Steps to Identify Regulatory Mechanism for Gene Expression

There are several possible ways the virus can regulate gene expression.

• Translational or transcriptional control
• Repressors
• RNAP modification
• Viral-specific polymerases

The experiment to determine which mechanism is used goes as follow:

• Identify Class I proteins
• Map location of each Class I gene by mapping RNA for each protein
• Make mutants in each Class I gene
• Analyze the phenotype of each mutant

To identify Class I proteins, proteins in T7-infected cells are pulse-labeled at various time post-infection. Next, the temperature-sesitive mutant experiment is performed. Temperature-sensitive mutants grow at the permissive temperature of 32°C; they do not grow at 39°C. Temperature-sensitive mutants in gene 1 do not express Class II and Class III genes at the non-permissive temperature. The methodology is as follows:

• T7-infect cells are pulse-labeled at various times post-infection.
• Profile of TS-infected cells at non-permissive temperature is compared to the profile of TS-infected cells at permissive temperature.

Are TS1 mutants blocked at the level of transcription? protein profile at non-permissive temperature? aAre ts1 mutants blocked at txnal level? test….infect cells with nhigh MOI at nonpermissive, label with ³H-uridine…extract RNA and hybridize to cloned DNAs of each class of gene….

There is an experiment designed to test this idea:

• Infect cells with high MOI at the nonpermissive temperature.
• Label with ³H-uridine.
• Extract RNA and hybridize to cloned DNAs of each class of gene.

TS1 mutants in gene 1 do not express Class II and Class III genes at the nonpermissive temperature. We design an experiment to see if there is a polymerase encoded in Gene 1 that is necessary to transcribe Class II and Class III genes. The experiment is as follows:

Gel Filtration: An extract of E. coli proteins is poured over a sizing column. Proteins go in and out of beads with different sized holes. Larger proteins elute first; smaller proteins elute later because they get trapped inside the beads. We find a 450kD cellular RNAP and the 98kD viral RNAP. the cellular RNAP has activity initially, but then the viral RNAP replaces it to transcribe the later genes. The conclusion: Gene 1 encodes a T7-specific RNAP responsible for transcribing Class II and Class III genes.

The temporal order of T7 gene expression is in the same order as the genes themselves, from left to right. There is a terminator for E. Coli RNAP before at the beginning of Class II and Class III genes. Class II and Class III promoters have a common DNA sequence different from Class I promoters. Class I promoters are transcribed by the E. Coli’s RNAP. Class II and Class III promoters are transcribed by the T7-encoded RNAP, which recognizes its own promoter sequence.

Summary of T7 Experiments:

• PAGE gel showed three waves of viral proteins.
• Temperature sensitive mutants were developed in Class I genes.
• The phenotype of gene I mutants was that there was no transcription of viral Class II and Class III genes at the non-permissive temperature.
• Extracts from WT infected cells show a viral specific RNAP eluting from an ion exchange column.
• Exctracts from TS1 infected cells at the non-permissive temperature do not show this viral RNAP activity.
• The conclusion is that gene 1 encodes a T7-specific RNAP responsible for transcribing Class II and Class III promoters.
• Class II and Class III promoters have a common sequence different from Class I promoters, which are transcribed by the cell’s RNAP.

This figure shows the activity of the cellular RNA polymerase and the activity of the T7 RNA polymerase as a function of column fractions. How do you think the activity of each of the RNA polymerases was measured? Viral RNAP activity replaces and is greater than cellular RNAP. Activity could be measured by labeling the promoters with different fluorescent genes. The degree of fluorescene would be proportional to RNAP activity.

Intro

Bacteriophage M13 has a 6kb circular ssDNA genome. It is filamentous (as opposed to icosohedral). Special proteins at the tips are involved in assembly, morphogenesis, adsorption and penetration. It infects F⁺ E. coli, but rather than killing host cells it just slows growth. Virions leak out from the cell. Thus, the virus is not very lytic.

Infection

M13 absorbs the tip of the F-pilus and injects its genome into the cytoplasm of the infected cell. Immediately after injecting the viral DNA into the cytoplasm, the circular ssDNA is converted into dsDNA. The dsDNA is transcribed by host RNAP into viral mRNAs. These mRNAs are translated by host cell ribosomes into viral proteins. Proteins then direct replication of viral DNA by host cell enzymes. Progeny virons assemble.

How is dsDNA converted to dsDNA?

• Strand synthesis following infection was blocked by treateing cells with E. coli RNAP inhibitor.
• Do you need pre-existing RNAP for ssDNA -> dsDNA? Find out if new viral gene expression is not needed for M13 genome replication.
  • Infect cells in presence of chloramphenicol (inhibits new protein synthesis).
  • Result: dsDNA is still made.
  • Conclusion: No new protein synthesis required for synthesis of viral dsDNA.
  • Transform naked viral ssDNA into cells.
  • Result: a small amount is converted to dsDNA.
  • Conclusion: capsid proteins not involved.

M13 ssDNA -> dsDNA

These studies revealed that the single stranded viral DNA becomes coated with E. coli Ssb (single-stranded DNA binding protein), except in one region where the sequence forms a hairpin structure. E. coli RNA polymerase can initiate RNA synthesis at this position on the M13 + strand because it is sufficiently double-stranded for RNA polymerase to use as a template. Once the – strand RNA primer is synthesized by RNA polymerase, it is extended around the circle of the + template strand by E. coli DNA polymerase III: Ssb is released as the – strand is synthesized. The RNA primer is then removed by digestion by the 5’ to 3’ exonuclease activity of DNA polymerase I. The primer is replaced by the DNA polymerase activity of DNA Pol I. Finally, the DNA ends are ligated together by DNA Ligase.

M13 DNA Synthesis

We are trying to take the ssDNA, get a capsid around it, and then get it out of the cell. We initially have circular dsDNA. pII knicks the + strand and binds to the end of the + strand. This allows DNAPIII and helicase together duplicate the strand to make another + strand. When pV accumulates late in infection, it binds to ssDNA and replaces the ssDNA binding protein. pVIII binds to the inner membrane. After pV coats the DNA, pVII and PIX binds to the morphogenic sequence. At the same time, pI and pIV compose a secretion apparatus in the membrane. pV interacts with pVIII and the DNA is passed through the secretion apparatus, with pVIII replacing pV. pIII and pVI are added to the tips of the DNA. At the end, there is one strand of DNA with pII, pIII, PVI and pVIII bound to it.

Proteins

• pI comes a secretion apparatus, it is involved in making a pore in the membrane.
• pII first knicks the dsDNA and binds to the end of the + strand. From that, DNAPolIII and helicase replicate the + strand.
• pIII (along with pVI) forms the rounded tip, and is the first protein to interact with E. coli.
• pIV (along with pI) form the secretion appartus, thus making a pore in the membrane. pI and pIV make a pore.
• pV displaces ssDNA binding protein. It interacts with (and is dispaced by) pVIII, thus guiding the virion toward and through the secretion appartus.
• pVI (along with pII) forms the rounded tip, and is one of the first proteins to interact with E. coli.
• pVII (along with pIX) bind to the morphogenic sequence. It interacts with pV and helps initiate phage secretion.
• pVIII lines the inner membrane. It interacts with (and replaces) pV as the phage exits the cell. The secreted virion is surrounded by pVIII rather than pV.
• pIX (along with pVII) binds to the morphogenic sequence. It interacts with pV and helps initiate phage secretion.

Questions

How do you initiate DNA replication from a dsDNA circle?
How do you separate ssDNA circles needed for further DNA replication from those to be packaged?
How do you put a capsid around the DNA and get it out of the cell?
How do you initiate transcription from the dsDNA circle?

• Protein II (pII) nicks the + strand.

How do you separate ssDNA circles needed for further DNA replication from those that are to be packaged?

• pV accumulated and binds displaced _ strand in place of El coli Ssb. = strand bound by pV does not serve as a template for – strand synthesis. M13 + strand replication…describe the cycle in detail.

At the origin of replication, a hairpin structure is formed that allows host cell RNAP to bind. RNAP lays down RNA primers in the. Possible ways for M13 to express genes: host cell zynes, virus encodes enczome packaged in capsid, encodes during lytic cycle

SSDNABindigProtein = SSB

M13 phage display libraries. It is a good template for doing DNA synthesis and mutagenis because of this ss circle and put primer as bridge and 15 mutations in the middle and mutate mutation in promoter or gene like put stop codon in gene.

Steps in phage display:
A peptide introcuced at N-terminus of major coat protein (gene VIII) by PCR mutagenesis
The linear PCR product

A billion porteins with slightly different protein shapes. As a protein peptide, it can bind to different things. By third process of sticking,, washing, eluting, growing…you can amplify specific peptide binding to protein.

Alternatives…mutage gene III not gene VIII. 2500 copies of gene VIII protein per phage and only 5 of gene III prtoein.

It has a long and unusual shape:

Like togaviruses and flaviviruses, coronaviruses have the following properties:

• + strand ssRNA genomes
• Use insects as secondary hosts
• Wide range and severity of diseases

Coronaviruses have a helical nucleocapsid. They also have an enormous genome (4-5 times larger than that of picornaviruses). While togaviruses use only two mRNAs to synthesize its proteins, coronaviruses use 7. Each coronavirus mRNA encodes for a different protins. Each coronavirus mRNA has the same 3′ sequence (including polyA), but each one begins at a different point on the genomic strand. Only the most 5′ gene is translated from each subgenomic RNA. We will analyze the replication cycle of Mouse Hepatitis Virus (MHV) for further detail:

Like togaviruses, coronaviruses are mainly distributed in the Americas, Africa, and Asia. They prefer topical, hot, and humid climates. Most of the viruses (especially α) use insects/mosquitos/ticks as 2° hosts. They use other animals as reservoirs to maintain virus in nature when 1º is not available.

Like coronaviruses and flaviviruses, togaviruses have the following properties:

• + strand ssRNA genomes
• Use insects as secondary hosts
• Wide range and severity of diseases

There are two classes of togaviruses:

The most important feature of togavirus replication is that is has a dicistronic genome. Two mRNAs are used, one to produce non-structural proteins and another to produce structural proteins. This is important because it is very efficient. Picornaviruses produce one polypeptide, which means that equal amounts of each protein are produced even though unequal amounts are needed. As a result, picornavirus replication is inefficient. Togaviruses, however, regulate synthesis of non-structural and structural proteins by using two mRNAs. In fact, there is 4 times as much subgenomic RNA as genomic RNA in cells infected with togavirus. Two classes of proteins are synthesiszed from different mRNAs, which allows temporal regulation and qualitiative replication. Genomic RNA can do two things: make non-structural proteins (such as replicase) by synthesizing the minus strand, or make structural proteins through synthesis of subgenomic RNA and synthesize positive strand RNA.

A minus strand is made that is complementary to the genomic plus RNA. This minus strand has two initiation sites for replicase: one for non-structural proteins and one for structural proteins. The site for non-structural proteins is at the 3′ end, and if replicase initiates there it will produce the plus RNA encoding non-structural proteins. The site for structural protiens is about mid-way through, and if replicase initiates there then a subgenomic (smaller than genomic) plus RNA will be synthesized. This will encode structural proteins.

Togavirus subgenomic RNA was discovered via the following experiment:

• Cells are infected with togavirus and incubated in labeled uridine
• This labeled uridine is incorporated into all RNA
• Actinomycin D shuts off synthesis of cellular RNA
• As a result, only viral RNAs are labelled
• This RNA is run on a gel, and 3 kinds of togavirus RNA are detected:
  • Genome (plus strand)
  • Negative strand
  • Subgenomic
• Further experiments revealed how the 2 mRNAs could be synthesized at different levels, and that each had only one ORF.

There are five structural proteins: capsid, E1 (composed of E2 and E3), and p62. The capsid protein autolytically cleaves itself from the other structural proteins. The capsid protein assembles with the genomic RNA into a nucleocapsid. E1 and p62 proteins contain a sequence which directs them into the lumen of the endoplasmic reticulum. They move to the Golgi and the trans-Golgi as if they were host cell proteins. They are also modified just like host cell proteins via glycosylation and further cleavage to p62, E2, and E3. Glycoproteins E1, E2, and E3 form a trimer in the cell membrane, which becomes the viral envelope. Incorporation of this trimer into the membrane is very important for the viral infectivity. Cytoplasmic tails of the viral glycoproteins are also required to bind to the nucleocapsid and ensure the membrane forms aorund the new viruses.

Human Immunodeficiency Virus (HIV) is the causative agent of AIDS. It is a retrovirus belonging to the lentivirinae family. HIV reverse-transcribes its RNA to DNA and then back to RNA.

HIV Infection

Leeches and mosquitoes are unable to transmit HIV because the virion is unable to replicate outside of a warm mammalian host. Due to HIV’s specific temperature and environment requirements, it is transferred between mammals.

Cellular requirements for productive HIV-1 replication:

• CD4 surface molecule
• Co-receptor expression
  • CCR5 – the majority of new infections are by viruses that utilize this co-receptor; regardless of the mode of transmission.
  • CXCR4 — approx. 50% of individuals will have viruses that switch to using this co-receptor. Associated with faster disease progression.
• Activated Cell: Cell must progress past the G1b phase of the cell cycle

Reverse Transcriptase (RT): Encoded by the pol gene, it does not have a proofreading function. As a result, it si very error prone. There is approximately one error “mutation” per round of replication.

Implications of RT error: Approximately one error is made per replication cycle. With up to 1×10⁹ viral particles made per day, it is speculated that every possible mutation is present. As a result, there is tremendous potential for generation of drug resistance. Mutations also allow HIV to escape from immune recognition. Mutations can confer a fitness advantage, be deleterious or make no difference at all to the viral fitness. Areas critical for viral function are conserved, although there is tolerance for variability in areas not as critical and especially those recognized by the immune system.

There is innate and adaptive immunity. Adaptive immunity involves two major types of cells:

So Why Don’t CD8+ T-cells Completely Control HIV?

• Loss of CD4+ T-cells compromises the ability of CTL to work.
• HIV mutation abolishes recognition of HIV by CTL.

How do CTL recognize HIV or other antigens?

• The T-cell receptor on CTL bind short antigen-derived peptides (9-10 amino acids).
• These peptides are presented by proteins on the surface of the cells called Human Leukocyte Antigens (HLA). These are the same proteins that make up your “tissue type”.

Human Leukocyte Antigens:

• Are encoded by the major histocompatibility complex (MHC) on chromosome 6 in humans.
• Class I antigens are found on all nucleated cells. CD8+ T cells recognize Class I antigens.
• Class II antigens are 1° on antigen Presenting cells (macrophages, dendritic cells and B cells). = DR, DP, DQ
• CD4+ T cells recognize Class II antigens.

Viral set-point is indicative of the rate of disease progression. The higher the viral load, the faster the disease progression. It peaks, lowers, plateaus, then increases before terminating.

Long-Term NonProgressors (LTNP)

• ~ 1% of HIV infected individuals
• Infected >10 years, CD4+ T-cells > 500/μl of blood, < 5 X 10³ copies of viral RNA/ml of blood, no clinical disease despite remaining untreated.
• Viral factors: attenuated virus e.g. Nef deletions
• Associated with HLA-B*57, HLA-B*27

HLA Association with LTNP

• Depending on the cohort, HLA-B*57 individuals make up to 85% of LTNP.
• The association with Class I suggests that CTL responses are involved.
• However, HLA can’t be the only factor.
• HLA-B*57 individuals are present among cohorts of “progressors” in the same frequency as the general population.
• HLA-B*57 homozygosity does not necessarily confer LTNP status.
• So why is HLA-B*57 so strongly associated with LTNP status?

HLA-B*57 LTNP Target Conserved Regions Of HIV-1 Derived Proteins

Does this confer better protection from disease progression?
Migueles et al.* have shown that HLA-B*57 progressors also recognize conserved regions of HIV-1 derived proteins. These experiments were all done years after the initial infection.

Is Timing Everything?
The viral set-point is an indicator of the rate of disease progression. The viral set-point is reached early after infection. Shouldn’t we be looking at immune responses early in infection? If LTNP are defined as not progressing to disease progression >10 years, how do you know who to study?

Multi-Center AIDS Cohort Study (MACS)

A cohort of HIV-infected men and at-risk men. Take epidemiological data and biological samples every six months for 20 years. Have individuals who seroconverted while under study. Have HLA-typed many of the men.

Questions We Are Addressing:

• Do CTL from LTNP target conserved regions within HIV earlier than Progressors?
• Early in infection, do CTL from LTNP target more conserved regions than Progressors?
• Is it multi-factorial? For example: maybe CTL all from HLA-B*57 target a high number of conserved regions, but LTNP have other responses that tip the balance in favor of the immune response? Antibodies? Innate immunity?

Results:

• There was no significant difference in the viral load between LTNP and Progressors at the first time-point post-infection. Oddly enough, while both CTL from LTNP and Progressors targeted conserved epitopes, overall, LTNP targeted more non-conserved epitopes than Progressors. Does this help?? During early HIV-1 infection 3/7 LTNP had CTL responses to Env compared to 0/7 progressors.
• 6/7 LTNP vs. 1/7 Progressors targeted an epitope in p24 (capsid).
• The 1 LTNP that did not target this epitope at the earliest time-point, targeted it soon afterwards. He retains that response today, 20 years post-infection. He also still responds to the same epitope in Env as he did 20 years ago.

Caveats:

• To measure responses, we are using peptides that represent the consensus sequences for HIV-1 clade B.
• Even though we have measured CD8+ T-cell responses early in infection, 3 to 9 months post-infection is a long time! Within weeks many other viral infections, like influenza, are won or lost.

Rhabdoviruses are rod- or bullet-shaped enveloped viruses with a single strand minus-sense RNA genome. The significant members are rabies virus and a well-characterized laboratory strain Vesicular Stomatitis virus.

Epidemiology Rabies is distributed in most of the world except Australia and Antartica. The epidemiology reflects that of animals in the community. Where canine rabies remains common, most cases of human rabies come from dog bites. In locations where dogs are vaccinated, most cases of rabies come from exposure to rabid wild animals. In 1992 there were 36,000 cases of rabies globally. In the U.S., there have been very few human cases of rabies. However, there were 20,000 cases of racoon rabies in the eastern U.S., in addition to cases from coyotes and bats. Prophylaxis, which is vaccination of domestic animals, has reduced incidence of rabies.

Pathogenesis Following introduction through a break in skin, mucosal surfaces, or respiratory tract, rabies virus replicates in muscle cells and then spreads to neurons of PNS and CNS. It produces severe, oftentimes fatal, CNS disfunction.

Clinical Manifestations The viral inoculum plays an important role in rate of clinical disease. A bite on exposed skin is much more likely to cause infection than a bite through thick clothing. Mutliple bites are more likely to result in infection than a single bite. The incubation period varies from a few days to over 19 years, although most (75%) of pateints become ill within 90 days of exposure. Initial symptoms are associated with other systemic viral infections, such as fever, headache, malaise, and disorders of upper repiratory and gastrointestinal tract. Neurologic complaints during this period include subtle changes in personality and cognition as well as pain at the exposure site.

Human rabies infections are divided into 2 forms: furious and paralytic.

Diagnosis Diagnosis is simple. If somebody has suffered a bite from a rabid animal and exhibits hydrophobia, then they have rabies.

Treatment The wound should be washed thoroughly with 20% soap solution to strip the viral envelope. A healthy dog or cat is observed for 10 days. If behavior is normal, the patient needs only wound care. If the animal exhibits symptoms of rabies, it is tested for rabies virus infection. Rabies immunoglobulin is injected equally into both the gluteus maximus and the would area itself.

Mosaic virus is caused by a variety of viruses which attack all members of the curcurbit family, but especially thrive on summer squash, cucumber and muskmelon plants. It is spread by a variety of methods and so is a serious disease for plants of the curcurbit family, including cucumbers, gourds, muskmelons, winter squash, summer squash, watermelons and pumpkins.

Mosaic virus damage first appears in the form of green leaves which look as if they are mottle or distorted. Often these leaves will also be curled upward, or appear as if their growth has been stunted. Typically these leaves will have yellowish spot on them, adding to their mottled appearance. If cucumber fruits are affected they will vary in color from light green to dark green mottled areas and some which pale to white. Affected areas of the curcurbit family plants may also be covered with warts or alternately the skins may be have faded and be very white and smooth.

Mosaic virus overwinters on a variety of plants including debris from curcurbit family plants which was not cleared from the garden, as well as catnip, pokeweed, motherwort, milkweed and wild cucumber plants. Aphids and cucumber beetles spread the disease as they feed going from infected plant to healthy plant. The prevalence of these insects once they have infested a garden can be damaging on it’s own, not to mention when these insects are spreading mosaic virus. The earlier in the season the disease is spread, the more plants will have severe damage from mosaic virus. Although mosaic virus can eventually kill off the curcurbit family plants, the main affect of this virus on the crop is that plants fruits will taste bitter, and therefore be inedible. However, it is good to know that plants which are infected after the fruit is already half grown typically do not turn out bitter.

No chemical control for mosaic virus, and plants need to be removed and destroyed promptly if they are infected with this viral disease. To control the spread of the disease by cucumber beetles and aphids, you will need to control these insect populations with a diazinon containing insecticide repeating the application as much as necessary in seven day intervals. Although there are mosaic virus resistant cucumber varieties, so far no resistant varieties of muskmelon and summer squash are available to plant.

It has only one RNA segment, meaning all genetic information is in one long RNA strand. It is an enveloped virus, which makes it very unstable. These envelopes are part of the cell membrane of the mamallian cell. The wall is unstable and easily dissolved, unlike polio which has a protein capsid and is very stable in the environemnt. Enveloped viruses tend to be very unstable because the bilayer is easily degraded. The viral attachment protein extends through the envelope, and without the envelope the virus is left with a very stable capsid that is uninfectious. It is unable to infect the mamallian cell because it lacks the viral attachment proteins in the bilayer.

Transmission Parainfluenza is transmitted via sneezing, virus shedding, and contact with eyes and nose. It gets into upper respiratory tracts and the trachea swell, resulting in croup. There is no vaccine for parainfluenza virus. It is mostly a pediatric virus.

Respiratory syncytial virus fuses membranes together to make enormous cells (fused aggregates). These aggregates get into alveoli of the lower respiratory tract, and the patient has trouble breathing out. It is more invasive than parainfluenza virus, as it goes deeper into the lungs, and is responsible for a lot of infant pneumonia. There is no vaccine.

Mumps virus is a member of the genus Paramyxovirus. It causes distinctive and generally benign system infections characterized by fever and parotitis (parotid inflammation, a salivary gland below the ear).

Epidemiology It is a disease of school-aged children worlwide. In unvaccinated populations, 92% of children have antibodies by age 15. A mumps vaccine was licensed in the U.S. in 1967, and it has reduced mumps infections to only 1,500 cases annually. Mumps is highly contagious. The virus infects epithelial cells of the upper respiratory tract, then spreads to regional lymphnodes. A viremia (bloodborne virion) spreads the virus to glandula and neural tissues.

Clinical Manifestations Up to 30% of mumps are asymptomatic. Symptoms include fever, malaise, and headache. After an 18 day replication phase of local replication and viremia, patients complain of ear pain and swollen salivary glands. CNS involvement is the most common extra-salivary manifestation, and occurs 10-30% of cases. It is 3-4 times more likely to occur in males than females for unknown reasons. Mumps CNS presents with high fever, vomiting, and headache lasting 48-96 hours. Maternal mumps infections during the first trimester of pregnancny may increase likelihood of spontaneous abortion.

Complications Mumps can effect gonads of both sexes. Testis inflammation (orchitis) is usually unilateral. Patients with mumps orchitis prsent with severe testicular pain and swelling, accompanied by high fever, nausea, vomiting, and headache. Testicular atrophy may follow orchitis in 35-50% of cases, but impotence or sterility is rare. Ovarian inflammation (oophoritis) occurs in 5% of postpubertal women with mumps. Patients typically report fever, nausea, and vomiting. Sequellae are uncommon, but impaired fertility can occur.

Prevention Children with mumps are usually isolated for 1 week after appearance of parotitis, even though this has dubious benefit since the virus is shed via respiratory secretions for several days before onset of clinical symptoms. In the U.S., the Jeryl-Lynn B strain of live virus is used for vaccination, following attenuation by serial passage in embryonated eggs. The mumps vaccine is a component of the MMR vaccine (measles, mumps, rubella).

Measles virus is a member of the genus Morbillivirus within the family Paramyxovirus. Measles virus differes from other members of the family in that it lacks neuraminidase. There is only one measles serotype, so recovery from natural infection confers lifelong immunity. Measles is one of five childhood exanthems, the others being rubella, varicella, roseola and fifth disease. Humans are the only known host for measles virus.

Epidemiology Measles is one of the most contagious human diseases. Vaccination has reduced the global incidence of measles, yet the World Health Organization reports there are still 45 million cases annually and 1.2 million deaths.

Transmission The principal mode of transmission is via large droplets of infected respiratory secretions inhaled during face-to-face exposure with coughing and sneezing individuals. This occurs during the catarrhal stage of the disease.

Pathogenesis Natural infection is initiated when measles virus reaches epithelial cells in the respiratory tract, oropharynx, or conjuctivae. During the first 2-4 days, the virus replicates locally and spreads via macrophages to draining lymph nodes, where further replication occurs. The virus then enters the bloodstream, producing a 1º viremia that spreads the virus throughout the reticuloendothelial system. Lymphoid hyperplasia occurs, and giant cells form due to cell fusion promoted by viral proteins. Further replication at these sites occurs, producing a secondary viremia of increasing magnitude that begins 5-7 days post-infection and spreads the virus to tissues throughout the body. During this 2 viremia, the virus is carried within leukocytes, more than 5% of which may be infected.

Clinical Manifestations Measles virus infection is rarely subclinical. The initial incubation period is clinically silent. Slight fever, malaise, and faint rash may occur in primary viremia. The prodromal stage of measles begin 8-12 days post-infect with fever, malaise, and anorexia followed by coryza (acute nasal congestion caused by secretion of mucus), conjunctivitis, sneezing, and cough. Catarrhal symptoms increase in intensity on or about the 5^th day. Coryza is intense, with profuse mucopurulent nasal discharge. There is palpebral conjuctivitis with lacrimation (abnormal and abundant shedding of tears). Severe coughing with a brass, barky quality ensues. 2-3 days before onset of rash, Koplik’s spots appear on inside of mouth. Kolplik describe them as 1-3 mm small irregular bright red spots with a minute bluish white speck at center. The rash begins 3-4 days after prodromal symptoms. The lesions appear behind the ear, on the forehead and on the upper part of the neck. They spread downward over the face, neck, and extremities, reaching the feet by the 3^rd day.

Clinical Diagnosis Measles can be diagnosed by isolates virus in cell culture from respiratory secretions, nasopharyngeal and conjuctiva swabs, PBMC, and urine as well as tissue biopsies. One can see giant cells characteristic of measles virus infection, or use specific antisera RT-PCR of the RNA.

Treatment Treatment of uncomplicated measles is symptomatic and includes bed rest, hydration, and antipyretics as needed. There is no antiviral therapy. The MMRV viaccine or a monovalent MV vaccine is used.

Small, infectious, obligate intracellular nonliving molecular parasite. Have ss or ds genome of DNA or RNA. Multiplication requires host cell. Progeny are the vehicle of transmission of virus. Icosohedral virus (simple/complex) and helical virus. Spindle viruses are specific to archaea. (+) RNA = mRNA; (-) RNA = complimentary mRNA. Viruses are filterable & passageable. Envelope is made of lipids & glycoproteins.

The repressor has a dumbbell shape. There is a DNA binding site, activating region, patch for tetramerization, patch for dimerization. With sufficient levels of repressor present, rightward transcription is repressed, transcription is activated and a stable repressed state is maintained. If repressor levels do not rise enough, repression is not established, rightward (and leftward) transcription ensues, repressor synthesis is not assured, replication and packaging and lysis follow. Lysogeny: repressor protein activating it’s own synthesis.

There are five steps in infection:

1. Attachment to host cell
2. Penetration of entire virion or injection of virion into host cell.
3. Synthesis of viral components (replication of viral genome & synthesis of proteins).
4. Assembly into virions.
5. Release of progeny

Attachment…basis of host cell specificity. Why do viruses attach to some cells & not others. When they attach to cells, they recognize a viral receptor even though the viral receptor is just a normal component of host cell’s surface. Such a component could be proteins, lipids, carbohydrates, etc. anything on exterior of cell surface. For example, bacteriophage T4 interacts with core polysaccarides of LPS on gram-negative bacteria.

There are two strategies for penetration: (1) in walled cells (prokaryotes, archaea & plants) cell walls are degraded, usually with lysozyme; (2) in wall-less cells (animal cells) the virion either fuses with cell membrane or undergoes endocytosis.

	 O
	 I		  O		  O		  O
	/|\\	->	..I..	->	..I..	->	..I..
------------------------------------------------------------------------------------out
							  ~
=========================================   ============= ~ ========================pdg
							  ~
----------------------------------------------------------~-------------------------in
							  ~

Not all phage have jointed tail fibers. After T4 interacts with core polysaccarides of LPS, it’s receptor, it then undergoes “retraction of tail fibers” & brings body in contact with membrane. Virus then releases lysozyme^-like protein which degrades peptidoglyca. Tail then contracts & DNA is injected

Lysogeny: This happens if there are not many cells or they are not very healthy. The phage lambda inserts it’s chromosome into the host genome, but remains latent & is present in all daughter cells. Induction causes lytic growth. If the cell is in danger, recA is activated and that cleaves lexA, which is like the lambda repressor. This allows it go into lysis. recA also represses other genes, read about the SOS signal.

Identifying a virus as a pathogen:

• Take sick patient
• Extract
• Inject extract into healthy patient
• Healthy patient becomes sick
• Culture extract
• If no growth, bacteria not responsible
• Filter extract so bacteria eliminated
• Inject into healthy patient
• Patient becomes sick
• Extract, filter, dilute, incubate in sterile NB: there should be no growth
• Inject that into healthy patient
• Extract from them, inject new healthy patient
• Repeat
• If potency remains constant, then a virus (not a soluble toxin) is responsible.
• Conclusion: viruses are filterable and passageable

The life cycle of a virus:

• Attachment/Adsorption (CHO, phospholipid as viral receptors)
• Injection of genome (Gram (-), add lysozyme; Gram (+), fuse with CM or endocytosis
• Lytic cycle (fast, host machinery transcribes and translates the viral genome), replicate viral genome, transcription & translation of lysozyme
• Lysogenic cycle (dormant, integration of viral genome into host genome)

Phage lambda is a virus that infects bacterial cells, first virus understood. It had all the right properties to help crack the secrets of molecular biology. But the guy who found it would not give it up! Yet if you work with a virus and you do not properly seal it, it gets everywhere. So they sent him a note asking for the virus, and when he sent the letter back saying no the virus was all over the letter! Hence, they dipped the letter in a salt solution and the lambda peeled off the letter. That is why it is lambda, for letter.

Flow of RNA & DNA for Viruses

Patients with two defective retinoblastoma (Rb) alleles invariably develop retinoblatomas, leading to its discovery as a tumor suppressor. E2F is a key host cell transcription factor in activating cellular genes required for synthesis of dNTPs, DNA polymerases, and other proteins required for cells to begin S-phase. Also, E2F binds and activates transcription from the adenovirus E2 promoter. The E2 region encodes viral proteins required for adenovirus DNA replication. In uninfected cells, retinoblastoma (aka Rb) is bound to E2F and inactivates it; in infected cells, Rb is not bound to E2F — the adenovirus protein E1A binds to retinoblastoma and removes it from E2F. Since E1A deactivates retinoblastoma by binding it and displacing it from E2F, it accomplishes the equivalent of mutations in both retinoblastoma alleles: there is no functional cellular retinoblastoma. SV40 and polyomavirus Large T-antigen also binds Rb; mutations of SV40 and polyomavirus Large-T that did not bind retinoblastoma did not transform cells. p110 is another tumor suppressor that was found to be absolutely identical to retinoblastoma.

p53 is a transcription factor that normally has a very short half-life; in response to dsDNA damage (due to radiation, for example) it stabilizes, activates and promotes transcription of certain genes by binding their control regions. Tumor tissues were analyzed via DNA sequencing, and the two p53 alleles were mutant in ∼50% of human metastatic cancers. Since it is mutant in so many tumors and normal in most healthy tissues, p53 is considered the most important tumor suppressor. All cells transformed by polyoma viruses or adenoviruses contain viral DNA integrated into one of their chromosomes. The integrated viral DNA expresses viral tumor antigens that inactivate the tumor suppressors Retinoblastoma and p53, stimulating the cells to enter S-phase even in the absence of normal physiogical signals that promote cell replication.