Initiating Coverage Comments

Investment Summary

Results for 3Q00 demonstrated a 19% increasein net income to $2,003,000 from $1,682,000 over the comparable period in 1999.For the nine months ending April 30, 2000 net income rose 36% to $5,040,000 from $3,705,000 versus the corresponding year earlier period.

·The Clinical Laboratories Division is generating $30 million in revenues annually, growing at a yearly rate of 10%.

·The Diagnostics (Life Sciences) Division is gaining increasing presence in a genomics-focused market that is expected to expand in tandem with the technology to annual sales in the range of $1-$4 billion.

·The Therapeutics Division is developing products for target treatment markets including:

Hepatitis B (HBV): (US & 15 member country European Union [EU]) in excess of $4.2 billion annually

HIV linked AID: (US alone) in excess of $18 billion annually

Graft versus Host Disease (GvHD): (US & EU) in excess of $2.75 billion annually

·The company’s proprietary life sciences product line for gene sequencing and genetic analysis is comprised of more than 400 patents (issued and pending) worldwide and are sold to the life science market globally. Enzo continues to expand its relationships supplying biological ‘gene juice’ material to chip makers for incorporation into silicon microchips (gene/DNA or biochips). The patent estate has been successfully defended numerous times and remedies applied. In each case Enzo has been the beneficiary of million dollar cash settlements and, as well, gained labeling agreements that force the licensee to include the Enzo Biochem, Inc. logo on that company’s proprietary substance label – hence the development of the ‘ENZO INSIDE’ identifying mark (see patent estate section).

·Phase II clinical trials of Enzo’s proprietary medicine EHT899 are underway, following a highly successful Phase I trial for the treatment of patients with chronic hepatitis B virus (HBV) thatresulted in an 80% favorable response rate. October 27-31 - Final Phase I data will be presented at the 51^st annual meeting of the American Association for the Study of Liver Diseases.

·Preliminary data from Enzo’s Phase I trial of HGTV43, its proprietary anti HIV-1 antisense gene therapy product was presented at the annual meeting of the American Society of Gene Therapy.The results demonstrated that the engineered cells remained in circulation, were active and remained present in the individuals’ CD4+ cells at 6 and 8 months.

·October 2, 2000 - The company reported that new data on the first individual treated in the Phase 1 clinical trial of HGTV-43, the company's HIV-1 gene medicine product, show that after nine-and-one-half-months Enzo engineered cells have successfully engrafted in the patient's bone marrow and were spawning new differentiated CD4+ cells designed to fight the virus.CD4+ cells have been shown to provide resistance to HIV.

Inside ‘ENZO INSIDE’—20+ years of scientific research, 410 patents issued and pending, extensive product pipeline, cash flow positive with growing profitability and debt free, the company is a leader in DNA/gene-based applied medical science.

The Company

Adenine

Enzo Biochem, Inc. is comprised of three operational divisions:

·Enzo Therapeutics

·Enzo Life Sciences (Diagnostics)

·Enzo Clinical Labs

Guanosine

Guanosine

The company embarked on its current expanded visionary applied science modality more than 10 years ago.Leveraging on its ongoing successful clinical laboratory’s profitable business base, management undertook to understand the nature of the diseases the presence of which the laboratory was determining. Long before it became fashionable Enzo’s investigators understood the gene to be a mine of information and that products for human use might be developed employing an informational model of the gene and its expressions. This was quite a visionary undertaking as at the time the norm consisted in thinking about the cell and its genomic structure as a medium in which biochemical interactions took place – specifically recombinant DNA was the dominant technology platform of the day. Management evolved the belief that gene modification and regulation would offer a robust modality in the quest for therapies with which to tackle a broad range of human ailments and diseases.This approach recognized that the molecules that comprise nucleic acids were more than storehouses of biologic information and could be employed as antiviral effectors.In consequence, Enzo’s Therapeutics Division strategically embarked on a two-tiered approach.

Thymine

Enzo researchers focused on the basic structural make-up of the nucleic acids and have been able to build a library of patents that describe and control the nature and essence of molecular binding at most of the optimal binding sites on the four nucleotides that comprise Deoxyribonucleic Acid (DNA):

·

Cytosine

The enormous advances in molecular biology were once inconceivable without the use of radioactivity for labeling and detection in nucleic acid analysis. For many years, it was unequivocally accepted that radiolabeled probes provided the highest level of sensitivity in detection of single-copy genes or rare mRNAs. However, there are major drawbacks to the use of isotopes, including short half-life times (some would no longer exist beyond minutes or hours), hazards to health and the environment, and extensive safety regulations for handling, storage, and disposal. The company was successful in developing alternatives that avoid the drawbacks of radioactivity but provide equal sensitivity and comparably convenient experimental procedures.

·Enzo’s global patent web includes separately issued patents that specifically cover labeling sites on each of the four nucleotides (Thymine, Cytosine, Adenine, and Guanosine) which comprise DNA and thus the ‘ENZO INSIDE’ label. They provide the company with comprehensive and distinctive area coverage throughout the nucleic acid continuum.Collectively, Enzo’s patent coverage extends to most labeling and detection positions on nucleotides for the efficient and convenient labeling of nucleic acids.

This basic structural work led Enzo scientists to identifying and utilizing nucleic acids – to deliver methods for regulating the functions of genes.This gene regulation has been referred to as genetic antisense. This antisense approach employs the Watson-Crick base pairing principle to permit therapeutic DNA to target a sensitive viral mRNA or even genomic DNA. Enzo has been able to use nucleic acids as antiviral agents, designing them to interfere with processes involving viral messenger RNA—that is to transcribe viral genes into mRNA or translate viral mRNA into proteins.

Until recently, the dominant technology in this arena was that of employing oligonucleotides which are synthetic, the manufacturing process being a large component of the expense in this costly procedure, administered locally with sustained dosing where large quantities are needed to affect the desired goals.This synthetic modality had an additional drawback, the proscribed doses can be toxic creating another whole problematic dimension to the protocol. By contrast, Enzo’s genetic modality is a therapy operating at the gene level. With this modality the insertion of a gene can have a positive effect, as in enabling the gene to express its natural protein as needed or a negative end outcome where the problematic gene is inhibited from producing the problem producing protein.This last is what is known as antisense. Its two major attributes are that the therapy is managed/produced by the gene and the impact is permanent. A further feature of the robust nature of this approach is one of the fundamentals of nature: that DNA to DNA binding is a constant irrespective of the disease. Therefore an approach such as Enzo’s, ought to find application to any disease because the approach focuses on the source of the problem rather than its expression.

A creature from the pre-computer era might serve as a good analogy by which to understand the antisense modality. The self-correcting typewriter would enable typos to be corrected by simply pressing a key and the typewriter would space back over the line, eliminate any trace of the incorrect letter and then one could type in the correct one creating the correct copy.

An extension on the success of this approach by Enzo is the recent approval and issuance of a US Patent for correcting genetic abnormalities at the single nucleotide level:

U.S. Patent No. 5,958,681 claims a method and materials suitable for correcting point mutations or small insertions or deletions of genetic material. By way of example, genetic abnormalities caused by single nucleotide alterations or small deletions could be corrected through the insertion or exchange of the correct or desired sequences into the genome of the cell where the error exists.

Normal Gene Expression

This new technology provides the company with an additional therapeutic platform and a uniquely specific approach for treating genetic-based diseases, in addition to gene therapy and gene modulation. This gene editing technique differs from other gene therapies in that other therapies typically require the complete insertion of an entire new gene to correct a nonfunctional or incorrectly functional gene.

Antisense Nucleic Acid Inhibits gene Expression

This invention could lead to creation of a normally functioning gene by correcting a mutated or altered gene sequence. It could correct, for example, an inborn error of metabolism for a condition such as hemophilia. Potentially, it could also be used to correct a mutated p53 gene that controls cell growth. In the mutated gene, cell growth is uncontrolled, likely resulting in a cancerous condition. In the corrected p53 gene, cell growth would be restored to its normal, non-cancerous state. The method and materials covered by the `681’ patent work by correcting the specific gene within cells by employing defined short gene sequences through a strategic application.

These sequences are held in place through formation of a triple helix complex. The principle of the invention is that when the short gene sequence containing the correct sequence is held in general proximity to the target DNA to be corrected, the probability of a double crossover or an editing event becomes quite high. That is that the triplex-forming DNA sequences bind to specific regions in the DNA in a sequence-specific manner so as to provoke cellular DNA repair. The elegance of this approach is embodied in that delivering the correct nucleic acid sequence to general proximity to the incorrect gene sequence inside the cell then stimulates the cell’s own distinctive DNA repair mechanisms to correct the deleterious condition.

The company’s therapeutics technologies are finding applications in a number of directly applicable arenas including the treatment of patients contending with such conditions as HIV infection, hepatitis, and Graft versus Host Disease.

Because detection of DNA can be made independent of the presence or absence of antibodies, presence of the virus can be determined much earlier after infection. This is clearly critical with such viral infections as AIDS.

Enzo’s Patent Estate is substantial with 200 patents approved and another 200 pending. In the diagnostics realm Enzo’s patents on the binding loci in each of the four nucleotides as described elsewhere in this report date back to the mid-1980’s. Enzo has an ongoing evolutionary approach to maintaining this estate as solid and robust as possible, by evolving new iterations and improvements to its framework so as to continually expand the patent duration and extension. There are patent improvements that constitute part of their library of potential patent developments both on existing patents and on prospective patents that are being kept in reserve to fit in to the ongoing scientific business development strategy that the company has forged. The nucleotide loci patent complex exists in at least 45 patents that cover the territory of the nucleotide binding sites. The company has defended its intellectual property in a number of infringement suits and has been successful in every exercise to date. In 1998 Enzo was granted a decision which netted the company a settlement of $21 million and an agreement that on every container label, where applicable, Boehringer Mannheim,GmbH is required to place the Enzo logo conspicuously present and equal in size to the manufacturers’ logo.Agreements are also in place withRoche Molecular Diagnostics, Ortho Diagnostics, Dako A/S, Nen Life Sciences, Inc., and Amersham International.

Spinning the patent web….

Innovation and development strategy keeps patents vibrant and robust beyond the standard 17.5 years of protection.

Other patents comprising the bulk of Enzo’s patent estate are similarly protected and in large part novel expressions of the Enzo scientific and research estate. Continual innovation permits ongoing support and expansion of initial patent protection.In any event, some of the patent estate, as on the continuum gene editing, genetic modification, and immune modulation is so new and radical that patent life is just commencing.It is our appreciation that patent protection will extend beyond the terms of reference for this analytical report and hence is firmly supportive of our projected revenue model. In the biochip and genetic sequencing area Enzo has agreements in place with Affymetrix, Inc., Gene Logic, Inc., and Li-Cor, Inc. where patent and product protection have been converted to ongoing commercial relationships.

‘non-radioactive tests use Enzo’s DNA probes to identify viruses and other infectious disease pathogens, and cancer markets…’

‘… detecting the presence of the AIDS virus in blood cells, the presence of hepatitis virus in serum and the presence of the tuberculosis organism in sputum.’

Enzo’s Life Sciences’ (Diagnostics) business continues to benefit from Enzo’s proprietary nucleic acid binding site patent web that Enzo enjoys. Leading pharmaceutical manufacturers and diagnostics labs that wish to attach molecules for labeling or other applications are required to negotiate with Enzo for the rights to access these locales on each of the nucleic acids as protected by the issued patents.Gene probes are emerging as the next growth technology in the diagnostics and testing market. The advantages that genetic probes bring to the industry include speed and that the diagnosis is processed in real time. Immunoassays, the technology that has been the gold standard, suffers from both a degree of inconsistency of outcome and the fact the process requires time to produce results.Gene probes, operating in real time, offer the healthcare community some significant advantages including: lower cost of use partly because of mechanization, specificity of treatment, precision, and for public health concerns, this technology can be distributed widely and easily.

Enzo’sDNA probe tests can be powerful diagnostic tools to find and identify viral and bacterial diseases, including sexually transmitted diseases, as well as cancer. Because they work at the genetic level, DNA probes have the potential to facilitate earlier and more definitive diagnoses. This is especially important in the diagnosis and testing of such problem diseases as AIDS. Antibodies to HIV, the virus that is causally linked to AIDS, often appear several months after initial infection. However, Enzo’s test can detect the HIV virus independent of the presence or absence of antibodies; presence of the virus can be determined much earlier after infection.

Enzo’sdiagnostics' market focus is primarily targeted at infectious disease diagnostics and genetic abnormality detection. The company has developed an integrated portfolio of proprietary technologies, each affording a variety of diagnostic applications.

Of particular importance is the patented technology for DNA that has been labeled with special non-radioactive signaling molecules. This pioneer technology has made possible the development of cohesive systems based on DNA for early diagnosis. These probes are similar in sensitivity to radioactive probes, yet have a longer shelf life and possess less health risk and disposal problems than do the radioactive ones. Enzo's work in this area has led to commercially viable product capabilities in what were previously esoteric laboratory tests. Enzo Diagnostics markets a broad range of products based on this technology.

Enzo's non-radioactive tests use DNA probes to identify viruses and other infectious disease pathogens, and cancer markers. The company's PathoGene® product line contains products which pinpoint under the microscope exactly which cells are infected and where in the cell the infection is located, making them especially well-suited for pathologists and cytotechnologists.

A second generation of nonradioactive DNA-probe based products, includes, among others, tests for detecting the presence of the AIDS virus in blood cells, the presence of hepatitis virus in serum and the presence of the tuberculosis organism in sputum. These tests, performed in a multiwell or ELISA plate can be readily automated and combine Enzo's proprietary microplate hybridization technology with the company's nonradioactive DNA probe technology. At the present these products are sold to the research community only. Enzo Biochem, Inc. has one of the largest libraries of disease specific targeted probes in the industry—more than 45 additional probes are being formatted into an automated diagnostic platform.

This approach and the resulting products, with the ability to identify microbial pathogens quickly, has the potential to displace current clinical testing methods which still use culturing and identification techniques developed more than a century ago.

Enzo's diagnostics’ business strategy has been to secure a technology position enabling the company to fully develop the potential of genetic-based diagnosis. The success of this approach is evidenced by the diversity and breadth of the company's patents and pending applications. Enzo has proprietary technologies for making products in a variety of formats, including solid-matrix hybridization, in situ products, and those that are solution based or in suspension. Moreover, a variety of technologies for detection systems has been devised. Enzo can now create tests that are detectable by visualization, spectroscopy, radiography, flow cytometry or fluorescence. Many of these detection systems can be readily automated. With Enzo's pioneering technological efforts, nonradioactive DNA detection has matured to a state where direct detection of pathogens in clinical samples can be accomplished in a timely and cost-effective manner.Enzo Diagnostics’ clinical diagnostic (amplification technologies) product line describes an annual market potential of $1.1 billion comprised of the following:

·

Enzo Biochem can offer tests that are detectable by visualization, spectroscopy, radiography, flow cytometry or fluorescence, which target market exceeds $1.1 billion annually.

Non-radioactive labeling of nucleic acids

· Labeling systems for nucleic acid arrays

·Membrane hybridization & detection

·In situ hybridization & detection systems

·BioProbe® labeled probes

·Signal generating systems

·Microplate hybridization assays

·Immunopathology reagents

With an annual revenue base of $30 million growing at a yearly rate of approximately 11%, the clinical labs division has been generating profits in the order of $3-$5 million and is limited only by the market’s uptake of Enzo’s attendant technology.

Enzo’s BioArray Labeling product line labels, detects, and identifies gene sequences under analysis by such methods as gene chip and other microarray methods.

EnzoClinical Labs, Inc. is a full-service clinical reference laboratory that serves the medical community through a network of patient service centers. Enzo Clinical Labs provides innovative high-quality service primarily to physicians as well as to hospitals, clinics, nursing homes and other clinical laboratories. The laboratory performs a complete range of clinical procedures from traditional testing, including routine screening, to advanced esoteric tests, contributing to the prompt and accurate diagnosis of disease. With 11 patient service centers the clinical labs division serves the greater New York City area.The labs division can efficiently process material delivered to its Farmingdale facility via courier regardless of its point of origin.With an annual revenue base of $30 million growing at a yearly rate of approximately 11%, the clinical labs division has been generating profits in the order of $3-$5 million.

Enzo Biochem, Inc. with its DNA-gene focused approach to diagnostics and therapeutics is a dramatic example of the tried and true business model: build a base, generate surplus, and utilize its core strengths on which expansion is achieved.With no debt, an historical cash flow positive framework, public since 1980, and with a management team that has directed its internal growth throughout its history, the company is poised for a quantum leap onto the highly visible therapeutic arena.

Significant Recent Developments

August 7, 2000The Japanese Patent Office, in an oppositional meeting, upheld Enzo’s patent No. 2,825,090 that covers a significant component of the company’s gene identification technology. This intellectual property covers an Enzo invention that permits direct quantitative measurement of the intensity of any signal inside a cell and thereby permits measurement of the amount of DNA present therein.

The World Health Organization estimates that approximately 2 billion people are currently infected by Hepatitis B (HBV).Of these, some 350 million are chronically infected and therefore at risk of death from liver disease.The virus accounts for 10% of chronic liver disease in the U.S. as well as a major proportion of liver transplants.

July 25, 2000Enzo Biochem, Inc. received notice of allowance for two broad-based patent applications; one from the U.S. Patent and Trademark Office covering broad applications in the field of nucleic acid technology and a second from the Canadian Intellectual Property Office for an Enzo patent for antisense RNA. Issuance of the Canadian patent is expected later this year and further strengthens Enzo’s position as a global holder of basic genetic antisense patents, which have been previously issued in Europe, Japan, and the U.S.These patents cover compositions and processes for trapping nucleic acid on solid supports, a process used in the majority of nucleic acid probe tests currently employed and add to Enzo’s substantial patent estate.

June 15, 2000Results for third fiscal quarter for 2000 ending April 30, 2000 were reported, demonstrating a 19% increasein net income to $2,003,000 from $1,682,000 for the quarter in 2000 over the same period in 1999. Total operating revenues rose to $12,579,000 compared to $12,193,000 for the corresponding period a year earlier. Per share earnings rose to $0.08 from $0.07 a year earlier, on both a basic and diluted basis. On a diluted basis average shares outstanding for the period amounted to 26,528,000 and 25,530,000 in the two respective quarters.

For the nine months ended April 30, 2000 total operating revenues rose to $35,755,000 compared to $33,718,000 for the previous year. Net income rose 36% to $5,040,000 from $3,705,000 in the corresponding period a year earlier. Net income per common share amounted to $0.20 ($0.19 on a fully diluted basis), as compared to $0.15 (basic and fully diluted) a year ago.

June 14, 2000Enzo Biochem, Inc. commences Phase II trial of treatment of Chronic Hepatitis B. The Phase I Trial results showed significant improvement in tested patients with no adverse effects. Enzo’s proprietary medicine EHT899 for the treatment of patients with Chronic Hepatitis B (HBV) was employed in the Phase I trial with an 80% success rate. Fifteen chronic HBV patients were treated orally with Enzo’s EHT899 viral protein for 20 weeks. Treatment resulted in a significant reduction in the viral load for the majority of patients and additionally, demonstrated a measurable improvement with regard to the damage to the liver stemming from HBV in selected patients.

June 2000As reported in Gastroenterology Volume 114 Number 4, Enzo’s contribution to basic research was furthered with the successful transplantation of human hepatocytes into a RagII mouse liver. This exercise established the creation of a mouse model which supports human cancerous livers and provides an animal model for HBV infection and replication, thereby reducing the transpecies gulf with the potential for reducing time and error in the drug development process.

ANTI-HIV-1 ANTISENSE GENE THERAPY

June 2, 2000The company announced preliminary results of Phase I clinical trials of its anti-HIV-1 antisense gene therapy product, HGTV-43.These results showed an unprecedented eight-month survival of the engineered cells in an HIV-infected individual in the group studied and the development of CD4+ cells expressing the HIV-1 antisense RNA within the cell.Such antisense cells have previously been shown to provide resistance to the virus. These results demonstrate that engineered stem cells have replicated and differentiated within the participants in the trial.

May 22, 2000Positive results were reported in a paper presented at a conference of leading medical scientists in San Diego, CA. These results indicated that the wasting response in humans as well as several forms of debilitating inflammation, among other side effects afflicting recipients of bone marrow transplantation, may be significantly alleviated using a new treatment developed by scientists at Hadassah University Hospital in conjunction with Enzo Biochem, Inc.

Cash flow positive and debt free, Enzo has grown from a clinical services and diagnostics company to a leading research and development company.

GRAFT VERSUS HOST DISEASE (GVHD)

Such side effects are part of graft versus host disease (GvHD), an undesirable immune response mounted against an individual receiving a bone marrow stem cell transplant. This disease can occasionally lead to death. There is currently no effective treatment for chronic GvHD. The paper detailing the Hadassah/Enzo studies using a mouse model system was presented by Yaron Ilan, M.D., of Hadassah University Hospital in Jerusalem, at the annual Digestive Disease Week Conference sponsored by several leading professional medical organizations. He reported on his collaborative research with scientists of Enzo Biochem, utilizing Enzo's immune enhancement technology.

This study demonstrated that oral administration of specific antigens effectively reversed the manifestations of chronic graft versus host disease. At the same time, the treated mice were shown to have undergone a favorable change in blood chemistry promoting a naturally occurring anti-inflammatory and anti-wasting response.

The results of this preclinical study demonstrate that Enzo's immune enhancement regulation technology could be effective in negating the effects of GvHD by enhancing the anti-inflammatory immune response mounted by the donor tissue or cells. The paper represented that the treatment was effective in ameliorating liver, bowel and skin manifestations of GvHD.

On March 15, 2000 the company announced its receipt of a method and materials patent for correcting point mutations or small insertions or deletions of genetic material. This covers an invention (employing a triple helix) that could lead to the creation of a normally functioning gene by correcting a mutated or altered gene sequence, and provides a further therapeutic platform to Enzo’s portfolio of technologies.

Investment Thesis

·Enzo Biochem, Inc. is an established and profitable biotechnology company. With more than 20 years of scientific research, a strong patent estate of more than 200 patents issued and another 200 pending, the company has developed an integrated technology portfolio that serves to support the company’s exceptional proprietary position. These resources serve also to propel the company to competitively capitalize on its strategic value. The technology platforms are at the cutting edge in concert with current market dynamics and include:

·DNA Identification

·Gene Regulation

·Immunological Regulation

·Gene Editing

·The company’s business development and growth has been managed on an internally driven model. Cash flow positive and debt free, Enzo has grown from a clinical services and diagnostics company to a leading research and development company. This internal engine has permitted the requisite growth without the financial and support crisis which typically plagues many drug research operations. Additionally management has been consistent in its focus and deliberately maintained its bearings. Senior management has been with the company for much of its history. We believe this to be a positive attribute in that they have demonstrated the capability to drive the operation onto different arenas while maintaining a focus on their core business.

·The cash flow positive contribution of the clinical laboratories business, and more recently diagnostics, continues to grow and further fuel development. The clinical laboratory services and the diagnostics products divisions have collectively been generating EBITDA rates of around 20% with a relentless consistency. Management will not be subject to the vagaries of market success and the trials and tribulations of dynamic growth. The target markets are as follows:

·There are approximately 53 million HIV infected individuals globally; the vast majority of these sufferers (more than 90%) live outside the developed world. For the purposes of our deliberations we have identified the U.S. market at approximately 950,000 individuals, a like number represented in the European Union. For our market evaluation purposes we have set the target market as primarily an U.S. market wherein the anticipated cost per treatment cycle per year is set at $20,000. We further estimate that should the World Health Organization entreat the developed world to supply such treatment to the under-privileged communities, this would have no effect on the net income of the company. Under such direction, we believe the effort would be run on a cost recovery basis, yielding only a slight positive impact resulting from the impact of production economies of scale. In the event, the expected U.S. market for such treatment is estimated to be $18 billion.

·

Hepatitis means and is ‘‘inflammation of the liver,” and the most

common cause is infection with one of 6 viruses, called hepatitis A, B, C, D, E, and G. All of these viruses can cause an acute disease with symptoms lasting several weeks including yellowing of the skin and eyes (jaundice), dark urine, extreme fatigue, nausea and vomiting, and abdominal pain. It can take several months to a year to feel fit again. Some of these viruses can cause a chronic carrier state in which the patient never gets rid of the virus, and many years later develops cirrhosis of the liver or liver cancer. Hepatitis B is a virus of this type and the most serious type of viral hepatitis. It is also the only type causing chronic disease for which a vaccine is available – but as a prevention and not as a cure.Hence the continuing need to find a treatment for chronic HBV sufferers.

The World Health Organization estimates that approximately 2 billion people are currently infected by Hepatitis B (HBV). Of these some 350 million are chronically infected and therefore at risk of death from liver disease that kills about one million persons each year. Hepatitis B is one of the major diseases of mankind, and is now preventable with safe and effective vaccines. Although the vaccine will not cure chronic carriers, it is 95% effective in preventing the carrier-state from developing. The virus accounts for 10% of chronic liver disease in the U.S. as well as a major proportion of liver transplants. Its incidence is believed to be even greater throughout Europe and the Mediterranean area, and even of greater impact in underdeveloped countries suffering from poor sanitation and health conditions. Supporting our evaluation we appreciate that there are approximately six million sufferers of the chronic form of HBV in the U.S. We do believe, however, that approval will come more swiftly in the European Union (EU) countries, partly because of the strategic decision to place the deterministic trials at the Hadassah Hospital Complex in Israel. We see this as a positive aspect of the bringing to market of Enzo’s treatment modality of HBV, in that there is a greater target market within the developed world that is the EU than there is in the U.S. We target the target market/population distribution to be: Europe (Western @1%, Central and Eastern, each @6%) and the Mediterranean region (@7%) as comprising an approximately 5% rate of chronic sufferers and consequently set the target market to comprise some 60 million individuals in aggregate. At a full pricing of $70 per treatment cycle (currently the data supports efficacy out through 20 weeks) we can establish a theoretical market of $4.2 billion.

·

The clinical laboratory services and the diagnostics products divisions have collectively been generating EBITDA rates of around 20% with a relentless consistency.

Enzo Life Sciences’ (comprised in genomics—gene research and sequencing, and clinical diagnostics) primary market focus is infectious disease diagnostics and genetic abnormality detection. The company has developed an integrated portfolio of proprietary technologies, each affording a variety of diagnostic applications. Of particular importance is the patented technology for DNA labeled with special non-radioactive signaling molecules. This pioneer technology has made possible the development of cohesive systems based on DNA for early diagnosis. The BioProbe® Systems are similar in sensitivity to radioactive probes, yet have a longer shelf life and possess less health risk and disposal problems. Enzo's work in this area has led to commercially viable product capabilities in what were previously esoteric laboratory tests. We have assessed Enzo’s BioProbe® technology including three major categories of product services. This is constituted in Gene Sequencing comprising a $100 million market; Bacterial ID and Viral load and molecular genetic Plate Assay in aggregate to constitute a $750 million market when mature; and Chemiluminescence that would likely comprise a $300 million mature marketplace. We have assigned market entry and success timing and rates in our revenue model that has been appended below (page 13).

Management

The management team has been together for much of Enzo’s existence. Unlike many biotechnology companies, this is the team that has brought the company to its current position and potential. They have accomplished this with an unusual aplomb and accompanying strict adherence to the notions of capital management and through internally generated development. This is a tried and true management group that has stood the test of 25 years of business growth.

This is a tried and true management group that has stood the test of 25 years of business growth.

DR. ELAZAR RABBANI (age 56) has served as Chairman and a Director of the company (President until 1996) since its organization in 1976. Dr. Rabbani received his BA degree from New York University in Chemistry and his Ph.D. degree in Biochemistry from Columbia University. He is a member of the American Society for Microbiology.

SHAHRAM K. RABBANI (age 48) has served as Chief Operating Officer, Secretary, and Treasurer of the company since November 1996, as Executive Vice President from September 1981 to November 1996 and as Vice President, Treasurer and a Director of the company since its organization. Mr. Rabbani received a BA degree in chemistry from Adelphi University.

BARRY W. WEINER (age 50) has served as President of the company since November 1996 and as a Director of the company since its organization. Mr. Weiner has served as an Executive Vice President of the company from September 1981 to November 1996, as a Vice President of the company from the company's organization to November 1996 and as Secretary of the company from March 1980 to November 1996. He was employed by Colgate-Palmolive Company, New York, New York from August 1974 until March 1980, when he joined the company on a full-time basis. Mr. Weiner received his B.S. degree in Economics from New York University and MBA from Boston University. Mr. Weiner is a Director of the New York State Biotechnology Association.

DR. NORMAN E. KELKER (age 61) has been a Vice President of the company since September 1981. Effective January 1, 1989, he was promoted to Senior Vice President. From 1975 until he joined the company, Dr. Kelker was an Associate Professor in the Department of Microbiology of the New York University School of Medicine. He holds a Ph.D. from Michigan State University.

DR. DEAN ENGELHARDT (age 60) has been Vice President since September 1981. Effective January 1, 1989, he was promoted to Senior Vice President. Prior to joining the company he was Associate Professor of Microbiology at Columbia University College of Physicians and Surgeons. He obtained his Ph.D. from Rockefeller University.

HERBERT B. BASS (age 52) is Vice President of Finance of the company. Prior to his promotion, Mr. Bass was the Corporate Controller of Enzo. Before joining Enzo in 1986, Mr. Bass held various positions at Danziger & Friedman, Certified Public Accountants, from 1979 to 1986, the most recent of which was audit manager. For the preceding seven years he held various positions atBerenson & Berenson, C.P.A's. Mr. Bass holds a Bachelor degree in Business Administration from Baruch College.

DR. BARBARA E. THALENFELD (age 60) is Vice President of Corporate Development and has been with Enzo since 1982. Prior to joining the company she held a NIH research fellowship at Columbia University. She received a Ph.D. from Hebrew University-Hadassah Medical Center and a MS from Yale University.

DAVID C. GOLDBERG (age 43) is Vice President of Business Development. Prior to joining Enzo in 1985, he was employed at DuPont NEN Products. He received a MS from Rutgers University and an MBA from New York University.

Risks and Concerns

As with all research and development activities that require regulatory approval, we reiterate that our models projecting revenues and the associated net estimates of outcomes are predicated on the company successfully negotiating the regulatory approval processes.Additionally, it is the nature of this industry that existing and unfolding technologies and applications of scientific method are continually being superceded by newly constituted developments, thereby rendering the competing technologies either redundant or moribund. In essence, there are no guarantees that any method or technology is or will be successful in this rapidly evolving and expanding marketplace even if they prove efficacious and utilitarian. This is a highly competitive environment and best or first does not necessarily warrant marketplace success.

With the intent of restraining exuberance we have assigned a market acceptance regime of 10% in the initial year that a therapy is on the market and a growth factor of 15% annually going forward from the second year.

Outlook and Valuation

The near term outlook supports our contention that the company will continue to generate positive cash flow and EBITDA rates consistent with its historical past.FDA trials bringing the clinical efforts on both the HBV and the HIV treatment modalities continue unfolding with approval target dates 24 months out into the future (final submissions tentatively set for 2002 for both indications).The new diagnostic products are expected to achieve market launch in the next 12 months (product releases spread over a wide period and differentially delivered – see Revenue Model).Our expectation is that at the very least the Micro Assay and Gene Sequencing products will have an impact in 2001.For a full appreciation of the product and market timeline and valuations please see the Revenue Model in Appendix I.

It is important to note that our revenue projections are consistent in the event that FDA approvals for prospective treatment candidates meet the criteria for approval and are, in fact, approved within the timelines we have ascribed.Other revenue streams including royalty/permissions, clinical laboratory services and diagnostic products are assigned valuations that reflect past practice and historical data.Needless to say, it is our contention that we are able to project forward given our assessment of both the developing marketplace and of management’s expected performance to meet the needs of the marketplace.

We have employed a discounted cash flow method to arrive at a current shareholder valuation. The 3Q2001 projected shareholder valuation that we identify as our 12-month target is based on extrapolating our discounted cash flow analysis as follows.We have employed a 12% discount rate coupled with a 7% terminal growth rate (reflecting the explosive rate of growth of a new treatment market currently classified as an unmet medical treatment marketplace), both of which we perceive to be very modest and conservative as a basis for these projections.The revenue streams that generated the net incomes on which this valuation is based also are fully conservative estimates of market size and of the products’ rate of market proportion gain.To provide a comparative sense of what Enzo’s current and prospective business productivity might be we list below a table of what the shareholder value would be under a range of market success perspectives.In each case we maintain the same discount rates for our discounted cash flow model.What we do alter are the market presence and capture variables.They are as follows:

Shareholder Valuation Scenario Table

Enzo Biochem, Inc. Product & Market Variables	HCFP/Brenner ModelCurrent Shareholder Value Per Share
Current Clinical Labs & Diagnostic Products ONLY	$18.65
+ Prospective Diagnostics including viral loading….	$39.75
+ HIV treatment USA only	$71.69
+ HIV USA only + HBV USA only	$80.66
+ HIV USA only + HBV (EU + USA only)	$102.16
+ HIV Global + HBV (EU + USA only)	$402.17
+ HIV Global + HBV Global	$612.47

NOTE:Global calculations for revenue streams for both HIV and HBV treatments adjust for the financial disparities between developing and undeveloped countries.In both cases the prospect of a World Health Organization (WHO) mandated treatment program is accommodated. Whatever the proscription, the net impact to Enzo Biochem, Inc. would be close to zero excepting the prospect of an advantage gained due to economies of scale at the production end.We anticipate that any involvement on the part of Enzo, with the delivery of these treatments to underdeveloped countries would be on a cost-recovery basis and as such would not have a net negative impact on Enzo’s bottom line.

We have selected the revenue stream we consider most likely to materialize within our set timelines as described in our revenue model.That is to say we expect the company to continue to perform strongly along its clinical laboratory services and diagnostic products lines in keeping with their historical profile.We expect, pending FDA and European Medicine Evaluation Agency (EMEA) approvals, the most likely scenario will have the HIV treatment approved initially in the USA and the HBV approval gained initially in the EU.In addition, it our contention that initial market acceptance of these therapeutic treatment technologies (as outlined in the Shareholder Valuation Scenario Table above) will be a modest 10% initially and that the market acquisition growth rate will be in the order of 15% per year for the initial period. That period extends to the outer limits of our model to the year 2006.

Additionally we have generated a profile of the company’s Enterprise Value (EV) and composed an EV/EBITDA ratio (see appended EV/EBITDA valuation sheet).The EV as estimated for Y2000 matches almost exactly to Enzo’s current market capitalization.The current EV/EBITDA ratio is estimated to be a 202 times multiple.By 2006 the company’s EBITDA is expected to grow to approximately 50 times its 2000 estimated value.These EV ratios support our free cash flow valuation as to the current shareholder values and future target ranges as described in the Discounted Cash Flow method (Appendix II) and is itself portrayed in Appendix V.

Accordingly we have assigned a target shareholder value for 3Q2001 of $111.

Financial Appendices

Science Primers

Normal Gene Expression

The DNA double helix, residing within the nucleus of the cell, contains two complementary strands of DNA. The magnified view shows how base pairing between complementary bases holds the strands together.

In transcription the information encoded in the base sequence in an unwound section of DNA is incorporated into a complementary RNA transcript (darker line). This transcript is constructed on the DNA template when complementary ribonucleotides base pair to the DNA bases. RNA transcripts encoding proteins, called messenger RNAs (mRNA), are transported through the nuclear membrane to the cell cytoplasm. In translation the base sequence in the mRNA directs the incorporation of specific amino acids (dark circles) into proteins. Protein formation occurs on protein factories called ribosomes.

Antisense Nucleic Acid Inhibits Gene Expression

Hybridization of an antisense nucleic acid (dashed line) with its mRNA complement interferes with protein synthesis

Gene Expression and Antisense Nucleic Acids

In gene expression the information encoded in the genes causes the production of specific proteins. Genetic information is contained within the chemical structure of DNA, short for deoxyribonucleic acid. The four different building blocks that comprise DNA are called nucleotides. The hereditary information is encoded in the linear sequence of nucleotides connected in a DNA strand. The first step of gene expression is called transcription because the information in DNA is transcribed into the nucleotide sequence of another nucleic acid, RNA or ribonucleic acid. In the second step of gene expression, the information in certain RNA transcripts, called messenger or mRNA, directs the construction of specific proteins from amino acid building blocks. This process is called translation because the linear array of nucleotides in the mRNA is "translated" into a corresponding sequence of amino acids to form the protein.

Nucleic acids, then, function in a variety of ways during which the information they carry is transmitted fairly precisely. In addition to its involvement in transcription, DNA also participates in exact replication, so that subsequent generations receive copies of all necessary heredity information. All the nucleic acid functions -- replication, transcription and translation -- depend upon the specific, reversible bonding that occurs between complementary nucleic acid strands. This bonding is best exemplified in the structure of DNA.

DNA has been dubbed the double helix because its structure consists of two long complementary nucleic acid strands that are spirally wound around each other.The four different nucleotides linked together in DNA differ only in their base components. In a chain of nucleotides, these bases protrude at regular intervals, like charms on a bracelet. Each of the four bases can bind to, or pair with, only one other base, called its complementary base. Complementary strands are ones in which the array of bases on one strand is exactly matched by an array of complementary bases on the other. Such strands can form base pairs all along their length, forming a double helix. This exact pairing process is called hybridization.

One strand of DNA can serve as a template upon which to construct a complementary strand. As complementary base pairing lines up nucleotides on the template strand, adjacent nucleotides can be linked to form a complementary strand. This is how DNA replication and RNA transcription occur. Base pairing not only creates faithful DNA replicas and faithful RNA transcripts, it also has a role in the translation of mRNA into protein. Hybridization between short segments of mRNA and the unique nucleic acids that carry each amino acid serves to correctly translate mRNA during protein synthesis.

While DNA is found in the double helix form, RNA transcripts are usually single stranded. Because the cell ultimately translates an mRNA strand, it is called the "sense" strand. A nucleic acid strand that is complementary to, and can hybridize with, at least part of a sense strand is called an "antisense"' strand. The hybridization of an antisense strand to a sense mRNA, can interfere with its translation to protein. Hence nucleic acids that are antisense to the transcript of a specific gene, such as a gene key to a pathogen or a deleterious human gene like those involved in certain cancers, could impair the expression of this gene, thereby disabling the particular disease state.

Advantages of an Antisense Approach

The main advantages that an antisense approach to therapy offers are specificity and point of attack. Antisense targets can be selected that are unique to the gene whose expression is to be controlled. Hence only that gene's expression is inhibited. This is especially important for diseases like viral infections and cancers that employ normal cell functions in the disease process. It has been difficult to devise therapeutic strategies against these diseases without also disabling normal cell functioning. Antisense nucleic acids targeted toward a gene that is diverting these normal cell functions, however, can specifically impair the disease state without affecting cell function. An antisense approach, therefore, has fewer side effects and offers real therapeutic promise for certain cancers and viral diseases.

Another advantage to an antisense approach is that it interferes at the source of the disease. That is, it interferes with the formation of unwanted proteins, rather than stopping these proteins from functioning. In addition, technically it is easier to define nucleic acid targets than it is to define protein targets. Information about gene structure is being amassed at an enormous rate, whereas information about protein structure is much harder to obtain. To illustrate, the genetic sequence of the human genome is expected to be completely known in fifteen years. It will be considerably longer, however, before it is understood what all it encodes, and longer still before the spatial structure of very many of the encoded proteins is known.

Ways Antisense Therapy can be Applied

Antisense nucleic acids can be applied in different ways. In one method short segments of antisense nucleic acids, termed oligonucleotides or "oligos" for short, can be administered as drugs. Such antisense oligos have been shown in cell culture to turn off the specific functions encoded by their sense target. Presumably antisense oligos will need to be administered during the acute phase of the disease.

In some cases it may be possible to insert a gene that will produce specific antisense nucleic acids in cells when and where they are required. This could be used in human therapy if a somatic cell type can be removed, transformed with an antisense gene, and reimplanted. Such antisense genetic engineering offers a possible therapeutic strategy for AIDS, caused by infection by the human immunodeficiency virus (HIV). To illustrate, the bone marrow of AIDS patients can be removed and transformed with gene producing RNA transcripts that are antisense to HIV nucleic acids. Bone marrow cells form the immune system cells that the HIV infects. Hence, when these antisense-HIV producing bone marrow cells are returned to the body, they could lead to immune system cells that resist infection by HIV.

Scientists are reporting positive results from their research and animal testing of genetic antisense for antiviral and cancer applications. Antisense technology is currently being investigated for the development of therapies involving HIV infections, glioblastoma, the k-ras oncogene in lung cancer and a variety of other disease states, as evidenced by the myriad of clinical studies listed by the National Institutes of Health Recombinant DNA Committee.

In cases where antisense genes can be introduced into the germ line, such as with plants and animals of agricultural importance, strains can be engineered that are inherently resistant to specific viruses. In addition, genes that are antisense to natural genes can be introduced into plants or animals to modulate the expression of these natural genes, thereby improving such characteristics as nutritional value, maturation time or aesthetic appeal. Already antisense genes have been engineered into tomato plants to yield less bruisable tomatoes and into floral varieties to change the flower coloration.

Commercial Opportunities of Antisense

Because an antisense approach is generally applicable to the regulation of any gene, it has broad commercial potential and can address markets of considerable magnitude. In addition to its usefulness in therapeutics, it can have a strong impact on agriculture and the bioprocess industry.

Therapeutics

The extreme specificity of antisense offers real therapeutic promise for cancer and viral infections, two types of diseases that have consistently eluded effective treatment, thereby opening up new markets of considerable size. By the end of the century it is estimated that greater than one million new cases of cancer will likely be diagnosed in the U.S. and more than a half million people will die of this disease. Last year greater than $2 billion was spent on cancer chemotherapeutic products and this market is growing around 25% per year.

Anti-viral drugs and vaccines are estimated to be about a third of the approximately $7 billion spent worldwide on anti-infectives last year, indicating both the difficulty in finding effective anti-viral therapy and their market potential. For AIDS alone the statistics are staggering. The Centers for Disease Control estimate that in the United States approximately 950,000 people (1 in 290 persons) are infected with HIV. Current estimates indicate that, worldwide, as many as 33 million persons may carry this virus. According to Public Health Service statistics, the cumulative number of reported AIDS cases is currently greater than 7.7 million and three quarters of these have been reported since 1993.

Agriculture

The use of antisense technologies to develop agricultural animals or crops that are resistant to viruses or other diseases also has considerable commercial significance. For example, chickens resistant to viral diseases such as Marek's Disease and Newcastle Disease Virus could strongly impact poultry production. It has been estimated that the annual cost of Marek's Disease to the U.S. poultry industry is $160 million due to vaccination costs and lost meat and egg production. On a worldwide basis this estimated cost is $940 million. Plant viral diseases are believed to reduce yields 10-20% across all plant crops. In the U.S. it is estimated that viruses are responsible for a loss of $1.5 to $2 billion in crop value each year, primarily in grains. Antisense could also be used to develop plants resistant to those fungi that require the expression of certain plant genes before they can successfully invade that plant. This could reduce the $350 million spent annually in the U.S. on fungicides.

Applications of this technology that alter the nutritional content or shelf life of foods would also have significant commercial impact. One possibility is reduced-cholesterol eggs. The value of this commodity in the U.S. has been estimated at $175 million annually. In plants antisense genetic technology can also be used to control genes that promote ripening and softening of fruits and vegetables, thereby providing considerable savings in picking, transport and storage as well as potentially expanding the seasonal availability of some fruits and vegetables. Similarly, antisense genetic technology could conceivably be used to improve the ratio of monosaturated fats to polyunsaturated fats in plant oils.

Bioprocessing Industry

With the advent of genetic engineering the number of commercially important materials being produced from fermentation and cell culture is increasing rapidly. In the pharmaceutical area alone, it is anticipated that U.S. revenues from products obtained by biological processes will total $12 billion by the year 2000. The yields of desired products can be improved with an antisense genetic strategy that inhibits interfering cellular reactions. This approach has already doubled the yield of Factor VIII from genetically engineered mammalian cells. The annual worldwide market for Factor VIII is estimated at $250 million.

On a worldwide basis products attainable from plant cell culture for use in medicinals, flavors, fragrances and agricultural chemicals comprise about a $750 million market. Important medicinal products in this category include corticosteroids and codeine. It is likely that the yield of these products can be improved by culturing cells in which antisense genes reduce competing pathways.

Antisense Nucleic Acids for Therapeutic and Other Applications hasbeen reproduced with the kind permission of Enzo Biochem, Inc. and is protected under the copyright laws of the United States of America and international conventions, and is the exclusive property of Enzo Biochem, Inc.All rights reserved.Enzo Biochem, Inc. 1998.

Overview

HIV disease is characterized by a gradual deterioration of immune function. Most notably, crucial immune cells called CD4+ T cells are disabled and killed during the typical course of infection. These cells, sometimes called “T-helper cells,” play a central role in the immune response, signaling other cells in the immune system to perform their special functions.
A healthy, uninfected person usually has 800 to 1,200 CD4+ T cells per cubic millimeter (cm) of blood. During HIV infection, the number of these cells in a person’s blood progressively declines. When a person’s CD4+ T cell count falls below 200/mm, he or she becomes particularly vulnerable to the opportunistic infections and cancers that typify AIDS, the end stage of HIV disease. People with AIDS often suffer infections of the intestinal tract, lungs, brain, eyes and other organs, as well as debilitating weight loss, diarrhea, neurologic conditions and cancers such as Kaposi’s sarcoma and lymphomas.
Most scientists think that HIV causes AIDS by triggering events that weaken a person’s immune function. For example, the network of signaling molecules that normally regulates a person’s immune response is disrupted during HIV disease, impairing a person’s ability to fight other infections. The HIV-mediated destruction of the lymph nodes and related immunologic organs also play a major role in causing the immunosuppression seen in people with AIDS.

Scope of the HIV Epidemic

Although HIV was first identified in 1983, studies of previously stored blood samples indicate that the virus entered the U.S. population sometime in the late 1970s. In the United States, 513,486 cases of people with AIDS had been reported to the Centers for Disease Control and Prevention (CDC) as of Dec. 31, 1995. Among these individuals, 319,849 had died by the end of 1995. AIDS is now the leading killer of people aged 25 to 44 in this country.
Worldwide, an estimated 15 million people had become HIV-infected through mid-1996, and 7.7 million had developed AIDS, according to the World Health Organization (WHO). Various projections indicate that, by the year 2000, approximately 950,000 people in the U.S. and 33 million people worldwide will be HIV-infected.

HIV is a Retrovirus

HIV belongs to a class of viruses called retroviruses, which have genes composed of ribonucleic acid (RNA) molecules. The genes of humans and most other organisms are made of a related molecule, deoxyribonucleic acid (DNA).

Like all viruses, HIV can replicate only inside cells, commandeering the cell’s machinery to reproduce. However, only HIV and other retroviruses, once inside a cell, use an enzyme called reverse transcriptase to convert their RNA into DNA, which can be incorporated into the host cell’s genes.

Slow Viruses

HIV belongs to a subgroup of retroviruses known as lentiviruses, or “slow” viruses. The course of infection with these viruses is characterized by a long interval between initial infection and the onset of serious symptoms.

Other lentiviruses infect nonhuman species. For example, the feline immunodeficiency virus (FIV) infects cats and the simian immunodeficiency virus (SIV) infects monkeys and other nonhuman primates. Like HIV in humans, these animal viruses primarily infect immune system cells, often causing immunodeficiency and AIDS-like symptoms. Scientists use these and other viruses and their animal hosts as models of HIV disease.

Organization of the HIV-1 Virion

Structure of HIV

The Viral Envelope

HIV has a diameter of 1/10,000 of a millimeter and is spherical in shape. The outer coat of the virus, the viral envelope, is composed of two layers of fatty molecules called lipids, taken from the membrane of a human cell when a newly formed virus particle buds from the cell.

Embedded in the viral envelope are proteins from the host cell, as well as 72 copies (on average) of a complex HIV protein that protrudes from the envelope surface. This protein, known as Env, consists of a cap made of three or four molecules called glycoprotein (gp)120, and a stem consisting of three or four gp41 molecules that anchor the structure in the viral envelope. Much of the research to develop a vaccine against HIV are focused on these envelope proteins.

The Viral Core

Within the envelope of a mature HIV particle is a bullet-shaped core or capsid, made of 2000 copies of another viral protein, p24. The capsid surrounds two single strands of HIV RNA, each of which has a copy of the virus’s nine genes. Three of these, gag, pol and env, contain information needed to make structural proteins for new virus particles. The env gene, for example, codes for a protein called gp160 that is broken down by a viral enzyme to form gp120 and gp41, the components of env.

Three regulatory genes, tat, rev and nef, and three auxiliary genes, vif, vpr and vpu, contain information necessary for the production of proteins that control the ability of HIV to infect a cell, produce new copies of virus or cause disease. The protein encoded by nef, for instance, appears necessary for the virus to replicate efficiently, and the vpu-encoded protein influences the release of new virus particles from infected cells.

The ends of each strand of HIV RNA contain an RNA sequence called the long terminal repeat (LTR). Regions in the LTR act as switches to control production of new viruses and can be triggered by proteins from either HIV or the host cell.

The core of HIV also includes a protein called p7, the HIV nucleocapsid protein; and three enzymes that carry out later steps in the virus’s life cycle: reverse transcriptase, integrase and protease. Another HIV protein called p17, or the HIV matrix protein, lies between the viral core and the viral envelope.

Life Cycle of HIV

Steps in Viral Replication

·Attachment/Entry

·Reverse Transcription and DNA Synthesis

·Transport to Nucleus

·Integration

·Viral Transcription

·Viral Protein Synthesis

·Assembly of Virus

·Release of Virus

·Maturation

Life Cycle of HIV

Entry of HIV into Cells

Infection typically begins when an HIV particle, which contains two copies of the HIV RNA, encounters a cell with a surface molecule called cluster designation 4 (CD4). Cells with this molecule are known as CD4 positive (CD4+) cells.

One or more of the virus’s gp120 molecules binds tightly to CD4 molecule(s) on the cell’s surface. The membranes of the virus and the cell fuse, a process that probably involves both gp41 and a second “fusion cofactor” molecule on the cell surface. Recent research by NIAID intramural and extramural researchers has identified two fusion cofactors for different types of HIV strains. Following fusion, the virus’s RNA, proteins and enzymes are released into the cell.

Although CD4+ T cells appear to be HIV’s main target, other immune system cells with CD4 molecules on their surfaces are infected as well. Among these are long-lived cells called monocytes and macrophages, which apparently can harbor large quantities of the virus without being killed, thus acting as reservoirs of HIV.
Scientists suspect that HIV also may infect cells without CD4 on their surfaces, using other docking molecules. For example, cells of the central nervous system may be infected via a receptor known as galactosyl ceramide. The role of HIV fusion cofactors in this process is currently under intense investigation.

Cell-to-cell spread of HIV also can occur through the CD4-mediated fusion of an infected cell with an uninfected cell.

Reverse Transcription

In the cytoplasm of the cell, HIV reverse transcriptase converts viral RNA into DNA, the nucleic acid form in which the cell carries its genes. Six of the nine antiviral drugs approved in the United States for the treatment of people with HIV infection—AZT, ddC, ddI, d4T, 3TC and nevirapine—work by interfering with this stage of the viral life cycle.

Integration

The newly made HIV DNA moves to the cell’s nucleus, where it is spliced into the host’s DNA with the help of HIV integrase. Once incorporated into the cell’s genes, HIV DNA is called a “provirus.” Billions of cells in an HIV-infected person may contain HIV DNA.

Transcription

For a provirus to produce new viruses, RNA copies must be made that can be read by the host cell’s protein-making machinery. These copies are called messenger RNA (mRNA), and production of mRNA is called transcription, a process that involves the host cell’s own enzymes. Viral genes in concert with the cellular machinery control this process: the tat gene, for example, encodes a protein that accelerates transcription.
Cytokines, proteins involved in the normal regulation of the immune response, also may initiate transcription. Molecules such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, secreted in elevated levels by the cells of HIV-infected people, may help to activate HIV proviruses. Other infections, by organisms such as Mycobacterium tuberculosis, may also initiate transcription.

Translation

After HIV mRNA is processed in the cell’s nucleus, it is transported to the cytoplasm. HIV proteins are critical to this process: for example, a protein encoded by the rev gene allows mRNA encoding HIV structural proteins to be transferred from the nucleus to the cytoplasm. Without the rev protein, structural proteins are not made.

In the cytoplasm, the virus co-opts the cell’s protein-making machinery—including structures called ribosomes—to make long chains of viral proteins and enzymes, using HIV mRNA as a template. This process is called translation.

Assembly and Budding

Newly made HIV core proteins, enzymes and RNA gather just inside the cell’s membrane, while the viral envelope proteins aggregate within the membrane. An immature viral particle forms and pinches off from the cell, acquiring an envelope that includes both cellular and HIV proteins from the cell membrane. During this part of the viral life cycle, the core of the virus is immature and the virus is not yet infectious. The long chains of proteins and enzymes that make up the immature viral core are now cleaved into smaller pieces by a viral enzyme called protease. This step results in infectious viral particles.

Drugs called protease inhibitors interfere with this step of the viral life cycle. Three such drugs—saquinavir, ritonavir and indinavir—have been approved for marketing in the United States.

Course of HIV Infection

Among patients enrolled in large epidemiologic studies in western countries, the median time from infection with HIV to the development of AIDS-related symptoms has been approximately 10 years. However, researchers have observed a wide variation in disease progression. Approximately 10 percent of HIV-infected people in these studies have progressed to AIDS within the first two to three years following infection, while 5 to 10 percent of individuals in the studies have stable CD4+ T cell counts and no symptoms even after 12 or more years.
Factors such as age or genetic differences among individuals, the level of virulence of an individual strain of virus, and co-infection with other microbes may influence the rate and severity of disease progression.
Viral burden predicts disease progression. Recent studies show that people with high levels of HIV in their bloodstream are more likely to develop new AIDS-related symptoms or to die than individuals with lower levels of virus. New anti-HIV drug combinations that reduce a person’s “viral burden” to very low levels may delay the progression of HIV disease, but it remains to be seen if these drugs will have a prolonged benefit. Other drugs that fight the infections associated with AIDS have improved and prolonged the lives of HIV-infected people by preventing or treating conditions such as Pneumocystis carinii pneumonia.

Transmission of HIV

Among adults, HIV is spread most commonly via a shared dirty needle by intravenous drug users (this is a controversial view) and during sexual intercourse with an infected partner. During sex, the virus can enter the body through the mucosal linings of the vagina, vulva, penis, rectum or, rarely, through oral sexual contact. The likelihood of transmission is increased by factors that may damage these linings, especially other sexually transmitted diseases that cause ulcers or inflammation.

Research suggests that immune system cells called dendritic cells, which reside in the mucosa, may begin the infection process by binding to and carrying the virus from the site of infection to the lymph nodes where other immune system cells become infected.

HIV also can be transmitted by contact with infected blood, most often by the sharing of drug needles or syringes contaminated with minute quantities of blood containing the virus. The risk of acquiring HIV from blood transfusions is now extremely small in the United States, as all blood products in this country are screened routinely for evidence of the virus.
Almost all HIV-infected children acquire the virus from their mothers before or during birth. In the United States, approximately 25 percent of pregnant HIV-infected women not receiving antiretroviral therapy have passed on the virus to their babies. NIAID-sponsored researchers have shown that a specific regimen of the drug zidovudine (AZT) can reduce the risk of transmission of HIV from mother to baby by two-thirds.
The virus also may be transmitted from a nursing HIV-infected mother to her infant.

Early Events in HIV Infection

Once it enters the body, HIV infects a large number of CD4+ cells and replicates rapidly. During this acute or primary phase of infection, the blood contains many viral particles that spread throughout the body, seeding various organs, particularly the lymphoid organs. Lymphoid organs include the lymph nodes, spleen, tonsils and adenoids and thymus.

During the acute phase of infection, the number of CD4+ T cells in the bloodstream decreases by 20 to 40 percent. Scientists do not yet know whether these cells are killed by HIV or if they leave the blood and go to the lymphoid organs in preparation to mount an immune response.

Two to four weeks after exposure to the virus, up to 70 percent of HIV-infected persons suffer flu-like symptoms related to the acute infection. The patient’s immune system fights back with killer T cells (CD8+ T cells) and B-cell-produced antibodies, which dramatically reduce HIV levels. A patient’s CD4+ T cell count may rebound to 80 to 90 percent of its original level. A person then may remain free of HIV-related symptoms for years despite continuous replication of HIV in the lymphoid organs seeded during the acute phase of infection.
One reason HIV is unique is that despite the body’s aggressive immune responses, which are sufficient to clear most viral infections, some HIV invariably escapes. One explanation is that the immune system’s best soldiers in the fight against HIV—certain subsets of killer T cells—multiply rapidly following initial HIV infection and kill many HIV-infected cells, but then appear to exhaust themselves and disappear, allowing HIV to escape and continue replication. Additionally, in the few weeks that they are detectable, these specific cells appear to accumulate in the bloodstream rather than in the lymph nodes, where most HIV is sequestered – the virus seems to mutate away from the immune cells.

HIV is Active in the Lymph Nodes

Although HIV-infected individuals often exhibit an extended period of clinical latency with little evidence of disease, the virus is never truly latent. NIAID researchers have shown that even early in disease, HIV actively replicates within the lymph nodes and related organs, where large amounts of virus become trapped in networks of specialized cells with long, tentacle-like extensions. These cells are called follicular dendritic cells (FDCs).FDCs are located in hot spots of immune activity called germinal centers. They act like flypaper, trapping invading pathogens (including HIV) and holding them until B cells come along to initiate an immune response.
Close on the heels of B cells are CD4+ T cells, which rush into the germinal centers to help B cells fight the invaders. CD4+ T cells, the primary targets of HIV, probably become infected in large numbers as they encounter HIV trapped on FDCs. Research suggests that some HIV trapped on FDCs remains infectious, even when cloaked with antibodies.

Once infected, CD4+ T cells may leave the germinal center and infect other CD4+ cells that congregate in the region of the lymph node surrounding the germinal center.

Over a period of years, even when little virus is readily detectable in the blood, significant amounts of virus accumulate in the germinal centers; both within infected cells and bound to FDCs. In and around the germinal centers, numerous CD4+ T cells are probably activated by the increased production of cytokines such as TNF-alpha and IL-6, possibly secreted by B cells. Activation allows uninfected cells to be more easily infected and increases replication of HIV in already infected cells.

While greater quantities of certain cytokines such as TNF-alpha and IL-6 are secreted during HIV infection, others with key roles in the regulation of normal immune function may be secreted in decreased amounts. For example, CD4+ T cells may lose their capacity to produce interleukin 2 (IL-2), a cytokine that enhances the growth of other T cells and helps to stimulate other cells’ response to invaders. Infected cells also have low levels of receptors for IL-2, which may reduce their ability to respond to signals from other cells.

Breakdown of FDC Networks

Ultimately, accumulated HIV overwhelms the FDC networks. As these networks break down, their trapping capacity is impaired, and large quantities of virus enter the bloodstream.
Although it remains unclear why FDCs die and the FDC networks dissolve, some scientists think that this process may be as important in HIV pathogenesis as the loss of CD4+ T cells. The destruction of the lymph node structure seen late in HIV disease may preclude a successful immune response against not only HIV but other pathogens as well. This devastation heralds the onset of the opportunistic infections and cancers that characterize AIDS.

Role of CD8+ T Cells

CD8+ T cells are important in the immune response to HIV during the acute infection and the clinically latent stage of disease. These cells attack and kill infected cells that are producing virus.
CD8+ T cells also appear to secrete soluble factors that suppress HIV replication. Three of these molecules—RANTES, MIP-1alpha and MIP-1beta—apparently block HIV replication by occupying receptors necessary for the entry of certain strains of HIV into their target cells. Researchers have hypothesized that an abundance of RANTES, MIP-1alpha or MIP-1beta, or a relative lack of receptors for these molecules, may help explain why some individuals have not become infected with HIV, despite repeated exposure to the virus.CD8+ T cells probably also secrete other soluble factors—as yet unidentified—that suppress HIV replication.

Rapid Replication and Mutation of HIV

HIV replicates rapidly; several billion new virus particles may be produced every day. In addition, the HIV reverse transcriptase enzyme makes many mistakes while making DNA copies from HIV RNA. As a consequence, many variants of HIV develop in an individual, some of which may escape destruction by antibodies or killer T cells. Additionally, HIV can recombine with itself to produce a wide range of variants or strains.

During the course of HIV disease, viral strains emerge in an infected individual that differ widely in their ability to infect and kill different cell types, as well as in their rate of replication. Scientists are investigating why strains of HIV from patients with advanced disease appear to be more virulent and infect more cell types than strains obtained earlier from the same individual.

Theories of Immune System Cell Loss in HIV Infection

Researchers around the world are studying how HIV destroys or disables CD4+ T cells, and many think that a number of mechanisms may occur simultaneously in an HIV-infected individual. Recent data suggest that billions of CD4+ T cells may be destroyed every day, eventually overwhelming the immune system’s regenerative capacity.

Direct Cell Killing

Infected CD4+ T cells may be killed directly when large amounts of virus are produced and bud off from the cell surface, disrupting the cell membrane, or when viral proteins and nucleic acids collect inside the cell, interfering with cellular machinery.

Syncytia Formation

Infected cells also may fuse with nearby uninfected cells, forming balloon-like giant cells called syncytia. In test-tube experiments at NIAID and elsewhere, these giant cells have been associated with the death of uninfected cells. The presence of so-called syncytia-inducing variants of HIV has been correlated with rapid disease progression in HIV-infected individuals.

Apoptosis

Infected CD4+ T cells may be killed when cellular regulation is distorted by HIV proteins, probably leading to their suicide by a process known as programmed cell death or apoptosis. Recent reports indicate that apoptosis occurs to a greater extent in HIV-infected individuals, both in the bloodstream and lymph nodes.
Uninfected cells also may undergo apoptosis. Normally, when CD4+ T cells mature in the thymus gland, a small proportion of these cells is unable to distinguish self from non-self. Because these cells would otherwise attack the body’s own tissues, they receive a biochemical signal from other cells that results in apoptosis. Investigators have shown in cell cultures that gp120 alone or bound to gp120 antibodies sends a similar but inappropriate signal to CD4+ T cells causing them to die even if not infected by HIV.

Innocent Bystanders

Uninfected cells may die in an innocent bystander scenario: HIV particles may bind to the cell surface, giving them the appearance of an infected cell and marking them for destruction by killer T cells.
Killer T cells also may mistakenly destroy uninfected CD4+ T cells that have consumed HIV particles and that display HIV fragments on their surfaces. Alternatively, because HIV envelope proteins bear some resemblance to certain molecules that may appear on CD4+ T cells, the body’s immune responses may mistakenly damage such cells as well.

Anergy

Researchers have shown in cell cultures that CD4+ T cells can be turned off by a signal from HIV that leaves them unable to respond to further immune stimulation. This inactivated state is known as anergy.

Superantigens

Other investigators have proposed that a molecule known as a superantigen, either made by HIV or an unrelated agent, may stimulate massive quantities of CD4+ T cells at once, rendering them highly susceptible to HIV infection and subsequent cell death.

Damage to Precursor Cells

Studies suggest that HIV also destroys precursor cells that mature to have special immune functions, as well as the parts of the bone marrow and the thymus needed for the development of such cells. These organs probably lose the ability to regenerate, further compounding the suppression of the immune system.

Central Nervous System Damage

Although HIV can infect monocytes and macrophages, they appear to be relatively resistant to killing. However, these cells travel throughout the body and carry HIV to various organs, especially the lungs and brain. People infected with HIV often experience abnormalities in the central nervous system. Neurologic manifestations of HIV disease, seen in 40 to 50 percent of HIV-infected people, are the subject of many research projects. Investigators have hypothesized that an accumulation of HIV in brain and nerve cells, or the inappropriate release of cytokines or toxic byproducts by these cells, may be to blame.

Role of Immune Activation in HIV Disease

During a normal immune response, many components of the immune system are mobilized to fight an invader. CD4+ T cells, for instance, may quickly proliferate and increase their cytokine secretion, thereby signaling other cells to perform their special functions. Scavenger cells called macrophages may double in size and develop numerous organelles, including lysosomes that contain digestive enzymes used to process ingested pathogens. Once the immune system clears the foreign antigen, it returns to a relative state of quiescence. During HIV infection, however, the immune system may be chronically activated, with negative consequences. As noted above, HIV replication and spread are much more efficient in activated CD4+ cells. Chronic immune system activation during HIV disease may also result in a massive stimulation of a person’s B cells, impairing the ability of these cells to make antibodies against other pathogens.

Chronic immune activation also can result in apoptosis, and an increased production of cytokines that may not only increase HIV replication but also have other deleterious effects. Increased levels of TNF-alpha, for example, may be at least partly responsible for the severe weight loss or wasting syndrome seen in many HIV-infected individuals.

Immune activation also seem to extend to the CD4+ T and CD8+ T main cell population which appears to be depleted systemically under conditions where both these cell types are killed off as a result. Thus the bank of cells conferring immune responsiveness is depleted resulting in a population well below the critical mass required for protection.

The persistence of HIV and HIV replication probably plays an important role in the chronic state of immune activation seen in HIV-infected people. In addition, researchers have shown that infections with other organisms activate immune system cells and increase production of the virus in HIV-infected people. Chronic immune activation due to persistent infections, or the cumulative effects of multiple episodes of immune activation and bursts of virus production, likely contribute to the progression of HIV disease.

Glossary

Apoptosis: Cellular suicide, also known as programmed cell death. HIV may induce apoptosis in both infected and uninfected immune system cells.
B cells: White blood cells of the immune system that produce infection-fighting proteins called antibodies.
CD4+ T cells: White blood cells that orchestrate the immune response, signaling other cells in the immune system to perform their special functions. Also known as T helper cells, these cells are killed or disabled during HIV infection.
CD8+ T cells: White blood cells that kill cells infected with HIV or other viruses, or transformed by cancer. These cells also secrete soluble molecules that may suppress HIV without killing infected cells directly.
Cytokines: Proteins used for communication by cells of the immune system. Central to the normal regulation of the immune response.
Cytoplasm: The living matter within a cell.
Dendritic cells: Immune system cells with long, tentacle-like branches. Some of these are specialized cells at the mucosa that may bind to HIV following sexual exposure and carry the virus from the site of infection to the lymph nodes. See also follicular dendritic cells.

Enzyme: A protein that accelerates a specific chemical reaction without altering itself.
Follicular dendritic cells (FDCs): Cells found in the germinal centers (B cell areas) of lymphoid organs. FDCs have thread-like tentacles that form a web-like network to trap invaders and present them to B cells, which then make antibodies to attack the invaders.
Germinal centers: Structures within lymphoid tissues that contain FDCs and B cells, and in which immune responses are initiated.
gp41: Glycoprotein 41, a protein embedded in the outer envelope of HIV. Plays a key role in HIV’s infection of CD4+ T cells by facilitating the fusion of the viral and cell membranes.
gp120: Glycoprotein 120, a protein that protrudes from the surface of HIV and binds to CD4+ T cells.
gp160: Glycoprotein 160, an HIV precursor protein that is cleaved by the HIV protease enzyme into gp41 and gp120.

Integrase: An HIV enzyme used by the virus to integrate its genetic material into the host cell’s DNA.

Kaposi’s sarcoma: A type of cancer characterized by abnormal growths of blood vessels that develop into purplish or brown lesions.

Killer T cells: See CD8+ T cells.

Lentivirus: “Slow” virus characterized by a long interval between infection and the onset of symptoms. HIV is a lentivirus as is the simian immunodeficiency virus (SIV), which infects nonhuman primates.

LTR: Long terminal repeat, the RNA sequences repeated at both ends of HIV’s genetic material. These regulatory switches may help control viral transcription.

Lymphoid organs: Include tonsils, adenoids, lymph nodes, spleen and other tissues. Act as the body’s filtering system, trapping invaders and presenting them to squadrons of immune cells that congregate there.

Macrophage: A large immune system cell that devours invading pathogens and other intruders. Stimulates other immune system cells by presenting them with small pieces of the invaders.

Monocyte: A circulating white blood cell that develops into a macrophage when it enters tissues.

Nucleotide: Any member of a class of organic compounds in which the molecular structure comprises a nitrogen-containing unit (base) linked to a sugar and a phosphate group. Nucleotides are of great importance to living organisms, as they are the building blocks of nucleic acids, the substances that control all hereditary characteristics. In the two families of nucleic acids, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), the sequence of nucleotides in the DNA or RNA codes for the structure of proteins synthesized in the cell.

Opportunistic infection: An illness caused by an organism that usually does not cause disease in a person with a normal immune system. People with advanced HIV infection suffer opportunistic infections of the lungs, brain, eyes and other organs.

Pathogenesis: The production or development of a disease. May be influenced by many factors, including the infecting microbe and the host’s immune response. Protease: An HIV enzyme used to cut large HIV proteins into smaller ones needed for the assembly of an infectious virus particle.

Provirus: DNA of a virus, such as HIV, that has been integrated into the genes of a host cell.

Retrovirus: HIV and other viruses that carry their genetic material in the form of RNA and that have the enzyme reverse transcriptase.

Reverse transcriptase: The enzyme produced by HIV and other retroviruses that allow them to synthesize DNA from their RNA.

Syncytia: Giant cells formed by the fusion of other cells.

Sources:National Institute of Allergy and Infectious Diseases, National Institutes of Health, Centers for Disease Control, Johns Hopkins Medical School, Adam.com, IntelliHealth.com.

The liver is the body's largest solid organ weighing about three pounds.It is a remarkable, intricate, responsive machine. All of the blood that passes through the intestines, rich with absorbed nutrients, must first pass through the liver on its way to the heart and lungs. Unlike other internal organs, such as the stomach or gall bladder, the liver is essential to maintaining life. Complimentarily, its cells are so extremely efficient, the entire organ is not needed to survive; humans can maintain a robust life with less than half of a liver.

Located in the upper right abdomen, the liver plays two major roles: It neutralizes toxins and wastes, and it synthesizes new chemicals essential for blood clotting and other functions. The liver contributes other vital tasks: it serves as a production facility where important substances, including most of the proteins in the blood, are made and then exported throughout the body.Also blood clotting is regulated from within the liver: the liver makes most of the factors that clot blood and initiate wound healing. When the liver's function is hampered by disease, the body's ability to recover from injury is severely impaired. Features of the liver’s contribution to the body’s health and welfare include:

·Proteins are processed in the gastrointestinal tract into small peptides and amino acids, then sent to the liver where they are reconfigured and/or metabolized. Drugs are absorbed in the stomach and bowel and are converted by the liver into forms the body can use, or they are broken down and excreted from the body.

·The liver stores glycogen, the storage form of glucose, which is readily at hand when the body needs a quick supply of energy.

·One of the liver's largest exports is bile, a detergent the liver makes to dissolve fat in the digestive tract, facilitating absorption. The bile flows through several channels or bile ducts, into the intestine.

Thus the liver acts as a massive filter, which detoxifies harmful substances, including potentially dangerous ingredients in food, and excretes the worst.

Interestingly, the liver is also the only solid organ in the body able to regenerate itself.If even as much as 70 percent of its tissue is damaged, by alcohol, drugs or a virus, such as hepatitis A, B, or C (all of which cause the liver to become inflamed) the liver manages its own recovery by creating new replacement cells and tissue.

As powerful and resilient as the liver is, it is particularly vulnerable to alcohol, which is a toxin. To the liver, a wine cooler or a can of beer is poison, although for most people, one drink has no lasting, harmful effects on the body. However, in combination with medication, or excessive alcohol intake, the effect can be additive. Over time, the liver will react and become inflamed and enlarged, a condition called alcoholic hepatitis; fatty liver, a condition in which globules of fat infiltrate the liver and harm its ability to function, may also develop. Over time, the liver can become scarred, labeled as cirrhosis. When too much tissue is destroyed, the liver can no longer handle the burden of toxins that constantly wash through it or make its important substances resulting in liver failure. Liver damage from alcohol or other toxins is often insidious netting negative results such as cirrhosis in people who have never experienced jaundice or suffered any other symptoms of liver damage. Although many substances can damage the liver, the symptoms of liver trouble are often similar. (E.g., a host of problems can result in jaundice.)These origination questions thereby require sophisticated diagnostic testing to determine the particular cause of damage.

Viral Hepatitis

There are at least six contagious viruses known to cause inflammation of the liver: Hepatitis A, B, C, D, E and G; the first three are the most serious in the United States. (Hepatitis D cannot take hold in the body unless a person is already infected with hepatitis B; thus, if not infected with hepatitis B, one is not at risk for hepatitis D.Hepatitis E, a health problem in East & South Asia, is extremely rare in North America, as is Hepatitis G.) Although their impact on the liver can vary greatly, hepatitis viruses B and C have the potential to develop into chronic hepatitis and to cause permanent liver damage.

Hepatitis A

Hepatitis A is responsible for about one-quarter of all cases of viral hepatitis in the United States. Unlike other forms of hepatitis, hepatitis A is most commonly transmitted in drinking water or food contaminated with fecal matter that contains the virus; high concentrations of the virus live in the feces of an infected person. Like many forms of food poisoning, this disease could be largely prevented by proper hygiene. Hepatitis A can also be transmitted by "deep" kissing, by anal sex and by using contaminated needles to administer intravenous drugs.

This virus is tenacious: at normal room temperature, it can survive for up to four hours in a speck of contaminated fecal material resident on skin, the outside of a diaper or—a common problem in daycare settings—on a hard surface such as a ball, cup or spoon. When contaminated sewage is dumped into fishing waters, shellfish can contract the virus. People who eat the tainted shellfish raw, or even swim in polluted waters, can contract the virus, as well.

Hepatitis A is especially common in the Middle East, South and Central America, Eastern Europe, Africa and Southeast Asia. Each year, the Centers for Disease Control and Prevention estimates some 150,000 Americans are infected with hepatitis A. Because the incubation period is two to six weeks, it is easy for the infection to be spread before any symptoms have developed.

Hepatitis A virus is seldom life threatening. In most people, it produces temporary influenza-like symptoms and jaundice, and runs its course. There is no specific treatment, and typically the body recovers within six months. The disease hardly ever results in serious complications to pregnant women or their unborn children. However, in a small percentage of cases, particularly in the elderly and in people already suffering from such liver problems as alcoholic hepatitis or cirrhosis, hepatitis A can produce direct or collateral complications.

Hepatitis B

Nearly 300,000 Americans contract hepatitis B every year; it is the most common cause of viral hepatitis. Hepatitis B is spread in infected blood and other bodily fluids (semen, vaginal secretions, saliva, open sores and breast milk). Note: It is NOT spread by casual contact; for example, from holding hands, being exposed to a cough or sneeze, playing with an infected child, "dry" kissing on the lips, or eating food prepared by someone who has the virus. An estimated 40 percent of those infected do not know how they acquired hepatitis B.

Because the incubation period can be one to six months, it can be difficult to pinpoint the source of exposure. Like hepatitis A, hepatitis B may be present in contaminated beaches and raw shellfish.As many as 10 percent of all people infected with Hepatitis B develop chronic hepatitis; young children and infants are particularly at risk of becoming chronically infected or of becoming carriers of the disease.

Most people manage to fight off the infection successfully within a few months, developing an immunity that lasts a lifetime. Thus, once cleared of the virus, the person will never again develop hepatitis B (although they could still develop another form of hepatitis). Immunity can be checked by a test called "anti-HBs," for "antibody to hepatitis B surface antigen." The test registers the presence in the blood of an antibody to the outside of the hepatitis B virus. If infectious hepatitis B was ever present, antibodies to the virus will always be present in the blood. Because there is a tiny risk that the anti-HBs test showed a false positive and that the virus could still be passed on, the individual will not be allowed to donate blood, plasma, tissue, body organs or sperm.

An estimated 1 million Americans are lifelong carriers of hepatitis B. If infected with hepatitis B for more than six months, then the person is considered a carrier — even if otherwise healthy and free of symptoms. This means that the virus can be transmitted to others by having unprotected sex, sharing needles, deep kissing, sharing food or drinks, or engaging in any other risky behavior. Because hepatitis B can be a sexually transmitted disease, it is particularly prevalent in young adults. It also means that the liver is more vulnerable than normal to injury.

If a woman is a carrier of hepatitis B and pregnant, there is an estimated 90 percent likelihood of passing on the virus to the unborn baby. Thus, all pregnant women should be tested for hepatitis B, and if positive, all their babies should be given immunoglobin and be vaccinated at birth to protect them from getting the virus. For reasons not understood, the hepatitis B virus spontaneously goes away in a very small percentage of carriers. Some people who are carriers develop chronic hepatitis.

According to the American Liver Foundation, in the United States hepatitis B is responsible for 5,000 deaths each year—from hepatitis itself, and from other damage to the liver, including liver cancer, cirrhosis and liver failure (all of which can be triggered by chronic hepatitis).

Hepatitis C

About 4 million Americans have hepatitis C; most of these people have never experienced any symptoms. Few realize they even have this virus, which historically was identified by what it was not.Until the late 1980s, it was called "non-A, non-B" hepatitis. A large number of Americans contracted hepatitis C from blood transfusions until the 1980s, when new purity tests dramatically lowered the risk of becoming infected from donated blood.Currently, about 180,000 Americans become infected with this virus each year; and, although it is usually silent, some scientists believe the annual death rate from hepatitis C will triple to 25,000 deaths over the next 20 years.

Although a small percentage of people infected with hepatitis C manage to clear the virus on their own, the majority develop a chronic infection. Most of these people have no symptoms and live normal, healthy lives. Less than one-quarter chronic hepatitis C sufferers eventually develop complications, including cirrhosis, liver cancer and liver failure. Chronic infection with hepatitis C is the leading cause for liver transplants.

Hepatitis C, like hepatitis B, is a blood-borne virus that can be transmitted through blood transfusions. Since 1990, however, screening tests have dramatically reduced this risk. It is most commonly spread through infected needles shared by intravenous drug users and, much less often, through "unprotected" sex (in which a condom is not used). Most people are diagnosed when a routine blood test reveals elevated liver enzymes or when a hepatitis C antibody (anti-HCV) test, given at the time of blood donation, is positive. This test can remain positive even several years after someone has recovered from acute hepatitis C — even if the body is no longer infected with the virus. Currently, there is no vaccine to prevent hepatitis C.

Symptoms

·Jaundice (yellowing of the skin or whites of eyes and/or a brownish or orange tint in the urine)

·Unusually light-colored stool (clay colored)

·Unexplained fatigue that persists for weeks or even months

·Influenza-like symptoms, such as fever, loss of appetite, nausea, and vomiting

·Abdominal pain

Prevention

There is much that can be done to prevent viral hepatitis. The rules that have become familiar because of efforts to prevent AIDS apply to hepatitis, as well: Don't use intravenous drugs or share needles; avoid "unprotected" sex (without a condom, and make sure to change condoms with each sexual act). Do not share chewing gum, drinks, razors, toothbrushes or pierced earrings with anyone (and if any part of the body is pierced or is getting a tattoo, ensure the needle is properly sterilized). If one must touch or clean up blood (or items with blood on them, such as tissues or tampons), wear disposable gloves. To clean an area with blood on it, use bleach (one part bleach to 10 parts water). To prevent hepatitis A, in addition to the steps listed above, wash hands frequently, particularly after using the bathroom or changing a baby's diaper. Also, avoid raw shellfish, which may be contaminated with hepatitis A or B.

Vaccines are available for hepatitis A and B. If exposed to the blood or body fluids of an infected person, a vaccine is needed immediately. Because most forms of viral hepatitis have an incubation period that lasts several weeks to months, there is still an opportunity to protect oneself from infection. To boost the immune system and help the body ward off the virus, an immunoglobulin shot is required.

Treatment

Treatment begins with steps to prevent infection, including immediate immunization if exposed to hepatitis A or B. (There is no vaccine for hepatitis C.)Temporary immunization is possible with an immune boosting shot called immune serum globulin (ISE), which should be given within two weeks of exposure to the virus.Bed rest—as much as possible—is necessary to speed recovery. Make every effort to eat a high-calorie, high-protein diet. If experiencing nausea, it can be difficult to get the calories needed to recover. Try eating as much as possible in the morning, when nausea tends to be lightest. Also, take extra care of the liver, which is now vulnerable to further injury. Do not drink alcohol; never mix alcohol with acetaminophen (found in Tylenol), the combination can harm the liver, and check with an informed physician before taking any medication. Six months is the mile marker for hepatitis B or C; if viral hepatitis lasts longer than this, it is then considered to be chronic hepatitis, and more aggressive treatment may be needed.

Sources:National Institute of Allergy and Infectious Diseases, National Institutes of Health, Centers for Disease Control, Johns Hopkins Medical School, Adam.com, IntelliHealth.com, The Scientist, Nature, Science.

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