Hepatitis C: A Brief Clinical Overview. ILAR (2): 107.
Hepatitis C is characterized by asymptomatic onset, is often discovered incidentally through blood tests obtained during routine physical examination or before blood donation. Spontaneous recovery occurs in about 20% of patients. Among those who remain chronically infected, an equal percentage progress to cirrhosis within 20 years, have stable nonprogressive disease, or progress more slowly over 40 to 6o years.
Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus of the family flaviviridae in company with the pestivirus and hepacivirus groups.
There are currently, 6 major genotypes of the Hepatitis C virus worldwide. For a virion, the Hepatitis C virus has a fairly high mutation rate of 0.003 nucleotide substitutions/genome site/year. This high rate of mutation alone or in conjunction with a heterogenous viral inoculum and subsequent host selectivity, leads to "quasispecies" within one individual over the course of a few short years. From the viewpoint of the individual, the significance of the quasispecies is seen in the inverse relationship to the number of these "species" and response to interferon. From a population point of view, this diversity coupled with selective pressure from the host immune system makes it difficult to identify a single or few antibodies that might be used for vaccine research and prophylaxis.
Hepatitis C infection occurs by contact with whole blood, blood products or body fluids with sharing of needles the currently most common mode of infection. Identified risk factors include heath care employment, unprotected sexual intercourse with multiple partners, low socioecconomic status, exposure to household members with a history of HCV and perinatal transfer, particularly in HIV-infected coinfected mothers.
Since HCV infection is essentially asymptomatic in immunocompetent persons, EIA assays for core proteins is the first step in diagnosis. Presence of these antigens does not confirm current infection but exposure. Confirmation of active infection is documentation of HCV RNA in the serum via polymerase chain reaction for both immune-competent and immune-compromised individuals.
Current therapy with combination of ribavirin and interferon, the therapy of choice, is only effective in 30-45% of infected individuals with genotype 1, the type responsible for the majority of infections. Phlebotomy to decrease iron load to the liver has not been beneficial.
African-Americans have twice the prevalence of caucasians; higher viremia and have a approximately 0.1 sustained response to combination therapy when compared to caucasians. However, they do not progress any faster in the disease than caucasian. These differences appear not to be due to HCV RNA levels; mode of acquisition of the virus; compliance with medication regimen or virus genotype.
Vaccine development is fraught with the asymptomatic nature of HCV infections; the relatively high mutation rate of HCV; the geographic differences in predominant HCV genotype and financial resources. Alternative treatment strategies include extracellular neutralization of virus; inhibition of viral entry and uncoating; inhibition of host or viral enzymes leading to impairment of intracellular replication; and control of hepatic inflammation.
No questions

New Animal Models of Hepatitis B and C. ILAR 42 (2): 127.
Chimpanzees have been used to study the pathogenesis of the viral infection and to conduct vaccine studies. liver disease in experimentally infected chimpanzees is characteristically mild, which makes it impossible to study the pathogenesis of cirrhosis or HCC. The tree shew (Tupaia) has been shown to be susceptible to HBV and HCV infections. However, this small animal model is relatively uncharacterized, and it has not provided any significant advantages over the chimpanzee.
Hepadnaviruses include HBV and agents that naturally infect selected hosts in the wild. The three best studied are the ground squirrel hepatitis virus, the woodchuck hepatitis virus, and the duck hepatitis B virus (DHBV). Infected ducks and woodchucks have been used for preclinical antiviral drug development. However, the lack of inbred ducks and/or woodchucks, as well as the lack of duck-and woodchuck-specific reagents to analyze the immune cells, remain obstacles in understanding the molecular and cellular basis of disease. In addition, neither of them develop cirrhosis, nor do infected ducks develop HCC.
The family Flaviviridae contains three genera; hepacivisuses (HCV), flaviviruses (yellow fever virus, dengue virus, Japanese and tick-borne encephalitis viruses), and pestiviruses (border disease and swine fever viruses, and the bovine viral diarrhea virus). HCV is related to these genera in terms of genome structure, the production of a polyprotein, the structure and function of the corresponding polypeptide, and their likely mode of replication. Unlike HBV, there have been no reports of closely related HCV-like hepaciviruses that naturally infect wild animals or that can be used to experimentally infect laboratory animals.
There is strong evidence to suggest that the pathogenesis of HBV infection is immune-mediated rather than by CPEs. HBV carriers and transgenic mice with sustained, high levels of virus replications have no evidence of liver disease. Also tissue culture systems that replicate HBV do not develop CPE. Flaviviruses tend to mediate pathology by CPE, although the case is not so clear cut with HCV. Some aspects suggest that pathogenesis of HCV could be by CPE, while other aspects suggest the likelihood of immune-mediated pathogenesis.
The development of transgenic mouse technology provided opportunities of HBV and HCV gene expression, replication, and disease. In 1985, research groups created transgenic mice that produced the HBV envelope or HBsAg particles. The findings that persistently high levels of HBsAg in human carriers and transgenic mice do not result in the development of liver disease, and that immunosuppression ameliorates chronic hepatitis, suggest that liver cell damage may be immune mediated. The immune-mediated nature of fulminant and acute hepatitis was implied by the finding that HBV replicates without triggering CPE in tissue culture cells. The development of CLD in HBV carriers is a major target for therapeutics, yet none of the HBV transgenic models available develop CLD because all transgenic mice are tolerant to the products of the transgene. In a recent report, transgenic mice supporting HBV replication were constructed using SCID mice. Because the T and B cells account for the bulk of specific antiviral immunity, these mice were not tolerant to HBV. Syngeneic splenocytes resulted in the development of chronic hepatitis, whereas a similar transfer of 50 million cells resulted in acute, resolving hepatitis. Although this model confirms the immune-mediated nature of HBV-associated liver disease, it will permit dissection of the immune components and virus antigen targets that contribute to pathogenesis.
The narrow host range and lack of suitable tissue culture systems for HCV have prevented a thorough understanding of the chronic liver disease. The use of transgenic models of HCV gene expression and replication revealed that the pathogenesis of HCV infection is immune mediated. Conditional expression of the HCV envelop and core genes was carried out in mice using the Cre/loxP system. The expression of both envelop and core proteins was detected in the liver within 1 wk after transgene activation. When transgene induction was repeated in T cell-depleted mice, there was normal histology and transaminase levels, suggesting the inflammatory response and hepatocellular damage were T cell mediated. By day 14 after transgene activation, core was cleared from serum and replaced by anti-core.
HBV and HCV gene expression, replication, and liver disease have also been studied in nontransgenic rodent systems. HBsAg expression has been demonstrated in the liver of adult rats intrahepatically injected with liposomes that carry an expression plasmid encoding the preS plus S polypeptides. Immunodeficient mice have also been used for transplantation of human hepatocytes to study infection and to evaluate antiviral compounds. Rag-2 immunodeficient mice (which lacked both T and B cells) were used for antiviral drug screening. However, none of the hepatocellular transplantation models are constructed to permit the development of immune-mediated liver pathogenesis.
There is considerable evidence that the pathogenesis of HBV and HCV infections is immune-mediated. Animal models that encompass a broader range of host-virus interactions should be useful for understanding the mechanisms of pathogenesis and for developing new therapeutics.
No questions

The Chimpanzee Model of Hepaitis C Virus Infections. ILAR 42 (2): 117.
The Chimpanzee (Pan troglodytes) is the only experimental animal susceptible to infection with hepatitis C virus (HCV) and is critical for ongoing research of HCV. There are currently no tissue culture systems or small animal models available to study HCV. HCV is a member of the Hepacivirus genus of the Flaviviridae family. HCV is a positive sense ss RNA virus. Infection can persist to lifelong disease including cirrhosis and hepatocellular carcinoma, and HCV is the leading cause of liver transplantation in the US.
2 cases of hepatocellular carcinoma have been observed in HCV infected chimps but fibrosis and cirrhosis has not been reported. The chimpanzee represents a non-selected population for the study of CV, as opposed to studies of human CV where subjects are selected because of known (typically decades-long) infection. Therefore the chimpanzee allows for the study of early infection. The chimpanzee is critical for studies of viral clearance because they have a high rate of viral clearance, analysis of immune response, and the development of vaccines for CV.
A retrospective study of infected chimpanzees at Southwest Foundation for Biomedical Research showed that 30-40% of infected chimpanzees are persistently infected as opposed to 85% of CV infected humans. This was thought to be either because of a difference in clinical course or the full clinical picture of human infection is not observed because of the way that humans are selected.
Because the percentage of persistently infected chimps was surprisingly low, a longitudinal study was done to look at kinetics of viral clearance and antibody loss. Based on results the chimps were grouped in three categories: Persistently infected animals, animals that cleared the infection with gradual loss of antibody over several years, and animals that cleared the infection with rapid loss of antibody. Extrapolation to the human population imply that there is the potential for the underestimation of human infection.
The Chimpanzee model of CV infection was instrumental on non-A non-B hepatitis, which led to the cloning of CV nucleic acid. Preliminary data shows that NS3-NS4 may be a good candidate because T cell responses to CV nonstructural proteins appear to coincide with viral clearance. The development of infectious DA clones of CV was dependent on the use of chimpanzee. Finally the development of micro array technology to probe the entire liver gene expression during the course of CV was dependent on the use of the Chimpanzee.
Critical environmental factors may be lacking in the chimpanzee model. For example, alcohol consumption and increased dietary iron levels are thought to play a role in the susceptibility and pathogenesis of HCV infection. The effects of alcohol consumption on HCV has not been studied in chimps. Preliminary studies have shown that iron loading did not influence viral load but did exacerbate liver injury in HCV-infected chimpanzees. The development of cDNA clones of HCV has lead to new research opportunities for HCV. However because of lack of tissue culture systems, the testing of mutant clones is sill restricted to the intrahepatic inoculation of chimpanzees with synthetic RNA.
Finally, the analysis of changes in liver gene expression in HCV infection is being studied with the use of DNA Micro array Technology. Other infectious diseases that this technology is being used include human immunodeficiency virus, human papilloma virus and human cytomegalovirus. Just remember that the Chimpanzee is the only model of HCV infection.

Questions:
1. What animal models of HCV exist?
2. TF: The rate of viral clearance is faster in the chimpanzee as compared to humans.
3. TF: Hepatic disease and pathology is the same for the chimpanzee and human.
4. Give 2 important environmental factors that are thought to play a role in HCV infection in humans. Have these factors been studied in chimps?
5. What part of HCV did this paper describe as a target for vaccine development?

Answers:
1. The chimpanzee is the only animal model for HCV. In fact no in vitro models exist either.
2. True. Viral clearance is faster in the chimpanzee than the human. This is a main reason the chimp is useful as a model for HCV.
3. False. Only HCV hepatocellular carcinoma has been observed. Fibrosis and cirrhosis has not been observed in the chimpanzee.
4. Alcohol consumption and Excessive dietary iron intake. Only preliminary studies of iron overload have been done. No studies of alcohol consumption have taken place.
5. Non-structural proteins: NS3-NS4.

Introduction. ILAR 42 (2): 073.
Based on seroepidemiological data, approximately 5.5 billion out of 6 billion people are estimated as having been infected with one of hepatitis viruses. Although progress has been made towards effective vaccines and therapeutics for hepatitis A and B, some urgency exists for the other viruses, especially hepatitis C virus. Although NHPs play a critical role in hepatitis viral disease research, there is a need for a better and more practical small animal model. The chimpanzee has been invaluable as an animal model for hepatitis-related research, especially in hepatitis C vaccine research and therapeutics development. However, the chimpanzee is endangered in the wild, and the use of these animals for research is restricted. The tamarin can be used as a good surrogate model for hepatitis C. Some non-NHP animal models for hepatitis research include the woodchuck model in hepatitis B infection studies. Some promising new approaches using transgenic mouse models are also becoming available. The tree shrew has been successfully infected with hepatitis B and C viruses and may prove to be useful for future research efforts.

Questions:
1. Match each hepatitis virus to its appropriate viral family or description.
A. Hepatitis A virus i. Flaviviridae
B. Hepatitis B virus ii. Circular RNA virus
C. Hepatitis C virus iii. Picornaviridae
D. Hepatitis delta virus iv. Caliciviridae
E. Hepatitis E virus v. Hepadnaviridae
F. Hepatitis G virus
2. Which of the hepatitis viruses are DNA viruses?
3. Which of the hepatitis viruses are RNA viruses?
4. Which of the hepatitis viruses requires hepatitis B virus as a "helper" virus for replications?
5. Give the scientific name for each of the following animals: chimpanzee, tamarin (Genus only) woodchuck, mouse, tree shrew (Genus only).

Answers:
1. A. iii.; B. v.; C. i.; D. ii.; E. iv.; F. i.
2. Hepatitis B virus is the only DNA virus among the hepatitis viruses.
3. Hepatitis A, C, delta, E, G viruses
4. Hepatitis delta virus
5. Chimpanzee - Pan troglodytes; Tamarin - Saguinus spp.; Woodchuck - Marmota monax; Mouse - Mus musculus; Tree shrew (Tupaia spp.)

Nonhuman primates: a critical role in current disease research. ILAR 42 (2): 074.
This review article emphasizes the critical role of nonhuman primates (NHPs) in biomedical research. It focuses on the most recent contributions that NHPs have made to the understanding, treatment, and prevention of important infectious diseases and chronic degenerative disorders of the central nervous system. The close phylogenetic relation of NHPs to humans not only opens avenues for testing the safety and efficacy of new drugs and vaccines but also offers promise for evaluating the potential of new gene-based treatments for human infectious and genetic diseases. There are more than 200 species of NHPs, but only about 30 are used in research yet they account for only about 0.3% of the animals required by the US research community. The process for acquiring laboratory primates has evolved significantly over several decades. With the exception of the importation of certain monkey species, commercial breeding colonies within the United States provide a large part of the NHPs needed for research.

AIDS The acquired immunodeficiency syndrome (AIDS) was first recognized in 1981 and has human immunodeficiency virus (HIV), has infected more than 34 million adults and children worldwide and has claimed more than 18 million lives. HIV acts by destroying helper CD4+ T-lymphocytes. For the present, success in developing an HIV vaccine depends on studying animals that not only are susceptible to infection but also progress to develop AIDS or AIDS-like symptoms similar to those in humans. Since the early 1990s, scientists have developed NHP models that can be naturally and experimentally infected with primate lentiviruses . Various simian immunodeficiency viruses (SIVs), infect many different species of macaque monkeys and cause an AIDS-like syndrome. More recently, simian-human immunodeficiency viruses (SHIVs), genetically engineered chimeras composed of an SIV core enclosed in an outer coat envelope protein of HIV, have revealed advantages over the SIV model. Many of these viruses retain the ability to cause CD4+ T-cell lymphopenia and an AIDS-like disease in chimpanzees and several monkey species; however, they express human surface proteins, which make them valuable as challenge viruses for evaluating HIV envelope-based vaccines.

Although progressive infection with HIV-1 can occur in some chimpanzees, chronically infected animals usually maintain normal numbers of CD4+ T-lymphocytes and do not become immunodeficient . The one exception stems from a report that a chimpanzee infected with three different isolates of type-1-HIV over a period of 10 years revealed a persistent decline in CD4+ T-lymphocytes that progressed to AIDS or an AIDS-like disease. Blood from this animal that was transfused into an uninfected chimpanzee induced a rapid depletion of CD4+ T-lymphocytes but did not cause clinical disease. Without disease as an endpoint, researchers can measure only the infection-blocking effect of candidate vaccines in chimpanzees.

Although progressive infection with HIV-1 can occur in some chimpanzees, chronically infected animals usually maintain normal numbers of CD4+ T-lymphocytes and do not become immunodeficient . The one exception stems from a report that a chimpanzee infected with three different isolates of type-1-HIV over a period of 10 years revealed a persistent decline in CD4+ T-lymphocytes that progressed to AIDS or an AIDS-like disease. Blood from this animal that was transfused into an uninfected chimpanzee induced a rapid depletion of CD4+ T-lymphocytes but did not cause clinical disease. Without disease as an endpoint, researchers can measure only the infection-blocking effect of candidate vaccines in chimpanzees.

Hepatitis C Hepatitis C Virus (HCV) is an enveloped RNA virus that causes acute and chronic liver disease. HCV has infected an estimated 3.9 million people in the United States and 170 million worldwide. Most people harbor HCV without clinical symptoms for many years before they develop serious complications such as cirrhosis and liver cancer. Approximately 15 to 25% of infected persons can clear the virus from their bloodstream within several weeks, whereas the remaining 75 to 80% develop a chronic infection. Treatment with interferon alone, or combined with the antiviral drug ribavarin, is not highly efficacious and can cause significant side effects. Consequently, there is a pressing need to develop vaccines aimed at preventing HCV transmission and eliminating chronic infection. In the absence of a reliable tissue culture system and a small animal model, a great deal of what is known about the nature of HCV infections has been learned through studies with chimpanzees--the only known nonhuman host for HCV. Chimpanzees can be infected with HCV and treated with potential antiviral drugs. They can also be immunized with various viral vaccines and challenged with HCV isolates to validate potential targets for the development of antiviral interventions.

A major breakthrough has been the successful construction of full-length, functional cDNA clones of HCV. The RNA transcripts of these clones initiated infection and produced disease when injected directly into the liver of chimpanzees. The virus clones now can be biologically amplified and used as challenge inocula in chimpanzees to develop a better understanding of the molecular biology and natural history of HCV infection. Two recent reports suggest that the ultimate outcome of an HCV infection may depend on a host's ability to mount a strong cellular and humoral immune response in the early phase of primary infection. One study conducted in chimpanzees demonstrated that HCV persistence is related to changes in the hypervariability region (HVR1) of the putative E2 envelope gene of HCV. The data suggest that the development of viral variants or quasispecies during the early phase of HCV infection predict whether the infection will be resolved or become chronic. One group of scientists has presented preliminary evidence that an HCV vaccine consisting of recombinant envelope proteins elicits neutralizing antibodies and stimulates the production of CD4+ cytotoxic T-lymphocytes during the acute phase, preventing chronic infection in chimpanzees. Another group has reported that immunization with E2 glycoproteins and peptides from the E2 hypervariable region of the virus is effective in chimpanzees.

Hepatitis B Hepatitis B virus (HBV) is also transmitted by contact with an infected person's blood or bodily fluids, mostly through the use of shared needles and high-risk sexual behavior. HBV infection produces a transient viremia that lasts about 4 to 8 weeks or becomes chronic. In 90% of infected people, the acute infection is clinically silent or produces flu-like symptoms, and the remaining 10% develop a chronic infection with a high risk of cirrhosis and hepatocellular carcinoma. Although vaccines generally became available in 1982, the infection and its sequelae remain a major cause of morbidity and death. Globally, close to 3 billion people are infected, 350 million become chronic carriers, and 1 to 1.5 million die each year. If the vaccine were more widely used, most cases of the disease could almost be prevented. Chimpanzees and, to a lesser extent, rhesus monkeys are the only animals that can be infected by human HBV genotypes. Chimpanzees develop circulating antibodies and increased enzyme levels indicating liver damage after virus inoculation and mount cellular immune responses to HBV, similar to those observed in acutely infected humans. Approximately 10% of young adults fail to respond to the commercially available HBV vaccine, prompting the demand for a vaccine that can stimulate strong cellular and humoral immune responses. Using chimpanzees, one group of researchers is testing a new recombinant vaccine that encodes for three viral surface proteins that stimulate strong cellular and humoral immune responses. Recently, a study conducted in chimpanzees has advanced our understanding of how the body protects against the spread of HBV without harming the liver. Researchers have shown that during a typical infection, elimination of HBV-DNA is accomplished by a tissue-sparing process rather than by the destruction of infected hepatocytes.

Malaria The World Health Organization estimates that 300 to 500 million new cases of malaria occur each year, claiming 2 to 3 million lives worldwide. In the last decade, considerable progress has been made in the development of malaria vaccines. Owl and squirrel monkeys are excellent models for testing vaccines because they can be infected with the two Plasmodium species (P. falciparum, P. vivax) that cause most human disease. The owl monkey is the primate model recommended by the World Health Organization for testing vaccines. Chimpanzees occasionally are used to produce infective parasites (sporozoites) for monkey challenge studies. Malarial parasites (protozoans) from the genus Plasmodium cause disease in many mammalian species, including humans, apes, monkeys, rodents, and birds. However, of the more than 100 Plasmodium species, only four infect humans. Plasmodium falciparum causes the greatest number of deaths and life-threatening complications. Currently, vaccines are being developed in NHPs to prevent the emergence and spread of malaria in the community and to reduce the severity of the disease and the risk of death in infected individuals. Researchers have been successful in identifying a number of surface proteins of the mosquito-stage parasite as suitable target antigens for transmission blocking vaccines, and candidate blocking vaccines are now being tested in human phase I safety and immunogenicity studies. These vaccines have been shown to be immunogenic in monkeys and chimpanzees, especially when they are administered with immune enhancers such as oligodeoxynucleotides. The goal of these vaccines is to prevent the invasion in hepatocytes or erythrocytes.

Respiratory Syncytial Virus Human respiratory syncytial virus (RSV1) is the most common cause of lower respiratory tract infections in infants, young children, and the elderly. Each year, RSV contributes significantly to deaths worldwide, including an estimated 4500 in the United States. Studies in nonprimate models cannot duplicate all forms of RSV disease. To date, the chimpanzee is the only experimental animal that is susceptible to RSV infection and develops a respiratory illness similar to the disease seen in human infants. Consequently, chimpanzees have become the model of choice for studying RSV disease and for evaluating live virus vaccine candidates against the disease. Chimpanzees used in these studies must be relatively young (less than 2 years of age) before they develop protective antibodies to RSV infections. Studies in animals and humans have shown that neutralizing antibodies can protect against RSV infection and illness. The technology exists to prepare live infectious RSV from cDNA, making it possible to design a recombinant RSV vaccine with known attenuating insertions, which is suitable for intranasal administration (Collins et al. 1999). The safety and efficacy of such vaccines can then be evaluated in seronegative chimpanzees. Recently, several candidate vaccines---a cold-passaged, temperature-sensitive RSV subgroup B, a live attenuated chimeric virus, and viral subunits of attenuated strains formulated with purified fusion and attachment proteins were shown to be immunogenic and to confer high levels of resistance on chimpanzees. Some of these live vaccines are being evaluated, or soon will be, in humans.

Periodontitis Periodontal or gum disease is the most common cause of bone and tooth loss in humans. Periodontitis is also a common health problem for several captive NHP species. Macaque monkeys (cynomolgus) develop dental calculus, plaques, and associated gingivitis, which often progress into periodontal disease. This species also manifests a subgingival flora characteristic of the anaerobic Gram-negative bacteria found in human periodontal pockets. NHP models allow scientists to manipulate variables in ways not possible in humans when studying the changes that occur in the progression of early gingivitis to periodontitis. Scientists have found evidence that the cluster of changes described above may be associated with increased risk of atherogenesis. Some investigators now believe that periodontitis may be an important risk factor for, or may be linked to, the initiation of the atherosclerotic process, which can lead to coronary heart disease. Investigators also are testing the effect of certain food additives that can be placed in the normal primate diet to retard calculus formation and reverse or prevent subsequent development of periodontal disease.

Bronchopulmonary Dysplasia and Respiratory Distress Syndrome Bronchopulmonary dysplasia (BPD) is a disabling and sometimes fatal chronic lung disease of newborn infants resulting from the abnormal development of the lungs. It occurs most often in premature babies who require mechanical ventilation and oxygen under pressure to survive respiratory distress syndrome (RDS) or hyaline membrane disease. RDS is caused by a deficiency or dysfunction of pulmonary surfactant, a substance produced by alveolar cells that coats the inner surface of the lungs. The surfactant acts by lowering the surface tension at the air-cell interface to promote the uptake of oxygen and excretion of carbon dioxide in expired air. RDS together with BPD account for much of the infant morbidity and mortality in the United States, with up to 10,000 cases of BPD occurring each year in surviving infants. Research with premature baboons has provided important information about lung growth and development in long-term survivors with BPD that could not have been obtained from studies with human babies. The results of studies in the preterm baboon have provided important basic findings and practical therapeutic information to decrease the severity of, or prevent, BPD. Administration of both natural and synthetic surfactants improves the clinical outcome of RDS and decreases the progression of BPD in premature baboons. The exact causes of BPD remain under investigation; however, studies in baboons are expected to complement human research and may ultimately lead to improved clinical management and greater survival for infants with these respiratory conditions.

Gene Transfer Human genetic engineering, or gene transfer experiments are is currently being investigated to provide alternative modalities for managing and correcting a number of inherited metabolic, infectious, and malignant diseases that are not amenable to current approaches. Gene transfer involves incorporating a gene into an individual either by the administration of naked DNA or DNA complexed to a harmless virus that carries genetic material into a target somatic cell genome, or by the introduction of cells manipulated to harbor foreign DNA. The recent completion of the Human Genome Project and the partial mapping of other mammalian genomes, including mice, rats, chimpanzees, and baboons, has led to the identification of genes for many human conditions, including those with animal counterparts. Because genetic conservation among mammalian genomes is extremely high, the mouse and the rat have been, and will continue to be, the most practical models for understanding disease processes and testing gene therapies. The close evolutionary relation of humans and NHPs argues strongly for the use of NHPs in certain preclinical studies. Now that the sequencing of the human genome has been achieved, there is a compelling argument for sequencing the genomes of other mammals, in addition to the mouse. The obvious candidates are the chimpanzee and the rhesus macaque. These models can be expected to yield the most valid assessment of the potential benefits and risks of gene transfer systems in humans. Gene transfer experiments are being actively pursued in NHPs to develop alternative treatments for a number of human infectious and genetic diseases

Brain Disorders NHPs are extremely valuable models for investigating the functions of the normal brain as well as brain-related disorders and disabilities. Annually, more than 90 million people in the United States are afflicted with brain related conditions, which include mental, neurological, and addictive disorders. The information gained from primate research is directly applicable to the human condition because NHPs share many features of brain biology and physiology with humans. Old and New World monkeys and many species of small primates are the animal models of choice for neurological studies because many of the associated regions of the cerebral cortex of these species are present and identifiable. Studies using NHPs have provided a more precise map of the circuitry and functions of the cerebral cortex than could ever have been achieved using other species, including humans. Monkeys can be trained to perform a wide variety of simple cognitive tasks involving perception, attention, and decision-making in return for positive rewards. While carrying out these tasks, the electrical activity of individual nerve cells can be measured and mapped using small microelectrodes positioned at specific locations within the brain. Microelectrodes are inserted through a penetrable window in the cranium that had been implanted earlier under anesthesia; and once in place, the electrodes cause no pain because the brain lacks pain sensors. Single-cell recording in awake, behaving animals is perhaps the most powerful approach to understanding the neural basis of all primate behavior. Studies involving NHP models have yielded many human health-related applications and a better understanding of auditory and visual impairment, learning and memory deficits, substance abuse, spinal cord and brain injury, aberrant social behavior, and mental illness.

Parkinson's Disease Parkinson's disease (PD) is the most common progressive neurodegenerative movement disorder of the central nervous system, primarily affecting older individuals (1% of the population over 55 years). Approximately one million people in the United States have PD, with 50,000 cases reported annually. The link between PD and the progressive degeneration of dopamine-producing nerve cells in the substantia nigra---a small structure located in the midbrain---was discovered in rabbits and later confirmed in several other animal species, including NHPs. Dopamine is a chemical that helps transmit signals to other nerve cells. This finding has led to the treatment of PD with levodopa or L-DOPA,1 which is converted into dopamine within the blood-brain barrier. Although the administration of L-DOPA remains the treatment of choice, the effects are not sufficiently lasting and lead to the development of dyskinesia, a condition characterized by spontaneous or uncontrolled movements. The administration of MPTP induces dopamine depletion in a wide variety of animal species. However, the behavioral changes induced in the MPTP-treated NHPs closely resemble those of the human disorder. Researchers have found that MPTP selectively destroys the substantia nigra in monkeys, marmosets, and baboons and produces the cardinal signs of parkinsonism. These NHP models are unique because no other mammalian species shows a resting tremor after destruction of the substantia nigra. Because the complexity of the central motor pathways of the primate brain closely resembles that of humans, NHPs have played a critical role in the development of effective and safe therapeutic interventions to slow or halt disease progression. Researchers are also evaluating a number of novel intervention strategies in MPTP-treated primate models

Alzheimer's Disease Alzheimer's disease (AD) is an irreversible, progressive brain disorder that affects nearly 4 million persons in the United States and more than 20 million worldwide. Approximately 10% of persons over the age of 65 are diagnosed with AD, as are nearly 50% of those older than 85 years. There is a close correlation between the chief symptoms of AD and the cerebral accumulation of plaques containing beta-amyloid protein. This process, in turn, leads to a decrease in the production of acetylcholine, a neurotransmitter that mediates cellular communication and contributes to the storage and retrieval of memory. Since the early 1990s, extensive studies in macaque monkeys have laid the groundwork for identifying the critical regions of the brain that are essential for memory and cognition. Aging monkeys also develop beta-amyloid plaques with neuronal loss and corresponding cognitive deficits like those seen in humans. Monkeys can be trained to perform certain memory-related tasks that permit the evaluation of changes in cognitive memory and emotional behavior during aging. Various treatment and prevention strategies can then be tested in these animals for their potential to enhance behavioral performance. Cholinergic drugs are being tested to improve task performance in aged rhesus monkeys. An exciting new development is a possible vaccine for AD. Mice treated with a vaccine composed of a synthetic protein fragment from beta-amyloid plaques, called AN-1792, not only kept beta-amyloid from accumulating but also eliminated preexisting deposits in the brain. Scientists believe that removal of the plaques could relieve the symptoms of AD. The vaccine was well tolerated when tested in several animal species, including monkeys and humans.

Questions:
1. Which of the following diseases are chimpanzee not used as models?
a. Respiratory Syncytial Virus
b. Human Immunodeficiency Virus
c. Hepatitis C Virus
d. Parkinson's Disease
2. Which NHP species is recommended by the World Health Organization for testing vaccines for Malaria?
a. Chimpanzee
b. Macaque
c. Owl Monkey
3. Which NHP species is used for Bronchopulmonary Dysplasia and Respiratory Distress Syndrome studies?
a. Baboon
b. Squirrel Monkey
c. Macaque

Answers:
1) d 2) c 3) a

Perspectives on Hepatitis B Studies with Chimpanzees. ILAR 42 (2): 085.
Both humans and chimpanzees originating in Africa have a high rate of positivity to the hepatitis B surface antigen
(HbsAg). De novo infections of chimpanzees by hepatitis B virus (HBV) have also been documented. Both chimpanzee positive
plasma and human HBV inocula can cause antigenemia and seroconversion in inoculated chimpanzees. The chimpanzee is thus an
established and useful animal model for HBV.
Chimpanzees have been used to characterize the biological properties of HBV. Previous studies have titrated known HBV
subtypes in chimpanzees and have determined that infectivity patterns resemble those seen in humans. Such studies are important
precursors for vaccine development and evaluation in the chimpanzee model. Two infection types that occur in both humans and
chimpanzees have been characterized - the acute self-limited form and the chronic carrier state. Hepatitis C infection can markedly
inhibit HBV infection in chimpanzees. An inverse relation between the titer of HBV inoculation and incubation periods before the
appearance of HbsAg has been demonstrated. This type of information is important when handling pooled plasma products for various
purposes.
Many vaccine studies have been done in the chimpanzee HBV model. Such studies help provide background for dose selection and
vaccination schedules to be used in subsequent clinical trials. Because no in vitro assay was available for detection of infectious HBV,
chimpanzee safety tests were the only way to ensure that lapses of Good Manufacturing Practice (GMP) did not result in contaminated
batches of plasma-derived HBV vaccines. Some plasma-derived vaccines contain the hepatitis B e antigen (HbeAg). This epitope's
importance is still under investigation. Recently, the use of "naked" DNA vaccines have been used as a new approach to immunization.
Such vaccines induce cell-mediated immunity and are attractive as approaches to multivalent immunogens. Immunizing strategies with
DNA-based vaccines are still under development.
The Biotest Company was the first manufacturer of human plasma derivatives to recognize the high-level risk of transmission of
hepatitis viruses via blood products. Various strategies for virus inactivation without loss of biological activity of the therapeutic blood
proteins have been evaluated in the chimpanzee model for human hepatitis infections. An example of one such strategy is "cold
sterilization" of a factor IX preparation by B-propiolactone and ultraviolet radiation. Other strategies involve various solvent-detergent
combinations (i.e. Tween 80 and ether, tri(n-butyl) phosphate and sodium cholate) for inactivation of blood-borne viruses.
HBV infection of captive chimpanzees are acquired in the wild and from humans. Recent PCR techniques have provided a sensitive
and accurate way to genotype HBV isolates. Human HBV strains fall into at least six genotypes. There is also a unique nonhuman
HBV genotype; a distinct chimpanzee HBV has co-evolved with chimpanzees.
The first large-scale use of chimpanzees in biomedical research was for HBV research. Many chimpanzees were initially exported
from West Africa. Wild chimpanzees are now endangered due to the expansion of logging, large-scale hunting, and agriculture of their
forest habitats. Since the mid-1980s, the US Fish and Wildlife Service and CITES has prohibited importation of wild caught
chimpanzees. Animals currently used for biomedical research must therefore be obtained from existing colonies or by breeding. In
1981, NIH sponsored a meeting on animal use alternatives in biomedical research, with chimpanzee use being one of the topics
addressed. A "Chimpanzee Bill of Rights" was proposed to address ethical issue with emphasis on the following points: 1) chimpanzees
cannot be captured in the wild; 2) chimpanzees must be maintained under humane conditions that satisfy their special needs; 3)
institutions performing chimpanzee research are responsible for the lifetime care of these animals under semi-free ranging sanctuary
conditions, if at all possible.

Questions:
1. What does CITES stand for? Name the different classifications for animals covered by CITES and what types of permits are required
for shipment of these different classes of animals.
2. What US agency enforces and interprets CITES for the US?
3. Which of the following types of studies are covered by Good Manufacturing Practice (GMP) regulations?
A. Safety studies in animal such as mean lethal dose (LD50).
B. Studies relating to stability, identity, strength, quality, and purity of test and/or control articles.
C. Studies of safety of regulated products on target animals.
D. Acute toxicity studies on a final product formulation.
E. Studies of a test article that are completed in 14 days or less.
4. What are the components of a primate environmental enhancement program according to the Animal Welfare Act?
5. What are the special considerations for primate environmental enhancement according to the Animal Welfare Act?
6. Which of the following statements is FALSE?
A. Hepatitis C infection can markedly inhibit HBV infection in chimpanzees.
B. The chimpanzee is an established and useful animal model for HBV.
C. Wild chimpanzees are an endangered species.
D. African chimpanzees have a low rate of positivity to the hepatitis B surface antigen (HbsAg).
E. There are at least 6 human HBV genotypes and one unique nonhuman primate HBV genotype.

Answers:
1. CITES - Convention on International Trade in Endangered Species of Wild Fauna and Flora. Over 150 party nations as of 2000
agree to abide by CITES. CITES is not law, but all signatories agree to abide by CITES. Appendix I: Involves species presently
threatened with extinction, which are or maybe affected by trade. All animal shipments require two permits - one import permit and one
export permit. Appendix II: Involves species not presently threatened with extinction but may become so unless their trade is regulated.
Animal shipments require an export permit from the country of origin. Appendix III: Involves species that do not fall under Appendix I or
II but are regulated for conservation purposes by a party nation. Animal shipments require an export permit from the country that listed the
species. Most NHPs fall into Appendix I or II.
2. Federal Wildlife Permit Office (FWPO), U.S. Department of Interior (DOI) acts as the U.S. Management Authority for CITES.
3. B is covered by GMP regulations. A and C-E are covered by Good Laboratory Practices (GLP) regulations.
4. Environmental Enrichment "GEES CRDE"
[Note: Developed in honor of Dr. Martin Kriete, NEI/NIH; DACLAM 1995]
Social Grouping
Environmental Enrichment
Special Considerations
Restraint Devices
Exemption
5. Groups of NHP's Requiring Special Considerations "Get A GRIIP"
Great Apes >110 lbs.
Restricted Activity
Infants/Juveniles
Individually Housed Without See/Hear Other NHP
Psychologically distressed (via behavior or appearance)
6. D. Both humans and chimpanzees originating in Africa have a high rate of positivity to the hepatitis B surface antigen (HbsAg).

The woodchuck model of hepatitis B virus infection. ILAR 42 (2): 089.
The woodchuck hepatitis virus (WHV) was the first of the mammalian and avian hepadnaviruses described after discovery of the virus of hepatitis B (HBV). Woodchucks chronically infected with WHV develop progressively severe hepatitis and hepatocellular carcinoma, which present as lesions that are remarkably similar to those associated with HBV infection in humans. The initial virological studies and studies of pathogenesis utilized woodchucks that had been trapped in the wild and had acquired WHV infection naturally. Research with wild woodchucks was complicated by lack of knowledge of their backgrounds (e.g. dietary history, exposure to parasites or environmental toxins, and source and duration of WHV infection). Breeding colonies of woodchucks have been established and maintained in laboratory animal facilities, and laboratory-reared woodchucks are superior for experimental studies of pathogenesis or hepatocarcinogenesis. It is possible to infect neonatal woodchucks born in the laboratory with standardized inocula and produce a high rate of chronic WHV carriers that are useful for controlled investigations. WHV has been shown experimentally to cause hepatocellular carcinoma, supporting conclusions based on epidemiological and molecular virological studies that HBV is an important etiological factor in human hepatocarcinogenesis. Chronic WHV carrier woodchucks have become a valuable animal model for the preclinical evaluation of antiviral therapy for HBV infection, providing useful pharmacokinetic and pharmacodynamic results in a relevant animal disease model. It also has been shown that the pattern of toxicity and hepatic injury observed in woodchucks treated with certain fluorinated pyrimidines is remarkably similar to that observed in humans that were treated with the same drugs, suggesting the woodchuck has significant potential for the preclinical assessment of antiviral drug toxicity.
======================
Notes: Chronic hepatitis B (HBV) carriers develop hepatitis that often progresses to cirrhosis, and HBV has been convincingly linked to the development of hepatocellular carcinoma. Four members of the Sciuridae family have naturally-occurring infections with hepadnaviruses (family Hepadnaviridae, genus Orthohepadnavirus), first described in woodchucks (Marmota monax) in 1978. The virus is an enveloped DNA virus measuring 42-45 nm in diameter.
In the woodchuck infected with WHV, the tumors are well-differentiated hepatocellular carcinomas, with hyperchromatic nuclei and prominent nucleoli. Metastasis outside the liver has not been reported in the woodchuck, in contrast to humans and some other animal models. Mean survival time is 29 months in infected woodchucks, and almost all develop hepatocellular carcinoma.
Other related Orthohepadnaviruses include (1) California ground squirrel hepatitis virus (Spermophilus beecheyi), (2) Arctic ground squirrel hepatitis virus (Spermophilus parryi), (3) and woolly monkey hepatitis B virus. In birds, genus Avihepadnavirus includes (1)duck hepatitis B virus (Anas domesticus), (2) heron hepatitis B virus (Ardea cineria), and (3) snow goose hepatitis B virus (Anser caerulescens).
In California ground squirrels, persistent viral infection is associated with chronic hepatitis and hepatocellular carcinoma, although tumor frequency is very low. Woodchucks can be experimentally infected with this virus. Domestic Pekin ducks worldwide are infected with the duck hepatitis B virus, and much about the virus' replication was discovered using this model system. Duck HBV does not seem to cause tumors.
Problems with wild-caught woodchucks included confusion of the liver lesions with parasitic lesions, particularly those caused by Ackertia marmotae and Capillaria sp. Cornell University has maintained a breeding colony since 1979. They have discovered that most woodchucks are able to clear infection and fail to become chronic carriers. This is similar to HBV in humans, in which <5% become chronic carriers. Immunosuppressing the woodchucks with cyclosporin A increases the rate of chronic carriers. It is theorized that very early infection, or perhaps vertical transmission, is needed for woodchucks to become carriers.
Unlike the rat or human situation, there is no sex predilection (males get more tumors) with woodchucks. This may be because for 8 months of the year, the testicles of male woodchucks are in an abdominal location and produce little or no testosterone (this is circannual reproduction).
There are two theories of how hepadnaviruses cause tumors. First, they may play a direct molecular role, for example by integration of DNA sequences into the host cell DNA, causing altered expression of genes regulating the cell cycle. Viral DNA is found in most tumors in HBV-infected patients and in WHV-infected woodchucks. However, mammalian hepadnaviruses do not contain oncogenes like those found in retroviruses, and up-regulation of the common proto-oncogenes does not occur. There appears to be synergy between activation of a gene called N-myc2 (which has no known physiologic function in woodchucks) and diminished expression of p53,a tumor suppressor. This work was done in mice that were transgenic for N-myc2 crossed with p53 null mice, which had markedly accelerated development of liver cancer. In ground squirrels, the gene of interest appears to be C-myc.
The second theory is that hepatic injury caused by viral infection provides an environment that enhances fixation of spontaneous mutations, rearrangements, or chromosomal translocations responsible for malignant transformation of hepatocytes. Chronic liver injury associated with cirrhosis frequently precedes development of tumors in HBV patients.
There may also be interaction between hepadnaviruses and aflatoxin. HBV transgenic mice are more susceptible to chemical carcinogens than are controls.
Finally, the woodchuck is a model for testing of antiviral drugs, such as nucleoside analogs and immune response modifiers. Acyclovir and azidothymidine have no antiviral effect in woodchucks, but lamivudine works well both in vitro and in woodchuck.

Questions:
1. Which of the following species does NOT develop hepatocellular carcinoma when infected with hepadnavirus?
a. Marmota monax
b. Spermophilus beechyi
c. Anas domesticus
d. Lagothrix spp.
2. Treatment with which drug significantly enhances tumor development in woodchuck hepatitis virus-infected woodchucks?
a. zidovudine
b. cyclosporin A
c. acyclovir
d. aspirin
3. Which of the following best explains the mechanism of hepatocellular carcinoma development in woodchucks?
a. synergy in multistage hepatocarcinogenesis between activation of N-myc2 and diminished expression of p53
b. linkage between woodchuck hepatitis virus infection and c-myc activation
c. synergy between aflatoxin B1 and woodchuck hepatitis virus infection early in life
d. synergy between exposure to chemical carcinogens and overexpression of HbsAg

Answers:
1. c
2. b
3. a

Animal Models of Hepatitis Delta Virus Infection and Disease. ILAR 42 (2): 103.
Hepatitis delta virus (HDV) is a defective, circular, negative-stranded RNA virus similar to unusual subviral plant pathogens. HDV requires as a "helper virus" hepatitis B virus (HBV) for its replication/transmission, and HBV-infected humans are the only established host. HDV's lipoprotein coat consists of surface antigens (HbsAg) contributed by HBV. The HDV genome is the smallest of the known human pathogens. HDV is taxonomically the sole member of the Deltavirus genus. Both the RNA genome and antigenome of HDV contain ribozymes that cleave the RNA at specific sites. These ribozymes are essential to the double rolling circle mechanism of HDV replication. Unlike other negative-stranded viruses, HDV does not encode a polymerase, and there is no known RNA-dependent RNA polymerase in humans, the only known natural host. It is thought that human host DNA-dependent RNA polymerase (Pol II) is used to recognize the viral RNA template. There is also a hepatitis delta antigen (HDAg) consisting of two forms, a small protein (HDAg-S) and a large protein (HDAg-L). HDAg-S is required for HDV RNA replication, and HDAg-L, although it inhibits RNA synthesis, is necessary for virion formation. HDV uses host RNA editing activities to manage the conflicting function of these two HDAg forms.
Due to the dependence of HDV on HBV, a hepadenovirus, the host range of HDV is limited to those species that can also support hepadenovirus replication. Relevant animal models are the chimpanzee and eastern woodchuck, natural hosts of human HBV and woodchuck HBV (WHV), respectively. HDV is difficult to study in human infection due to the short period of peak viremia in acute infection and the variable levels of viremia in chronic infection. Frequent plasmaphereses during peak viremia in the chimpanzee model provided enough source material to allow biophysical and biochemical characterization of HDV. Source material from the chimpanzee model also permitted development of serological and hybridization-based assays for the detection of HDV RNA, HDAg, and anti-HD in clinical samples. Both coinfection, the simultaneous infection of an individual with HBV and HDV, and superinfection, HDV infection of an HbsAg-positive individual, have been documented in chimpanzees. Coinfection results in moderately severe hepatitis with either unimodal or bimodal elevation of serum alanine aminotransferase activity (serum marker of liver damage). In unimodal disease, both hepatitis B core antigen and HDAg are expressed in infected hepatocytes at the same time, whereas in bimodal disease, one or the other is expressed in the second episode. Superinfection results in more severe disease and HBV markers are suppressed during the acute HDV phase. Serial passage of HDV in chimpanzees results in increasingly more severe acute hepatitis. Although many features of human HDV infection are reproduced in this model, chimpanzees usually do not develop the more progressive disease pattern seen in HDV worldwide. It is not known whether HBV and HDV genotype interactions can influence disease course. HBV-infected chimpanzees have also been used to assess the safety of blood products.
Although there is no evidence that HDV infection occurs in natural WHV infection of woodchucks, human origin HDV can be transmitted to WHV-carrier woodchucks and can cause acute or chronic hepatitis in these animals. The liver is the only organ involved despite WHV replication in extrahepatic tissues. In this model, HDV might have the capacity to be latent for a short period and be expressed if the helper hepadenovirus arrives subsequently. This observation is also true in the chimpanzee model but its relevance to human clinical infection is unclear. The WHV/woodchuck model has been used for the development of immunization strategies against HDV. There is some evidence that immunogens based on HDAg may modulate experimental infection. The model has had limited use in drug development of chronic HDV infection due to the rapid progression of endstage disease in experimentally derived WHV carriers. Current research uses WHV strains in which disease progression is slower than the standard WHV inoculum.

Questions:
1. Give the genus of HDV and the genus and species of the chimpanzee and the woodchuck.
2. Name two rodent models for Hepatitis B virus. Which one is also associated with hepatic carcinoma?
3. Which of the following statements are FALSE with respect to HDV infections?
A. HDV is a defective, circular, negative-stranded RNA.
B. HDV genome is the smallest of the known human pathogens.
C. It is thought that human host RNA-dependent RNA polymerase is used to recognize the viral RNA template.
D. Relevant animal models of HDV infection include the chimpanzee and eastern woodchuck.
E. Chimpanzees usually do not develop the more progressive disease pattern seen in HDV worldwide.

Answers:
1. HDV - Deltavirus genus; Chimpanzee - Pan troglodytes; Woodchuck - Marmota monax.
2. Woodchuck, California Ground Squirrel (Spermophilus beecheyi). Woodchuck develops hepatocellular carcinomas.
3. C. It is thought that human host DNA-dependent RNA polymerase (Pol II) is used to recognize the viral RNA template.

Molecular clones of hepatitis C virus: applications to animal models. ILAR 42 (2): 139.
This article summarizes the study of Hepatitis C (HCV) and the ability to increase the types of animal models through the use of molecular clones of Hepatitis C genes inserted into model systems. Hepatitis C is a chronic infection affecting approx. 3% of the world's population. It can not be propagated in cell cultures. The chimpanzee is the only nonhuman primate proven susceptible to HCV, and thus has been the primary model for study of the pathogenesis and management of this disease. From this model, the HCV genome has been characterized and cloning of the genome has allowed the development of transgenic mice models for the study of HCV pathogenesis. By studying expression of specific viral protein products in mice, the structure-function or specific genome sequences can be evaluated. For over 20 years, the chimpanzee has been the model used for research on HCV, however these animals are rare, expensive, and difficult to house. HCV infection can not be propagated in mouse tissues, however transgenic mice containing cloned HCV sequences are useful in understanding the mechanisms of HCV pathogenesis. The HCV genome, first cloned in 1989, is composed of a positive-sense single-stranded RNA molecule. Six major HCV genotypes have been identified in a single genus in the family Flaviviridae. The structure is diagramed on page 140. For replication, an anti-sense RNA intermediate is developed and serves as the template for synthesis of genomic RNA. The genetic variability of HCV, in particular the hypervariable region I of the E2 envelope glycoprotein, facilitates chronic infection by hindering a protective antibody response. Current treatments are limited to interferon-based therapies and ribivarin, which help <25% of patients. Development of infectious molecular clones of HCV may allow development of in vitro culture systems for HCV. For example, a subgenomic HCV replicon has been grown in human liver cells. The positive-strand HCV RNA serves as the mRNA for synthesis of the HCV polyprotein. Several studies have shown that molecular clones can be infectious in the chimpanzee model. HCV genotype I appears to cause more severe disease than other types. Also, the composition and length of the 3' UTR may affect viral replication in vivo. Studies of various clones have shown a spectrum from silent infection to active disease and hepatitis. There is extensive discussion of the various genotypes of HCV and the roles of different polyproteins in replication of the virus and production of disease. HCV clones have been shown to not only infect chimpanzees, but to produce new infectious virions. Thus, clones can be used to study infectivity and disease severity. Additional studies of HCV mutations will also add to knowledge of the mechanisms of HCV persistence. Alternative nonhuman primate models include the tree shrew (Tupaia belangeri chinensis), approx. 1/3 of animals receiving a pooled virus source became viremic and sero-converted to anti-HCV positive, there were transient increases in liver enzymes such as ALT levels. The tree shrew may represent an alternative model after studies of the pathogenesis of disease in this species are completed. The chimp model usually does not develop chronic disease to the extent seen in humans. Transgenic mice have been developed that express HCV proteins from tissue-specific promoters and have proven to be useful models for characterizing pathogenesis due to HCV protein expression. The Cre/loxP system is used to alter gene expression in the liver of transgenic mice. Mice have been developed that express low levels of complete HCV polyprotein under transcriptional control of the albumin promoter. Other mice express HCV structural proteins only. Mice can be used to study development of steatosis, a common finding in human chronic HCV infection. Studies have shown that expression of HCV structural proteins may augment the apoptosis signal transduction pathway in hepatocytes. In vitro studies have shown that HCV core protein can serve as a modulator of gene expression from a number of cellular promoters and may be a co-factor in development of hepatocellular carcinoma. HCV structural protein expression has been demonstrated in lung, intestine, kidney, testes, heart, thymus, spleen, and salivary glands in addition to liver tissue. E1/E2 envelope protein expression produced pathology of lacrimal and salivary glands similar to Sjogren's Syndrome which has also been associated with human HCV infections. Use of new models and molecular clones of the full viral polyprotein or sub-products will be useful in the future for study of HCV including pathogenesis, immune responses of the host, pathogenicity of the various genomic varieties of HCV, etc. The ability to use transgenic mice and even cell cultures for some of this work will decrease the need for nonhuman primate models. Use of mice is expected to play a pivotal role in understanding the molecular mechanisms of HCV-related liver pathology. The use of alternative nonhuman primate models such as the tree shrew in place of the chimpanzee may also make research on this disease more practical than in the past.

Questions:
1. Which of the following is the primary animal model for HCV studies?
A. Mouse
B. Tree Shrew
C. Chimpanzee
D. Woodchuck
2. What discovery has allowed the development of new HCV models?
A. The genome of the virus
B. Ability to clone the virus or parts of it
C. Transgenic technology
D. All of the above.
3. Name the family containing Hepatitis C Virus.
A. Herpesviridae
B. Flaviviridae
C. Parvoviridae
D. Hepatoviridae
4. Which of the following are now being considered as models for HCV?
A. Transgenic mice
B. Squirrels
C. Tree Shrews
D. Squirrel monkeys
E. A and D
F. A and C
G. All of the above
5. HCV is what type of virus?
A. DNA
B. RNA negative-strand
C. RNA positive-strand

Answers:
1. C 2. D 3. B 4. F 5. C

GB virus B as a model for hepatitis C virus. ILAR 42 (2): 152.
The agents recognized as causing human viral hepatitis include hepatitis viruses A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV). For decades, nonhuman primates have served to propagate these viruses and to model the liver damage from hepatitis virus infection. Hepatitis viruses A through D replicate in the chimpanzee, which has served as the major nonhuman primate model for these viruses. GB viruses A and B (GBV-A and GBV-B) are members of the Flaviviridae family and are isolated from tamarins injected with serum from a human hepatitis patient. Along with a related human virus, GB virus C, or alternatively, hepatitis G virus (GBV-C/HGV), the three viruses represent the GB agents. Of the three viruses, GBV-B has been proposed as a potential surrogate model for the study of hepatitis C virus (HCV) infections of humans because the relatedness of the two viruses as well as the similar tropism for the liver supports the use of GBV-B as a model for HCV.
In the 1960s and 1970s, Deinhardt and coworkers injected marmosets (Saguinus) and tamarins (Callithrix) species, with sera from patients with hepatitis in an attempt to pass the human hepatitis viruses to these small (approximately 0.5 kg) New World primates. The viral agents, termed GB (a patient whose initials are G.B.) virus A (GBV-A) and GB virus B (GBV-B), showed similarity to the Flaviviridae family. GBV-B is phylogenetically most closely related to HCV and causes an acute, self-resolving hepatitis in tamarins as indicated by an increase in alanine aminotransferase and changes in liver histology. The shared protease substrate specificities suggest that GBV-B may be useful in testing antiviral compounds for activity against HCV. Although there are numerous similarities between GBV-B and HCV, there are important differences in that HCV frequently causes chronic infections in people, whereas GBV-B appears to cause only acute infections. The acute versus chronic course of infection may point to important differences between the two viruses that, along with the numerous similarities, will make GBV-B in tamarins a good surrogate model for HCV.
GBV-A appears to be a common primate virus, with viral genomes having been identified in Saguinus, Callithrix, and Aotus species (owl monkey). GBV-A causes chronic infections although it is believed not to cause hepatitis in tamarins by itself. The second GB agent identified, GBV-B, is clearly distinct from HCV but phylogenetically is most closely related to HCV and GBV-B alone is sufficient to cause hepatitis. GBV-A and GBV-B are considered tamarin viruses based on their restricted host range. However, the lack of additional isolations of GBV-B from tamarins in the wild raises questions as to whether tamarins are the natural host for GBV-B. Two other GB agents, termed GBV-C, and hepatitis G virus (HGV) are now often termed GBV-C/HGV. Recently, a similar virus has been identified in chimpanzees. Interest in GBV-C/HGV is great because this human virus has been found in 1 to 2% of the US general population using GBV-C/HGV-specific primers and is known to cause persist-tent infections. With its broad distribution in the human population, it is unlikely that GBV-C/HGV causes significant levels of severe disease.
Although similarities between GBV-B and HCV are impressive, there are also some important differences including the propensity for HCV to induce chronic infections. The percentage of people developing chronic HCV after infection has been estimated to be as high as 85%. However, recent chimpanzee studies showing less than 40% persistence as well as a human study showing less than 50% persistence suggest that the 85% persistence rate for humans may be an overestimate due to higher infection and viral clearance rates than previously appreciated. GBV-A and GBV-C/HGV, but apparently not GBV-B, cause chronic infections. Chimpanzees that have cleared an HCV infection can then be rechallenged with a homologous challenge inoculum of limited variability to determine the protection afforded to chimpanzees after clearance of the initial HCV infection.
Hepatitis viruses are notoriously difficult to grow in cultured cells. Although the availability of nonhuman primate models of hepatitis diseases is of great value, the lack of suitable tissue culture system has lagged because hepatocytes frequently undergo rapid dedifferentiation upon isolation and plating. The development of such tissue culture systems would reduce the need for viral hepatitis studies in nonhuman primates and help preserve these valuable animals. The ability to infect primary hepatocytes isolated from naive tamarins provides the basis of a neutralization assay. Since their identification in 1995, rapid progress has been made on the analysis of the GB agents, including determination that GBV-B is the causative agent of GB-associated hepatitis in tamarins. Although progress has been impressive, there are still many questions concerning GBV-B and its relation to HCV.

Questions:
1) Name the 3 major nonhuman primate species used for hepatitis research?
2) Which hepatitis agent is the most closely related to the Hepatitis C virus?
a) Hepatitis B Virus
b) GB virus B
c) GB virus A
3) Which of the following viruses ONLY causes an acute hepatitis?
a) HCV
b) HBV
c) GB virus A
d) GB virus B
4) What percentage of humans infected with HCV become chronic carriers?
a) 85%
b) 50%
c) 40%
5) True or False? Hepatitis G virus (GB-C) has been found in 1-2% of the U.S. population?

Answers:
1) Chimpanzee, Tamarin, Marmoset
2) B
3) D
4) A
5) True

Animal models of hepatitis A and E. ILAR 42 (2): 161.
The best models (at this time) for hepatitis A include chimps, tamarins (Saguinus mystax, S. labiatus), and owl monkeys and for hepatitis E include macaques (cynos and rhesus), chimps, owl monkeys, and swine.
Hepatitis A and E are both acute, self-limiting diseases spread by non-enveloped viruses, fecal-orally. This is as opposed to C and D which have chronic infections, enveloped viruses, and blood transmission. Heptitis A (HAV) is a picornavirus, passed through undercooked shellfish or occult contamination with feces. Also called "campaign jaundice." Hepatitis E (HEV) was once considered a calicivirus but is now unclassified. It has been reported to be transmitted via water.
Approximately 80% of the world's population has been exposed to HAV and approximately 30% have been exposed to HEV.
Course of HAV infection in animal model: Oral administration is followed by viral detection in the liver. Soon afterward, virus is found in the blood, bile, and feces. Onset of clinical disease (usually mild) signals diminished relication of the virus and first detection of the humoral immune response. Shedding can last 2-3 weeks, but risk to conspecifics is minimal after the peak of disease (in first week). Histologically see hepatic lesions similar to those seen in other hepatitis infections: focal necrosis, Kupffer cell proliferation and ballooning degeneration, and apoptosis of hepatocytes. See more necrosis and mononuclear cell inflammation of the periportal tract in HAV.
Transmission of HEV has been confirmed in rats in regions where HEV has a high prevalence. Many rats have HEV antibody, but isolation of actual virus has not been successful. This is an intriguing finding because rats may serve as a reservoir of infection for man.
Clinical course of HEV infection: After exposure, regardless of route, seen evidence of infection in the liver. Soon after, see virus in blood, feces and bile. Peak shedding occurs prior to onset of clinical disease. Long incubation period (but time frame not given). Histology similar to HAV, with formation of "pseudogland" in the hepatocyte plates.
The animal models of both diseases have allowed the development of candidate vaccines to help prevent infection in humans.

Questions:
1. Which of the following are animal models for HAV?
a. Chimps
b. Rhesus macaques
c. Owl monkeys
d. Tamarins
e. All of the above
f. All except one (name which one if you choose this)
2. Which of the following are animal models for HEV?
a. Chimps
b. Rhesus macaques
c. Owl monkeys
d. Tamarins
e. All of the above
f. All except one (name which one if you choose this)
3. T/F HAV and HEV are responsible for chronic hepatitis in humans. 4. T/F HAV and HEV infections are very rare.

Answers:
1. f (rhesus)
2. e
3. False - acute, self-limiting infections
4. False - HAV has 80% incidence, HEV has 30% incidence.