Acute or chronic non-A, B, C, D, E hepatitis is being studied for biological, serological or molecular evidence of transmissible agents. Fulminant non-A to E hepatitis remains a diagnostic enigma and may represent one or more previously unrecognized diseases. We are attempting to discover the etiology of this disease. Evidence for the existence of an additional water-borne hepatitis virus has come from our seroepidemiologic studies in India, Egypt and Saudi Arabia. Hepatitis E virus may be emerging as a greater public health problem than previously thought. We are studying its epidemiology in developing and industrialized countries worldwide. Serologic evidence of infection of swine with hepatitis E virus (HEV) was obtained. A new and unique HEV strain was recovered from infected swine and characterized. It was shown to have a worldwide distribution. Seroepidemiological studies of swine handlers and matched blood donors have shown an excess of antibody to HEV in swine handlers, suggesting that the virus may be zoonotically spread. In addition, throughout FY 2009 and 2010, we have been evaluating the significance of antibody to HEV (anti-HEV) in domestic animals that are part of the human food chain, especially cattle, sheep and goats. Anti-HEV has been found in all of the species, although not to the extent that it is found in swine. Rarely, swine are an important zoonotic source of hepatitis E, especially in industrialized countries, but these don't account for the high prevalence of anti-HEV in Islamic and Jewish cultures. In collaboration with XJ Meng, we are determining the susceptibility of goats to infection with the recognized HEV strains and attempting to recover HEV-like agents from young goats that are being intensively monitored for serologic and molecular evidence of infection. Similar serologic evidence for infection of wild rats with HEV has also been obtained and the infecting agent has been identified. To date we have successfully transmitted the agent from rats trapped in Los Angeles to laboratory rats of the same species (Rattus norvegicus). However, transmission was difficult, suggesting that the virus replicates at low titer. In 2010, using PCR primers developed for detecting a new rat HEV in Germany, we were able to recover sequence from the Los Angeles strain of rat HEV that we had transmitted to other rats previously. The sequence was very similar to the sequence of rat HEV recovered in Germany. We have confirmed transmission of rat HEV to other rats and we have determined both the infectivity titer and the PCR titer of rat HEV in several clinical samples from infected rats, including feces, serum and liver tissue. We have documented that both the infectivity titer and the PCR titer of the virus is relatively low in these clinical materials, thus providing an explanation for difficulty in consistent transmission. We have also confirmed by PCR that human strains of HEV were not transmissible to laboratory rats and that the rat HEV was not transmissible to rhesus monkeys, a surrogate of man. The low virus titers detected in rats, the difficulty in transmitting the virus to rats and the inability to transmit the rat virus to a nonhuman primate species that is susceptible to infection by human and swine HEV strains leads us to conclude that the rat virus probably does not pose a threat of zoonotic infection to humans. Modern techniques of molecular biology have been used to discover new viruses in recent years. These are now being applied to sera from patients with transfusion-associated or community-acquired hepatitis in a search for new hepatitis viruses that may cause up to 2% of such hepatitis in the US and up to one-third of hepatitis in developing countries. In addition, a significant number of cases of chronic hepatitis, cirrhosis and liver cancer remain undiagnosed. In an attempt to increase the sensitivity of virus discovery, we are applying microarray technology to attempts to transmit new agents to chimpanzees, the only species other than man that is susceptible to all five recognized human hepatitis viruses. Preliminary results are promising. Similarly, approximately one half of nonbacterial gastroenteritis cases have no recognized etiology. In collaboration with the Epidemiology Section, LID, we are applying the same approaches to attempts to identify new gastroenteritis agents. In FY 2009, while evaluating the innate and adaptive immune responses of chimpanzees that had been experimentally infected with hepatitis E virus (HEV), we discovered an aberrant innate immune response in two chimpanzees that had been infected with HEV from an outbreak of hepatitis E in Pakistan. Based on extensive experience with innate and adaptive immune responses to all five recognized human hepatitis viruses in chimpanzees, we postulated that the response in these animals was to a second agent replicating in the liver. By attempting transmission from the second innate immune response episode to new chimpanzees, we demonstrated evidence for an infection not related to hepatitis E in the original infection. It has been associated with only minor biochemical evidence of hepatitis, but that is also true for HEV infection of chimpanzees. We plan to determine if this putative agent produces more severe infection on serial passage and whether it can be associated with hepatitis or other liver disease, such as liver cancer. Liver cancer is a common sequela of infection with three of the five recognized human hepatitis viruses. In 2010, we attempted to identify an etiologic agent in clinical materials from one of the chimpanzees with evidence of infection with a possible new hepatitis agent. Clinical samples were tested on the "virus Chip" being utilized at the time under the NCI contract with SAIC. Although, based on sequences detected, several viruses were found to be infecting the chimpanzee, none was found to be unique to the acute phase sample but not the pre-infection sample. Subsequently, both pre-infection and acute phase plasma samples from the chimpanzee were subjected to "next-generation" deep sequencing with 454 technology. Both sequences of known virus families (in some cases the same as those detected by the virus Chip) and unique sequences were detected. These are currently being subjected to in-depth analysis to determine if they include one or more previously unrecognized viruses. In 2011 we have continued to refine extraction and amplification techniques for the identification of new hepatitis viruses from primate-derived clinical materials, in anticipation of additional "next-generation" sequencing experiments. Known viruses of known titer have been used as controls to optimize procedures. In other collaborative studies, the epidemiology of emerging norovirus infections among children were studied in stored clinical samples obtained between 1975 and 1991. Evolutionary analysis revealed that norovirus genotype GII.3 viruses evolved at a rate comparable to that of other small RNA viruses and that these viruses evolve at a relatively steady state. Understanding the evolutionary dynamics of prevalent noroviruses is relevant to the development of effective prevention and control strategies.