key: cord-0857565-jznp3n1n authors: Bradley, Daniel W. title: The agents of non-A, non-B viral hepatitis date: 1985-04-30 journal: Journal of Virological Methods DOI: 10.1016/0166-0934(85)90047-3 sha: f73656630365b37ec358f10523d3e76f5d1c9c8b doc_id: 857565 cord_uid: jznp3n1n Abstract Recent studies have provided physicochemical and electron microscopic evidence for the existence of two distinct agents of posttransfusion non-A, non-B (NANB) hepatitis. One of these agents is chloroform-resistant and is not associated with the formation of unique ultrastructural structures in infected liver. The other agent is CHCl3-sensitive, induces the formation of characteristic hepatocyte cytoplasmic tubules, and interferes with concurrent HAV or HBV infection in experimentally inoculated chimpanzees. The tubule-forming agent (TFA) has also been shown to pass through an 80 nm capillary pore membrane filter, suggesting that it is a small enveloped (or lipid-containing) virus. The TFA can also be recovered from low titer (⩽ 105 infectious doses/ml) chronic-phase chimpanzee plasma by use of a multi-step purification procedure that assumes the agent is a small enveloped RNA virus with an approximate buoyant density of 1.24 g/cm3 and a sedimentation coefficient of 200–280 S. The apparent lack of nucleic acid homology between the NANB-TFA and HBV further suggests that the NANB-TFA is either Togavirus-like or belongs to another or as yet undefined class of RNA or DNA virus. agent is CHCI,-sensitive. induces the formatlon of characteristic hrpatocyte cytoplasmlc tubules. and interferes with concurrrnt HAV or HBV Infection in experimentally inoculated chimpanzees. The tubuleforming agent (TFA) has also been shown to pass through an X0 nm capillary porr membrane filter. suggesting that It is a small enveloped (or lipid-containing) wrus. The TFA can also br recovered from IOU titer ( 1 yr) may also demonstrate interference with HAV superinfection. This finding indicates that even presumed low-level replication of a NANB tubule-forming agent is sufficient to cause significant interference with at least one other hepatotrophic virus. Finally, we and others (Bradley, 1983b; Tsiquaye, 1983) have also shown that acute NANB-PTH also interferes with the replication of HBV in HBsAg carrier chimpanzees, as judged by the decrease in surface antigen titer and serum HBV DNA polymerase activity during the acute-phase of NANB disease. Coinfection of chimpanzees with HBV and the NANB-TFA has also been found to delay, moderate, or obviate the appearance of serologic markers of HBV infection (Brotman, 1983; G. Dolana, pers. comm.) . It is now clear that acute and persistent NANB-PTH infections are capable of interfering with at least two different hepatotropic viruses, however, the mechanisms or effecters responsible for this phenomenon have not been identified. One of the most unusual, and perhaps revealing, aspects of experimental NANB-PTH in chimpanzees is the finding of unique ultrastructural alterations in hepatocytes of acutely and persistently infected animals (Shimizu, 1979; Bradley, 1980 Bradley, , 1981 Bradley, , 1984 Pfeifer, 1980) . These changes are primarily confined to hepatocyte cytoplasm (Fig. 1) and include the formation of peculiar convoluted membranes (ERc), 150 to 300 nm diameter tubules comprised of double-unit membranes enclosing an as yet uncharacterized osmiophilic substance, and dense reticular inclusion bodies. Most of these structures appear to result from proliferated smooth endoplasmic reticulum (SER) in response to infection with the NANB-TFA (Fig. 2) . Bundles of granular microtubules may also be observed in some infected hepatocytes; these structures are dissimilar to the more commonly described smooth microtubules in that they appear to be comprised of 25 nm diameter stacked disks (Fig. 3) . The dense reticular inclusion bodies referred to above are fibrillar-granular masses of highly convoluted, densely stained materials that may, in fact, be comprised of microtubules coated with an amorphous, osmiophilic substance. Many of these structures contain multiple foci with radiating, strand-like attachments to the surrounding ER (Fig. 4) . These latter structures are somewhat similar to the viroplasmic foci, or virus factories, described for mouse hepatitis virus, an RNA-containing coronavirus (David-Ferreira and Manaker, 1965) or influenza virus (Compans and Choppin, 1973) . Although no convincing evidence for a virus-specific association of nucleic acid (RNA) with the NANB dense reticular inclusion bodies has been documented, it is worth noting that one group of investigators (Shimizu et al., pers. comm.) has found that EBV-transformed peripheral lymphocytes from a NANB-infected chimpanzee produce an IgM antibody that specifically binds to these structures. It is presently unclear whether this antibody is recognizing a virus-or host-specific antigen. (Bradley, 1984; X 18,850) . tis, yet none has been irrefutably linked to the transmission of disease. The putative virus-like particles (VLPs) were shown to range in diameter from 22 nm to 70 nm and include some that were morphologically similar to picornaviruses (Bradley, 1979; Yoshizawa, 1980) , hepadnaviruses (Hantz, 1980) , and Togaviruses (Coursaget, 1979) . More recent studies (Gerety et al., pers. comm.) further suggest that the NANB-TFA may be retrovirus-like based on the finding of an apparent specific association of reverse transcriptase activity with NANB infectivity. The CHCl,-sensitivity of the NANB-TFA (see below) and the documented propensity of acute disease to progress to chronic infection would appear to lend some support to the above assertion. Although no candidate agent of posttransfusion NANB hepatitis has been serologitally linked to the transmission of human disease, several properties of one or more virus-like agents can be inferred from the results of our recent animal transmission studies. The agent associated with the induction of hepatocyte cytoplasmic tubules is sensitive to CHCI, treatment and is most probably an enveloped or lipid-containing virus (Bradley, 1983a (Bradley, , 1984 Feinstone, 1983) . The NANB-TFA has also been shown to pass through an 80 nm sharp cut-off polycarbonate membrane filter (Bradley, in press) indicating that it is a relatively small, enveloped (or lipid-containing) virus. The NANB-TFA has recently been recovered from chronic-phase chimpanzee plasma of presumed low titer by use of an extensive purification procedure that assumes the agent is a small, enveloped RNA virus with a buoyant density of 1.24g/cm3 in CsCl and a sedimentation coefficient of at least 200 S (Bradley, in press ). Studies in our laboratory (Fields, 1983) and elsewhere (Fowler, 1983; Prince, 1982) rule against any similarity of the NANB-TFA to HBV, since nucleic acid hybridization assays, even under conditions of low stringency, have not revealed the presence of a significant sequence homology between these agents. Because HBV (a hepadnavirus) is the only currently known enveloped DNA virus that will pass an 80 nm filter, we have considered the possibility that the NANB-TFA is an enveloped RNA virus. Of the known enveloped RNA viruses, only those belonging to the Togavirus class will readily pass through an 80 nm membrane filter. Although our findings presently suggest the NANB-TFA is Togavirus-like, we cannot exclude the possibility that this agent belongs to another or as yet undefined class of RNA or DNA virus, such as a small retrovirus. We have also recently presented evidence for the existence of a second agent of NANB-hepatitis that is resistant to CHCl, treatment (Bradley, 1983a) . This agent can be recovered from either chronic phase plasma or acute-phase liver homogenates by a multistep procedure designed for the purification of picornaviruses. This second presumed viral agent of posttransfusion NANB hepatitis does not induce the formation of unique hepatocyte tubules and ERc, however, extensive hepatocyte cytoplasmic vesiculation and vacuolation have been observed in infected chimpanzee liver biopsy specimens during the acute phase of disease. The latter ultrastructural changes are similar in character to those found in cells infected by picornaviruses. These combined findings are in agreement with our previous recovery of 27 nm virus-like particles from a proven-infectious factor VIII concentrate (Bradley, 1979) and from the livers of chimpanzees in the first and second primate passage of the factor VIII-derived NANB hepatitis agent(s) (Maynard and Bradley, 1981) . Finally, both NANB hepatitis agents appear to be associated with persistent viremia, and presumably persistent hepatic disease, since chronic-phase plasma from a factor VIII-infected chimpanzee was found to contain both CHCl,-resistant and CHCl,-sensitive agents. The physicochemical and pathogenetic properties of one major agent of human PTH, the NANB-TFA, are summarized in Tables 1 and 2, respectively. Can be pelleted from plasma (assumes agent has an S,,, u of 2200 S) (5) Agent can be recovered from chronic-phase plasma bya'multi-step procedure used for the purification of small, enveloped RNA viruses (Bradley, 1984) probably relate to the low titer of circulating virus and relative deficiency of potent NANB IgG or IgM antibody. In regard to the former issue, most investigators have found that human or chimpanzee inocufa generally have titers between IO2 and IO4 chimpanzee infectious doses (CID) per ml. For example, our factor VIII agent has a titer of less than 10' CID/ml (Bradley, 1983a) ; the NANB 'F' strain characterized by Feinstone and co-workers (1981) (a chronic phase human plasma) has a titer of less than lo2 CID/ml; also, a fibrinogen preparation described by Yoshizawa and co-workers (1982) has a titer greater than lo2 but less than IO4 CID/ml; an acute phase 'H' strain agent, another plasma described by Feinstone, has a titer of lo6 CID/ml. Chronicphase plasma from one of our NANB carrier chimpanzees has recently been shown to have a titer of between lo4 and lo5 CID/ml (unpubl. findings) and to cause biochemical and electron microscopic evidence of disease within 4 wk after inoculation into a susceptible animal. Finally, Tabor and Gerety (pers. comm.) have found that one of their NANB inocula has a titer of approximately IO* CID/ml. Even if we assume that there are 100 times more defective than infective virions, only the specimen with lo6 CID/ml may contain detectable antigen, provided a high-titered antibody is used in an optimized immunoassay procedure. If chronic carriers of NANB hepatitis normally have circulating virus titers of less than lo5 ID/ml, and if excess or soluble virus-specific antigen is not present, then serologic tests of considerably greater sensitivity will be required for the successful detection of particulate viral antigen. What are the possible alternatives to the development of a conventional serologic test for NANB-PTH? Table 3 lists several newer approaches, many of which are antibody independent in nature or make use of a disease-related phenomenon, such as viral interference. One of the most promising techniques involves the possible detec- tion of disease-specific antibody produced by transformed lymphocytes (see above). If verified, this procedure could theoretically allow for the identification of NANB-infected blood donors by serologic analysis of in vitro transformed lymphocytes obtained from these individuals. Viral Hepatitis: A Contemporary Assessment of Etiology. Epidemiology, Pathogcnesis and Prevention Viral HepatItisand LiverDlsease Vtral Hepatitis: 1981 International Symposium Advances in Hepatitis Research. cd. Chisari. F Viral Hrpatitls: 2nd International Workshop. cds. Hopkins, R.and Fie1ds.S. (Nuclear EnterprIes Viral. I I Viral Hepatitis and Liver Disease. cds Ultrastructure of Animal Viruses and Bacterlophagrs: an Atlas Viral Hepatitis: 1981 International Symposium Viral Hepatitis: 1981 Intcrnatlonal Symposium