ACLAD NEWSLETTER Vol. 16, No. 2

American Committee on Laboratory Animal Diseases

Fall 1995

Editor: Stephen S. Morse, Ph.D.
The Rockefeller University 1230 York Avenue, Box 2
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e-mail: morse@rockvax.rockefeller.edu
For: Items for the Newsletter, general comments

Editorial Assistant: Joan Bailie
Section of Comparative Medicine
Yale University School of Medicine
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New Haven, CT 06510
Telephone: (203) 785-2507 FAX: (203) 785-7499
For: Changes of address; questions about mailing or dues =================================================
ACLAD ANNUAL BUSINESS MEETING (note day!): Sunday, October 15, 5-6 PM, Chesapeake A Room, Baltimore Hyatt Hotel ACLAD SCIENTIFIC PROGRAM: Tuesday, October 17, Baltimore, 8 A.M.--Noon Rm. 307, Convention Center Seminar: Rodent Parvovirus Infections WALLACE P. ROWE LECTURER: Donald E. Mosier, PhD, MD ======================================
CONTENTS IN THIS ISSUE OF THE NEWSLETTER:

Program for Annual Meeting

Benjamin J. Weigler: B Virus Transmission in Rhesus Monkeys

Lisa J. Ball-Goodrich, Elizabeth Johnson, Frank Paturzo, and Robert Jacoby: Molecular Studies of RV-UM and Rat Parvovirus-1 (RPV-1), a Member of a Newly Recognized Parvovirus Serogroup

Aron Lukacher: Susceptibility to Polyoma Virus-Induced Tumors Conferred by an Endogenous MMTV Superantigen

Robert J. Powell, Shaoguang Wu, Robert G. Tuskan, David W. Pascual, John Van Cott, Jerry McGhee, George K. Lewis, and David M. Hone: A Live Salmonella Vaccine Vector Expressing HIV-1 Antigens: Potential as an Oral HIV-1 T Cell Vaccine

Kazutaka Ohsawa, Yoji Watanabe, and Hiroshi Sato: Hantaan Virus Persistently Infects Marmoset B-Lymphoblastoid Cell Line

Acknowledgments & Announcements

NEXT ISSUE OF THE NEWSLETTER: SPRING 1996. PLEASE SEND CONTRIBUTIONS FOR THE NEXT ISSUE BY FEBRUARY 1, 1996. ========================================================== ("Page 3") ANNUAL BUSINESS MEETING AND ACLAD SCIENTIFIC PROGRAMS at AALAS, Baltimore, October 15 & 17 *** (Note: The Business Meeting is on Sunday evening, October 15) ACLAD ANNUAL BUSINESS MEETING Sunday, October 15 5-6 P.M. Chesapeake A Room Hyatt Hotel

ACLAD SCIENTIFIC PROGRAM Annual Seminar and Wallace P. Rowe Lecture Tuesday, October 17 8 A.M.--Noon Rm. 307, Convention Center

SEMINAR: RODENT PARVOVIRUSES 8-11 A.M. Moderators: Drs. Joseph E. Wagner and Kimberly S. Waggie. Dr. David G. Besselsen (Univ. of Missouri): The molecular and cellular biology of rodent parvoviruses, I. Dr. Lisa Ball-Goodrich (Yale): The molecular and cellular biology of rodent parvoviruses, II. Dr. Abigail L. Smith (Yale): The immunobiology of rodent parvovirus infections. Dr. Robert O. Jacoby (Yale): The pathobiology of rodent parvovirus infections. Dr. Lela K. Riley (Univ. of Missouri): The diagnosis of rodent parvovirus infections. 11 A.M.-NOON: WALLACE P. ROWE LECTURE Donald E. Mosier, PhD, MD Small Animal Models for AIDS TRAINEE PROGRAM: 12-2 P.M.: Lunch for speakers and trainees, Columbia/Frederick Room, Hyatt Hotel All Trainees are cordially invited to join the speakers for lunch and postprandial discussion.

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A REVIEW OF B VIRUS TRANSMISSION IN RHESUS MONKEYS

by Benjamin J. Weigler Laboratory Animal Resources
Department of Companion Animal & Special Species Medicine, College of Veterinary Medicine North Carolina State University, Raleigh, NC 27606

B virus (cercopithecine herpesvirus 1, Herpesvirus simiae) is a zoonotic agent of significant concern to individuals working with nonhuman primates or their tissues. Approximately 40 cases of B virus disease, 70% of which were fatal, have been confirmed in human beings since the virus was first described in 1933. Asian Old World monkeys of the genus Macaca are the natural hosts for B virus, and all macaques should be considered infected unless specifically documented otherwise. One recent report suggested that B virus disease is typically more severe in rhesus than in cynomolgus monkeys, and field strains of the agent from these two species have been differentiated through restriction endonuclease analysis. Early reports describing the clinical findings associated with B virus epizootics in captive colonies of cynomolgus appear to contradict this latter hypothesis, however. Experimental studies and rare instances of inadvertent natural cross-species transmission of B virus have also demonstrated the agent's potential to replicate and sometimes cause disease in other primates as well, including cebus monkeys, marmosets, DeBrazza's monkeys, African green monkeys, patas, and colobus monkeys, although the diagnostic testing was incomplete in some reports.

The biology of B virus parallels that of the herpes simplex viruses (HSV) of human beings in many ways. Both agents are capable of establishing lifelong latent infections in the trigeminal and lumbosacral sensory ganglia following initial virus entry and replication in oral-facial and genital epithelial tissues, respectively. Factors which promote reactivation from latency and subsequent re-shedding of B virus from epithelial sites near the portal of entry (i.e., recurrent infections) remain uncertain to date, but evidence from the related HSVs suggest that stress, peripheral tissue damage, immunosuppression, and certain drugs can play important roles. In the majority of cases, seroconversion follows B virus exposure in macaques by approximately 1-2 weeks, and high titers of humoral antibodies usually persist indefinitely, at least in immunocompetent monkeys. Recent advances in the diagnostic technology for B virus, including well-established standardized serological assays, genomic and polypeptide characterization of B virus isolates in tissue culture, and polymerase chain reaction (PCR) technology for detection of B virus genomic DNA, have allowed for epidemiologic studies and individual case investigations that now greatly minimize the potential for misdiagnosing human beings and macaques.

Other previous problems in early attempts to assess B virus transmission have been the sampling methods used, wherein populations have not been tested in a manner which enables unbiased generalizations of results to other similar groups of monkeys. In a recent study, use of a systematic sampling scheme along with an ELISA antibody assay in a cross-sectional study design for three outdoor breeding corrals of rhesus monkeys (n=146) allowed for unbiased population-based inferences regarding the importance of age as a risk factor for B virus infection. Monkeys younger than 2.5 years old (approximately the age of puberty for that colony) were only 22% seropositive, compared to 97% for older monkeys in that retrospective assessment, while the prevalence of B virus positivity varied from 52% to 76% across corrals. Neither the sex nor social dominance ranking of monkeys tested were found to be important risk factors for infection.

Age has long been suspected as important to B virus epidemiology, in that older monkeys have consistently been antibody-positive more often than younger monkeys. It is important to recognize, however, that age is only a proxy variable for the types of behaviors and contacts that expose uninfected monkeys to the virus over time, so it has little direct value for improving our understanding of virus transmission. The observation that an age-related effect appeared to maximize around puberty, together with a relatively higher frequency of B virus isolations from genital tissues and their associated neuronal ganglia, has led some researchers to conclude that most B virus transmission was venereal. However, these conclusions were potentially confounded by other possible risk factors that routinely occur in conjunction with breeding activity, such as grooming and aggression in pair- or group-housed environments.

The hypothesis of venereal transmission formed the basis of a recent 16-month prospective cohort study in a large breeding corral (n=157), in which different exposure variables were monitored in conjunction with repeated antibody testing (for seroconversions) and repeated epithelial swab samples for virus isolation from oral, conjunctival, and urogenital mucosa over time. A behavioral sub-study was included to provide season-averaged estimates of activity budgets for different behavioral exposure factors (grooming, play, aggression, sexual mounts) as monkeys aged, relative to B virus infection. The biologically meaningful outcome of time-to-infection in this study was evaluated through multivariable Cox proportional hazards regression analysis, which quantitatively assessed the instantaneous risk associated with individual exposure variables, while simultaneously adjusting for all other factors in the model. Cox regression is useful for prospective time-to-event studies, particularly when there may be losses to follow-up (censoring) and when some of the exposure factors change in magnitude over time (e.g., seasonal breeding behavior). The resulting epidemiologic measures of association from Cox regression models are hazard rate ratios (HRR), which have an interpretation similar to that of the more familiar relative risks.

In this analysis, the data indicated that cage mate aggression was the primary risk factor for B virus infection in the study cohort (HRR=4.7, 95% CI=1.9-11.5), followed by breeding (HRR=3.9, 95% CI=1.1-14.4). Thus, those monkeys that were bitten and scratched were nearly five times more likely to become infected during the same time period than were monkeys that were not attacked. A significant association, but of lower magnitude, also held for the breeding variable. These findings argued against a predominant role for venereal transmission of B virus in the study population. Like all epidemiologic research, whether the strength of these associations hold constant for all group-housed macaques can only be ascertained through similar evaluations of other populations.

Prospective designs allow for direct calculation of incidence rates, and the cohort study results indicated that the enclosure-wide cumulative incidence for B virus infection was approximately 15% and 10% during the first and second breeding seasons, respectively, or approximately three new cases per month on average overall. However, infectious B virus was directly isolated from only 14 of 5036 epithelial swab specimens obtained throughout the study, at least eight of which were shown to represent recurrent infections since they came from seropositive monkeys. The difficulty in isolating infectious virus from asymptomatically infected monkeys has been observed by others, and will hopefully be circumvented in part by new, more sensitive molecular approaches to virus diagnosis. Notably, only one of the 47 incident cases observed through repeated serological and virological diagnostic testing occurred in conjunction with outwardly apparent disease (unilateral conjunctivitis and small ulcer on upper lip), emphasizing the need for diligence in personal protective practices when working with macaques.

A third study was designed to assess the possible differential distribution of B virus DNA in head-region (trigeminal) versus genital-region (lumbosacral) ganglia of individual monkeys using a newly-developed polymerase chain reaction assay, in conjunction with epidemiologic predictors of infection. In this case, the design was a case-series of necropsy specimens (n=49), since neuronal tissues were only available for harvest during post-mortem examination. Terminal blood samples for B virus antibody testing via ELISA and Western blot were also obtained. Using antibody status as the outcome, and exposure factor information from computerized colony records, multivariable logistic regression was used to retrospectively assess different risk factors for infection in terms of meaningful odds ratios, while simultaneously adjusting for other variables in the model.

In this study, breeding history again emerged as a significant risk factor for infection (odds ratio=1.64, 95% CI=1.21-2.23), while the history of aggressive attacks was not recorded and could not be included in this investigation. Another useful epidemiologic measure, the population attributable risk (etiologic fraction), was determined to be 22.7%, based upon the odds ratio estimate for breeding along with the prevalence of breeding exposures in the study population. This implies that 77.3% of B virus transmission occurred via other portals of entry, and that less than one-fourth of all B virus cases in the study population would disappear if the venereal route were fully eliminated as a risk factor. These conclusions were drawn from serological test results in conjunction with epidemiologic assessment of different risk factors using pre-existing colony records. The PCR testing of the ganglia harvested at post-mortem examination provided similar evidence, in that 22.9% of the lumbosacral ganglia pools tested positive for B virus DNA, compared with 12.8% of the trigeminals. These concordant biologically independent assessments add great confidence to the conclusions regarding the secondary role of breeding in B virus transmission.

In summary, several epidemiologic studies have shown that the venereal portal of entry is important in B virus transmission, but that other routes (e.g., biting and scratching) predominate on a population-wide basis. This is particularly true in group-housed environments, where daily opportunities exist for virus exposure through several routes and behaviors. Pre-pubertal monkeys are susceptible to infection via these other routes, while the added risk of venereal transmission minimizes the likelihood of monkeys remaining uninfected through puberty, at least for B virus infected colonies that allow for normal monkey-to-monkey interactions. Male and female monkeys are apparently at equal risk of infection in group-housed facilities. Furthermore, despite its outward appearance for promoting breeding opportunities or aggressive interactions, social dominance rank did not predict risk of B virus infection in any analysis. Finally, it should be noted that human infection with B virus is also inherently multifactoral, with the sporadic nature of human case reports complicating efforts to quantitatively assess different risk elements of the causal pathway. Monkey-to-human transmission of the agent requires the presence of infectious titers of virus in monkey tissues or fluids that are presented in a manner which allows for virus replication in the human host, a set of circumstances which is generally unpredictable for any individual encounter. The number of such cases in past decades should warn individuals working with macaques to take precautionary measures at all times and to heed the advice of published guidelines in testing and follow-up of known or suspected B virus exposures.

References

1. B Virus Working Group. 1988. Guidelines for prevention of Herpesvirus simiae (B virus) infection in monkey handlers. J. Med. Primatol. 17:77-83.

2. Boulter, E.A. 1975. The isolation of monkey B virus (Herpesvirus simiae) from the trigeminal ganglia of a healthy seropositive rhesus monkey. J. Biol. Standard. 3:279-280.

3. Holmes, G.P., L.E. Chapman, J.A. Stewart, et al. 1995. Guidelines for the prevention and treatment of B-virus infections in exposed persons. Clin. Infect. Dis. 20:421-439.

4. Lees, D.N., A. Baskerville, L.M. Cropper, et al. 1991. Herpesvirus simiae (B virus) antibody response and virus shedding in experimental primary infection of cynomolgus monkeys. Lab. Anim. Sci. 41:360-364.

5. Palmer, A.E. 1987. B virus, Herpesvirus simiae: historical perspective. J. Med. Primatol. 16:99-130.

6. Weigler, B.J., J.A. Roberts, D.W. Hird, et al. 1990. A cross-sectional survey for B virus antibody in a colony of group housed rhesus macaques. Lab. Anim. Sci. 40:257-261.

7. Weigler, B.J. 1992. Biology of B virus in macaque and human hosts: a review. Clin. Infect. Dis. 14:555-567.

8. Weigler, B.J., D.W. Hird, J.K. Hilliard, et al. 1993. Epidemiology of cercopithecine herpesvirus 1 (B virus) infection and shedding in a large breeding cohort of rhesus macaques. J. Infect. Dis. 167:257-263.

9. Weigler, B.J., F. Scinicariello, and J.K. Hilliard. 1995. Risk of venereal B virus (cercopithecine herpesvirus 1) transmission in rhesus monkeys using molecular epidemiology. J. Infect. Dis. 171:1139-1143.

10. Whitley, R.J. 1990. Cercopithecine herpes virus 1 (B virus). In: Fields, B.N., and D.M. Knipe, eds. Fields virology, 2nd ed. Vol 2, pp. 2063-2075. New York: Raven Press.

11. Zwartouw, H.T., and E.A. Boulter. 1984. Excretion of B virus in monkeys and evidence of genital infection. Lab. Animals 18:65-70.

12. Zwartouw, H.T., J.A. MacArthur, E.A. Boulter, et al. 1984. Transmission of B virus infection between monkeys especially in relation to breeding colonies. Lab. Animals 18:125-130. ****************************************************
MOLECULAR STUDIES OF RV-UM AND RAT PARVOVIRUS-1 (RPV-1), A MEMBER OF A NEWLY RECOGNIZED PARVOVIRUS SEROGROUP

by Lisa J. Ball-Goodrich, Elizabeth Johnson, Frank Paturzo, and Robert Jacoby
Section of Comparative Medicine Yale University School of Medicine, New Haven, CT 06473

RPV-1 is a newly recognized virus isolated from a naturally infected rat colony. Sera from RPV-1 infected rats did not inhibit agglutination of erythrocytes by either H-1 virus or RV. Therefore, it is the prototype virus of a fifth rodent parvovirus serogroup, the other four being represented by RV, H-1 virus, MVM, and MPV. RPV-1 was compared with the UMass strain of RV (RV-UM) at the genomic level. Cleavage of the two viruses by restriction enzymes was compared by Southern blot analysis. Most restriction patterns, even those by enzymes which cleave in conserved regions of the genome, were different for the two viruses, and the RV probe used to detect the DNA hybridized poorly to the RPV-1 samples. We cloned the RV-UM and RPV-1 genomes, sequenced from nucleotides 1086-3373, and compared both the nucleotide sequence of and putative viral proteins for RV-UM and RPV-1. When nucleotide identity was compared for the entire region sequenced (NS and VP), RPV-1 and RV-UM are only 78.2% identical, a lower level of identity than is found between RV-UM and H-1 (88.3%), and similar to the identity between RV-UM and the mouse parvoviruses MVMi (80.2%) or MPV-1 (79.4%). These results explain the substantial differences in restriction enzyme cleavage between RPV-1 and RV, and the reduced hybridization of the RV probe to RPV-1 DNA. The nucleotide identity in the capsid-coding region of the genome was 74%, and the VP1 amino acid similarity and identity were 85% and 77.7%, respectively. These results confirm that RV-UM and RPV-1, biologically different agents in vivo, are also antigenically and genetically distinct viruses. (Presented at the International Parvovirus Workshop, Montpellier, France, September 1995) *************************************************************** SUSCEPTIBILITY TO POLYOMA VIRUS-INDUCED TUMORS IS CONFERRED BY AN ENDOGENOUS MOUSE MAMMARY TUMOR VIRUS (MMTV) SUPERANTIGEN

by Aron Lukacher
Department of Pathology Emory University School of Medicine, Atlanta, GA 30322

Polyoma virus can be a powerful oncogenic pathogen in the mouse, its natural host. When inoculated into newborn mice, this DNA virus is capable of inducing a large variety of tumors derived from epithelial and mesenchymal cell lineages (1). Multiple tumors typically develop in individual animals, some grossly evident as early as five weeks after infection.

Susceptibility to tumors induced by polyoma virus varies among inbred mouse strains. This variability is controlled by products of MHC as well as non-MHC genes (2). In crosses between MHC- nonidentical strains differing in tumor susceptibility, resistance correlates with dominant inheritance of the resistant H-2 haplotype. Surprisingly, the opposite pattern of inheritance of susceptibility is found in crosses between MHC-identical strains. In crosses between the highly susceptible C3H/BiDa mouse and the highly resistant but MHC-identical (H-2k) C57BR/cdJ mouse, polyoma tumor SUSCEPTIBILITY is inherited in an autosomal DOMINANT manner. Genetic analysis clearly demonstrates that this dominant susceptibility is conferred by a single gene from the C3H/BiDa parent (3). This gene, provisionally designated PyvS, does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody response, or affect intracellular events essential for productive infection or cell transformation by the virus (3). Whole body irradiation renders C57BR/cdJ mice fully susceptible to polyoma induced tumors, indicating an immunological basis for this strain's resistance. These findings raised the possibility that PyvS encodes a Mouse Mammary Tumor Virus (MMTV) superantigen (SAg) that confers susceptibility on C3H/BiDa mice by deleting precursors of polyoma-specific T cells.

Inbred mouse strains generally carry several MMTV proviruses (Mtv) in their germline. The 3'-LTR (long terminal repeat) of the MMTV genome contains an open reading frame encoding a type II transmembrane glycoprotein with superantigen activity. Unlike conventional peptide antigens, which occupy a specific groove in MHC proteins on the cell surface, SAg's find to class II MHC proteins outside this groove and engage the variable domain of the beta-chain (V-beta) of the T cell receptor (TCR). As endogenous proteins, Mtv SAg's cause intrathymic deletion of thymocytes expressing V-beta-reactive TCR's. Because T cell activation is required for the MMTV life cycle, deletion of SAg reactive T lymphocytes may represent an evolutionary strategy to protect against horizontal transmission of MMTV (4). By altering the peripheral T cell repertoire, however, Mtv SAg's incur the potential risk of eliminating T cells required for protection from unrelated infectious agents.

The possibility is confirmed by the following evidence that PyvS is the endogenous MMTV superantigen Mtv-7 (5). Tumor susceptibility in (C3H/BiDa x C57BR/cdJ) backcross mice cosegregates with the Mtv-7 provirus. Genotyping of backcross mice using markers of simple sequence repeat polymorphisms flanking Mtv-7 shows no evidence of recombination between PyvS and Mtv-7. Historically, all inbred mouse strains possessing high polyoma tumor susceptibility are H-2k and carry the Mtv-7 provirus; no other Mtv provirus correlates with susceptibility. Inheritance of Mtv-7 shows perfect concordance with absence of peripheral V-beta 6+ T cells. Furthermore, strongly biased usage of V-beta 6 by polyoma-specific CD8+ cytotoxic T lymphocytes in C57BR/cdJ mice implicates T cells bearing this Mtv-7 SAg-reactive V-beta domain as critical anti-polyoma tumor effector cells in vivo. These results indicate identity between PyvS and Mtv-7 SAg, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's T cell repertoire.

References

1. Dawe, C.J., R. Freund, G. Mandel, K. Ballmer-Hofer, D.A. Talmadge, and T.L. Benjamin. 1987. Variations in polyoma virus genotype in relation to tumor induction in mice: characterization of wild type strains with widely differing tumor profiles. Am. J. Pathol. 127:243-261.

2. Freund, R., T. Dubensky, R. Bronson, A. Sotnikov, J. Carroll, and T. Benjamin. 1992. Polyoma tumorigenesis in mice: evidence for dominant resistance and dominant susceptibility genes of the host. Virology 191:724-731.

3. Lukacher, A.E., R. Freund, J.P. Carroll, R.T. Bronson, and T.L. Benjamin. 1993. PyvS: a dominantly acting gene in C3H/BiDa mice conferring susceptibility to tumor induction by polyoma virus. Virology 196:241-248.

4. Golovkina, T.V., A. Chervonsky, J.P. Dudley, and S.R. Ross. 1992. Transgenic mouse mammary tumor virus superantigen expression prevents viral infection. Cell 69:637-645.

5. Lukacher, A.E., Y. Ma, J.P. Carroll, S.R. Abromson-Leeman, J.C. Laning JC, M.E. Dorf, and T.L. Benjamin. 1995. Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen. J. Exp. Med. 181:1683-1692. ****************************************************************
A LIVE SALMONELLA VACCINE VECTOR EXPRESSING HIV-1 ANTIGENS: POTENTIAL AS AN ORAL HIV-1 T CELL VACCINE

by Robert J. Powell, Shaoguang Wu, Robert G. Tuskan, David W. Pascual*, John Van Cott*, Jerry McGhee*, George K. Lewis, and David M. Hone+
School of Medicine, University of Maryland, Baltimore [+Corresponding author] and *Department of Oral Biology, University of Alabama at Birmingham

Although correlates of protective immunity to HIV-1 in humans have not been fully elucidated, it is becoming clear that an effective HIV-1 vaccine should stimulate broadly neutralizing antibody and HLA class I- and class II-restricted T cell mediated responses in the mucosal and systemic compartments. One approach towards the goal is to develop a combination vaccine comprised of a component to optimally induce HIV envelope-specific neutralizing antibody and a component to induce potent HLA class I- and class II-restricted T cell mediated responses. We believe that attenuated live oral Salmonella vaccine vectors are an attractive tool to serve as the latter component. One objective of our group, therefore, is to optimize the capacity of our live oral Salmonella-HIV vaccine vectors to elicit HIV-specific HLA class I- and class II-restricted T cell immunity against multiple HIV antigens, in the mucosal and systemic compartments. Toward this end, we constructed a chimeric Salmonella OmpA outer membrane protein that is fused to sequences encoding most of the HIV envelope protein, gp120. This fusion protein, called OmpA::tgp120, forms an approximately 70kD protein that is located in the outer membrane of the Salmonella vector. Recently, we initiated experiments to characterize T cell responses in mice immunized with the Salmonella OmpA::tgp120 vector vaccine. These preliminary findings indicate that mice immunized with a single oral dose (10^9 cfu) of Salmonella bearing OmpA::tgp120 display measurable gp120-specific T cell proliferation in splenocytes 14 days after immunization. In addition, these mice displayed high levels of cytotoxicity for BC fibroblasts expressing HIV envelope but not for BC fibroblasts expressing beta-galactosidase. Control mice immunized with the Salmonella vector alone did not develop measurable gp120-specific proliferative and cytotoxic responses. Further, we have found evidence that oral immunization of mice with Salmonella strains expressing OmpA::tgp120 results in marked mucosal immune responses. Thus, mice immunized orally with Salmonella bearing OmpA::tgp120 developed impressive gp120-specific IgA producing B cells in the lamina propria and mesenteric lymph nodes. In contrast, negative control mice immunized with the Salmonella vector alone did not develop significant levels of gp120-specific IgA producing B cells in these sites. Collectively, these results provide encouragement that Salmonella vectors will be a safe and inexpensive means for delivery of HIV antigens to, and the elicitation of HIV-specific immunity in, the mucosal and systemic compartments. **************************************************************
HANTAAN VIRUS PERSISTENTLY INFECTS MARMOSET B-LYMPHOBLASTOID CELL LINE

by Kazutaka Ohsawa, Yoji Watanabe, and Hiroshi Sato
Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan

Hantaviruses (HV), members of the genus Hantavirus, family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is mild to severe disease transmitted from HV infected rodents to humans and characterized by fever, hemorrhagic manifestations and renal disorder. Activation of the complement pathways and appearance of the immune complexes in sera were reported in some of patients with HFRS [2, 15, 16]. These reports suggest that immune response mediate a pathogenic mechanism for HFRS in humans. It seems that some HFRS patients are liable to shift to subclinical symptom and to continue high anti-HV antibody for a long period [9]. Whereas, wild and laboratory rodents easily obtain persistent infection without the obvious general symptoms [1, 12, 14]. Some peritoneal cells consist of large majority of macrophages and macrophage-like cell lines of rodents and human are susceptible to HV infection and replication [10, 18]. In HFRS patients, the T and B lymphocytes are found to be infected with HV [3], and therefore HV may show tropism toward immuno-reactive cells, as macrophages and lymphocytes, in rodents and human. However, susceptibility to HV in vitro has been left unsolved with lymphocytes or lymphoid cell lines. Elucidating the mechanism of HV infection in lymphoid cell lines may lead to more understanding of the epidemiology of HFRS.

To evaluate the susceptibility of lymphoid cells to HV infection, we used the ten different cell lines, that is Vero/E6, B95a derived from a cotton-top marmoset, Saguinus oedipus, CGM1, JM, Jurkat, MOLT-4, AT(L)5KY, LYM-1, BW5147, and L1210. We inoculated three HV strains propagated initially in Vero/E6 cells;. prototype Hantaan 76-118 (HTN) and Seoul types B-1 and SR-11. An IFA assay with anti B-1 antibody for 1st serum was used for the detection of viral antigen. Cell specimens were prepared on 1, 2, 4, 7, 14 (no cell passage; P/0), 60 (P/2), 180 (P/6,7), 270 (P/12-18), and 365 (P/18-23) days post inoculation (dpi). Specific fluorescence was observed in the B95a cells inoculated with HTN on and after 4 dpi, and was only in a small number of cells (0.1%). The number of immunoreactive cells increased with days, but not all cells were immunoreactive (60%), even up to 365 dpi at passage 18. HV strains, possible to propagate in B95a cells, were restricted to HTN. The HTN infection did not cause CPE to B95a cells. While all HV strains proliferated in LYM-1 cells, the growth was not followed up more than 14 days, due to cytolytic infection with the viruses. None of the HV strains were propagated in the other lymphoid cell lines tested, i.e., in CGM1, AT(L)5KY, BW5147, L1210, Jurkat, JM, and MOLT-4. In Vero/E6, fluorescent cells were observed on and after 1 dpi, almost all cells exhibiting reactivity at 4 dpi (>80%). This characteristic was continued up to at least 365 dpi at passage level of 23. We observed many cells with stipple-like fluorescence in cytoplasm of B95a, LYM-1, and Vero/E6. The amount of virus antigen in the three cell lines was fount to be same level, since IFA intensity using the our standard antiserum of the rat anti B-1 strain was virtually the same.

Using a modified immunoperoxidase procedure [13], avidin-biotin peroxidase complex (ABC) method, we also estimated the infectivity titer of HV suspensions by focus-forming assay. All the initial HV strains showed high infectivity with Vero/E6, although only HTN virus replicated in B95a cells. But the infectivity titer of initial HTN strain with B95a was only 4.0 x 10^4, in contrast to that with Vero/E6. The B95a-cultured supernatant on 365 dpi contained infective virus to both B95a and Vero/E6, indicating a persistent HTN infection in B95a cells. In comparison with infectivity titers of initial and persistently-infected HTN, the infectivity against Vero/E6 declined according as serial cultures of infected B95a, but rose against B95a. SR-11 and B-1 strains cultured through B95a showed no infectivity with both B95a and Vero/E6 in all generations until 365 dpi, at passage level of 18.

We observed that B95a and LYM-1 cells as well as Vero/E6 cells were susceptible to HV infection and replication; contrary to our expectations, the B95a cells failed to propagate the SR-11 and B-1 virus. B95a cells are transformed with the Epstein-Barr virus (EBV) and maintain EBV release. CGM1 and AT(L)5KY cells transformed with EBV were not susceptible to HV replication, which reject a view that all EBV transformed cells acquire the capacity to maintain virus replication.

Some researchers have reported human lymphocytes infected with HV in vivo. The T and B lymphocytes of sixteen patients with HFRS were found to have been infected by HV during the early period of infection, i.e., the febrile, hypotensive, and oliguric stages [3]. Tang et al. reported that lymphocytes of HFRS patients differ in HV infection rates, these being reported as 8.5% of T cells and 31.3% of B cells, and the rate also varying with the lymphocyte subset, being less than 0.5% for CD25+, 6.1% for CD4+, and 10.5% for CD8+ cells [11]. Many lymphocytes particularly B cells seem to be susceptible to HV, therefore HV infection with B95a is worthy of attention. B95a cells have been reported to be susceptible and useful for the isolation and the replication of wild measles virus, lapinized rinderpest virus, and canine distemper virus [4, 6, 7]. HTN of HV strains was added to those B lymphotropic virus type in B95a tropism.

LYM-1 cells, unlike B95a cells, are slender and spindle-shaped, morphologically distinct from lymphocytes. It is possible that LYM-1 cells are derived from reticular cells in spleen lymph nodes and/or from the endothelial cells of vessels rather than from lymphocytes. Macrophage and peritoneal cells are susceptible to HV infection and replication [10, 18]. Furthermore, the major targets for HV are considered to be endothelial cells and interstitial cells of veins [8, 17]. These reports suggest that HV replicates in many cell types of lymphoreticular and reticuloendothelial systems, which may be closely related to condition of the HFRS patients with fever, hemorrhagic manifestation, an activation of complement pathways, and an appearance of the immune complexes. In this study, some B lymphocytes might be nominated for one of the secondary targets of HV.

In our present study, it was demonstrated that persistent HTN virus infection was supported not only by the Vero/E6 cell line but also by the B95a cell, for at least 365 days. To our knowledge, this is the first in vitro observation that indicates HV replication in lymphoid cell lines. The B95a and LYM-1 cell lines may be useful for investigating lymphoid cell HV interactions and revealing the pathogenic mechanism for HFRS in humans.

References

1. Arikawa, J., Takashima I., Hashimoto, N., Takahashi, K., Yagi, K., and Hattori, K. 1986. Arch. Virol. 88:231-240.

2. Cosgriff, T. M. and Lewis, R. M. 1991. Kidney Int. 40:S72-S79.

3. Gu, X.-S., You, Z.-Q., and Meng, G.-R. 1989. Mater. Med. Pol. 21:103-105.

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Sincere thanks and appreciation to Drs. Benjamin J. Weigler, Robert Jacoby, Aron Lukacher, David Hone, and Hiroshi Sato for contributing articles and other items for this issue of the Newsletter! (Table of Contents for this issue is on "page 2") SCHEDULE OF ACLAD EVENTS FOR ANNUAL MEETING IS ON "PAGE 3". ***BUSINESS MEETING (note day!)*** Sunday, October 15, 5-6 PM, Chesapeake A Room, Hyatt Hotel ***ACLAD SCIENTIFIC PROGRAM*** Tuesday, October 17 8 A.M.--Noon Rm. 307 Convention Center, Baltimore SEMINAR: RODENT PARVOVIRUS INFECTIONS WALLACE P. ROWE LECTURER: Donald E. Mosier, PhD, MD

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