Genetic resistance to lethal flaviviral encephalitis

Summary The replication of Banzi virus, a flavivirus, was comparedin vitro andin vivo in tissues of congenic mice genetically resistant (C3H/RV) or genetically susceptible (C3H/He) to lethal infection. Ultrastructural changes in brains of resistant and susceptible adult mice following intracerebral or intraperitoneal inoculation of virus also were compared.Banzi virus replicated equally well in monolayer cultures of infant and adult brain, stimulated and non-stimulated macrophages and embryonic cells from both strains of mice. Similarly, no significant differences were found between strains in virus growth in brain, spleen or thymus of peripherally-inoculated infant mice. In intracerebrally-inoculated adult mice, virus titers in brains of resistant mice were consistantly lower than in susceptible mice.Visualization of virus particles was dependent on virus concentration in tissues. The changes in brain tissues of both strains of mice were similar, differing only in the time of onset which was noted two days later in C3H/RV mice than in C3H/He mice.These results indicate that, in the case of Banzi virus, the phenotypic expression of genetically-determined resistance to lethal flavivirus infection cannot be attributed primarily to the ability of host cells to support virus replication.With 10 Figures     

Infant rat model of attenuation for recombinant influenza viruses prepared from cold-adapted attenuated A/Ann Arbor/6/60.

The pathogenicity of 6 wild-type influenza A viruses and 21 recombinant strains prepared from wild-type viruses and cold-adapted A/Ann Arbor/6/60 virus for infant rats was determined. Thus, the titers of virus present in the turbinates and lungs of virus-infected animals was measured serially for 5 days after intranasal infection, and the ability of virus strains to promote subsequent systemic bacterial infection by Haemophilus influenzae was measured at 48 h after virus infection. The results obtained were assessed with reference to the genetic constitution of the virus strains and to virus virulence for volunteers. The results showed that virulent viruses grew to relatively high titers in rat turbinates and significantly promoted systemic infection by H. influenzae. In contrast, attenuated strains grew to lower titers and failed to promote systemic H. influenzae infection. For the strains tested, the results showed clear differences for attenuated and virulent strains, and the model was a reliable indication of virulence for humans. Although the virulent strains tended to grow to higher titers in rat lungs than did attenuated strains, exceptions were found, and this measurement could not reliably discriminate virulent and attenuated virus strains. The results suggest that infant rats can be used to assess the virulence of cold-adapted recombinant influenza virus strains, and thus, they can facilitate the development of such strains for vaccine production.     

Coxsackievirus B4 heterogeneity: effect of passage on neutralization and mortality

We have compared two CB4 isolates for virulence, tissue tropism, and antigenic drift using monoclonal antibodies. Both isolates replicated in C57B1/6 and Balb/c mice. The human isolate Edwards, recovered from a fatal case of encephalohepatomyocarditis, produced lethal infection in adult animals. Lethal infections were associated with high viral titers in visceral organs but not with the presence of specific neutralizing epitopes. Virulence seemed stable upon passage, and also the avirulent JVB isolate retained its phenotype. Mock infection and recovery experiments demonstrated the stability of these characteristics. Neither the tissue from which the virus was isolated nor the cell line used in isolation significantly reduced virulence. However, antigenic variation among isolates was abundant. Thus, the set of monoclonal antibodies employed here may not be appropriate positive markers for virulence. This study suggests that CB4 virulence is stable upon extended in vitro passage and limited in vivo passage and that isolation site and method may not select for or against virulence. It is therefore possible that laboratory adapted strains of CB4, although antigenically different from freshly isolated specimens, may still retain these properties responsible for virulence present in low passage isolates and may be, with regard to virulence, very similar to freshly isolated specimens.     

Experimental chemotherapy of rabies. Preliminary results

The effect of 17 immunosuppressive, cytostatic or antiviral agents on the course of illness in weanling conventional mice infected intracerebrally (i.c.) or intramuscularly (i. m.) with lethal or sublethal doses of rabies virus strains isolated from fox and small wild rodents was studied. Potentiation or suppression of virulence by the same compound with some virus strains depended on the route of infection. The best protective effect on i.c. and i. m. infections was achieved with iododeoxyuridine (IUdR) and Actinomycin D on i.c. and with 6-azacytidine and Imuran on i.m. infections, respectively.     

Suboptimal Humoral Immune Response against Influenza A(H7N9) Virus Is Related to Its Internal Genes

Influenza A(H7N9) virus pneumonia is associated with a high case fatality rate in humans. Multiple viral factors have been postulated to account for the high virulence of the virus. It has been reported that patients with influenza A(H7N9) virus infection have relatively low titers of neutralizing antibodies compared to those with seasonal influenza virus infections. In this study, we compared serum hemagglutination inhibition (HI) and microneutralization (MN) antibody titers of mice challenged with wild-type A(H7N9) viruses [H7N9(Anhui) and H7N9(Zhejiang)], an A(H1N1)pdm09 virus [pH1N1(2009)], and a recombinant A(H7N9) virus with PR8/H1N1 internal genes (rg-PR8-H7-N9). All mice infected by H7N9(Anhui) and H7N9(Zhejiang) developed serum HI antibodies at 14 days postinfection (dpi) but no detectable MN antibodies, even at 28 dpi. A low level of neutralizing activity was detected in H7N9(Anhui)- and H7N9(Zhejiang)-infected mice using fluorescent focus MN assay, but convalescent-phase serum samples obtained from H7N9(Anhui)-infected mice did not reduce the mortality of naive mice after homologous virus challenge. Reinfection with homologous A(H7N9) virus induced higher HI and MN titers than first infection. In contrast, pH1N1(2009) virus infection induced robust HI and MN antibody responses, even during the first infection. Moreover, rg-PR8-H7-N9 induced significantly higher HI and MN antibody titers than H7N9(Zhejiang). In conclusion, the internal genes of A(H7N9) virus can affect the humoral immune response against homologous viral surface proteins, which may also contribute to the virulence of A(H7N9) virus.     

Variation Between Strains of Hamsters in the Lethality of Pichinde Virus Infections

Infection by Pichinde virus, a member of the arenavirus group, was studied in Golden Syrian hamsters (Mesocricetus auratus) with regard to possible mechanisms of resistance to virus infection in adult hamsters. Two hamster strains were found to differ in their susceptibility to lethal Pichinde virus infection. LVG/Lak randomly bred hamsters were found to be 100% susceptible to low doses of Pichinde virus during the first 6 days of life, but after 8 days of life, mortality was uncommon. Peak virus titers in the serum of animals infected at 3 days of life were 4 logs greater than in animals infected at 12 days. MHA/Lak inbred hamsters, in contrast, were found to be susceptible to lethal virus infection both as newborns and as adults. Peak virus titers of greater than 10(8) plaque-forming units/ml were observed in serum 8 days after infection of adult MHA hamsters as compared with less than 10(3) plaque-forming units/ml in the serum of adult LVG hamsters. Cultured primary kidney cells and peritoneal macrophages from either hamster strain supported Pichinde virus replication equally well in vitro. Antibodies to the complement-fixing antigens and to antigens at the surface of virus-infected cells were produced by both strains of hamsters. Cyclophosphamide immunosuppression rendered adult LVG animals susceptible to lethal infections, and virus grew to high titers in the treated animals. These findings suggest that immunological factors that appear early in life in LVG hamsters and are deficient in MHA hamsters limit Pichinde virus infection. Unlike previously reported arenavirus diseases, the observations suggest that death is produced by a direct viral effect and not through immunopathological mechanisms.     

Genetic control of resistance to Mycoplasma pulmonis infection in mice.

The differences in susceptibility of various inbred strains of mice to a highly pathogenic strain of Mycoplasma pulmonis CT (T2) has been known for some time. We assessed the genetic control of resistance to T2 infection. Tracheolung lavage samples and lungs of mice were assessed for T2 organisms after intratracheal injection of T2. We found that H-2b (C57BL/6 (B6) and H-2k B10.BR mice were resistant, whereas H-2b A.By, H-2k C3H/Bi, H-2k C3H/HeJ (C3H), and H-2b BALB.B mice were susceptible. We also typed individual B6C3F2 mice for H-2 and for resistance to T2 and observed that resistance to T2 infections is controlled by a single dominant gene not linked to H-2. Histologic examination revealed severe lung lesions typical of M. pulmonis infections in susceptible C3H mice, in contrast to minimal lung lesions in resistant B6 mice. No significant titers of local or systemic antimycoplasma antibodies were detected in either resistant or susceptible mice at 5 days postinfection. Macrophages taken from uninfected B6 or C3H mice failed to inhibit growth of T2 in vitro. However, macrophages from B6 mice did inhibit growth of T2 much better than C3H macrophages when harvested on day 5 of infection. Thus, there is an association between activation of macrophage bactericidal function and genetic resistance to growth of T2 organisms.     

Pathogenicity of different PR8 influenza A virus variants in mice is determined by both viral and host factors

Experimental mouse models were used to compare virulence and reproduction rate of three mouse-adapted variants of the PR8 influenza A virus strain. We observed large differences in pathogenicity in two mouse strains. The PR8M variant was lethal in DBA/2J mice but not in C57BL/6J mice, whereas PR8F and hvPR8 variants were lethal in both mouse strains. High lethality of PR8M in DBA/2J correlated with high viral load at early time points after infection and spread of the virus into alveolar regions. Also, higher viral loads and mortality in mice infected with PR8F resulted in a higher number of infiltrating leukocytes. 3D-protein structure predictions of the HA indicated amino acid sequence alterations which may render the HA cleavage site in PR8F more accessible to host proteases. Infection of C57BL/6J mice with a re-assorted PR8 virus revealed that the HA gene is the main determinant of virulence of the PR8F variant.     

The major site of murine K papovavirus persistence and reactivation is the renal tubular epithelium.

K virus, a murine papovavirus, produces a lethal pneumonia in newborn mice. Animals surviving acute illness develop a persistent infection which reactivates under conditions of immunosuppression. The present study was conducted to identify the cell populations which support persistent K virus infection and to determine the cell populations in which this persistent infection is reactivated during immunosuppression. Mice inoculated by the oral route with 100 50% newborn mouse lethal doses (LD50) of K virus at 14 days of age were followed over a period of 7 months. The distribution of infection was studied by virus assay, immunohistochemistry, and in situ nucleic acid hybridization methods. Viral replication during the acute phase of infection was confined to pulmonary and systemic vascular endothelial cells, as well as to scattered, apparently lymphoid cells within spleens. Beginning 2 months after inoculation, however, specific hybridization for K virus nucleic acids was detected in rare renal tubular epithelial cells, and by 6 months after inoculation renal tubular epithelial cells represented the major site of viral persistence. Positive cells were frequently present in groups of two or more, and a minority of positive cells also expressed viral capsid (V) antigen. Immunosuppression with cyclophosphamide resulted in reactivation of infection, with highest titers of virus being detected in kidneys and with increased numbers of renal tubular epithelial cells expressing viral capsid antigen. Capsid antigen was also detected in rare endothelial cells in kidneys, livers and lungs of these immunosuppressed mice. Although K virus behaves as an endotheliotrope during acute infection, the major site of K virus persistence and reactivation, the renal tubular epithelial cell, is similar to that involved during persistent infection by polyoma virus in mice, SV40 virus in monkeys, and BK and JC viruses in man. The observation that persistently infected renal tubular epithelial cells occur in groups of two or more and occasionally express capsid antigen suggests that virus may persist as a productive infection which is confined by antiviral antibody but maintains itself by cell-to-cell-spread. The present study represents the first instance in which the cell populations which support infection by a member of the polyomavirus subgroup in its natural host have been defined during acute, persistent, and reactivated infection.     

Potentiation of coxsackievirus B3 infection in adult mice pretreated with a gold salt.

In mice treated with sodium aurothiomalate (myocrisin), prior to infection with Coxsackievirus B3, 90% of the animals died by the 11th day postinfection (p.i.). A mortality of 10% was noted in mice receiving myocrisin only, and no deaths occurred in animals infected with virus alone. The highest amount of virus was recovered from the pancreas of myocrisin-treated mice on day 3 p.i. This was over 500-fold higher than the virus titer found in the pancreas of mice infected with virus only. Generally the titer of virus present in different organs was higher at every point in drug-treated animals as compared to intact mice infected with the virus. A high and persistent viremia was present in myocrisin-treated mice; in contrast a low viremia followed by virus clearance from the blood was observed in intact mice infected with the virus. The antibody response was studied in intact and myocrisin-treated mice infected with the virus. In both groups, no neutralizing antibodies were detected on days 1, 2, and 3 p.i. On day 7 after infection, the titers of antibodies were 1:16 and 1:12 in intact and myocrisin-treated mice, respectively. Administration of hyperimmune anti-Coxsackievirus B3 serum 6 hours after infection protrected in myocrisin-treated group of mice against lethal disease. The results of these studies suggest that (1) antibodies alone may not be sufficient to limit the spread and persistence of virus in natural infections and (2) in the absence of any apparent histopathological differences the increased multiplication of Coxsackievirus B3 could be the cause of death in myocrisin-treated mice.     

Susceptibility of inbred mice to rickettsiae of the spotted fever group.

A mouse strain susceptible to lethal infection with Rickettsia conorii was required for testing vaccine efficacy and for studying the immunology and pathogenesis of infection. Among 20 strains of inbred mice inoculated intraperitoneally with the Malish strain of R. conorii, the C3H/HeJ mouse strain was the most susceptible, with a 50% lethal dose of approximately 10 PFU. Infection of all mouse strains resulted in a measurable antibody response; the highest titers correlated with the greatest degree of rickettsial replication as measured by plaque assay of infected spleen homogenates. Inoculation of C3H/HeJ mice with 5.0 log10 organisms of strain Malish by the subcutaneous route did not result in lethal infection. The Casablanca and Moroccan strains of R. conorii were not lethal for C3H/HeJ mice and, in addition, produced plaques in L-929 cells morphologically distinct from those produced by the Malish strain. The only other spotted fever group rickettsia tested which produced a lethal infection in C3H/HeJ mice was Rickettsia sibirica. Sublethal infection with any of the spotted fever rickettsiae tested protected against lethal infection with R. conorii. These data established a lethal challenge system for examining the protective efficacy of spotted fever immunogens and presented evidence of biological variation among strains of R. conorii.     

Differences in resistance to Trypanosoma musculi infection among strains of inbred mice.

Inbred strains of mice were inoculated with Trypanosoma musculi, and the course of the ensuing parasitemia was followed. The mouse strains fell into three groups: those displaying high and moderate (fivefold less) parasitemia and C57BL/6 (B/6) mice which had exceptionally low infections. To gain insight concerning the mechanisms responsible for interstrain variations in infections, several types of experiments were performed. Comparison of the ability of spleen cells from the various strains to provide the growth-promoting substances required by T. musculi for growth in culture revealed that B/6 cells were deficient; this suggested one mechanism for regulating parasite infections. Exposure of C3H (high parasitemia) and B/6 mice to graded levels of ionizing radiation revealed that B/6 mice have much greater innate resistance to infection than do C3H mice. The effects of treating mice with silica dust or mercaptoethanol indicated that relative resistance to infection is not primarily associated with macrophage activity or limited growth-promoting substances. We conclude that variations in immune responsiveness to parasite antigens (probably not associated with the H-2 complex), possibly in concert with variations in a non-immunological mechanism, account for interstrain variation in resistance to T. musculi infections.     

Immunosuppression-induced susceptibility of inbred hamsters (Mesocricetus auratus) to lethal-disease by lymphocytic choriomeningitis virus infection

Summary The role of the immune response in the pathogenesis of lethal and non-lethal lymphocytic choriomeningitis virus (LCMV)-infections of young adult Syrian golden hamsters (Mesocricetus auratus) of different strains was examined using immunosuppressive treatment with cyclophosphamide or with whole-body gamma-irradiation. In all hamsters, the LCMV strains, WE and Armstrong (ARM), caused systemic infections and induced comparable serum LCMV-antibody titers. However, lethal wasting-disease occurred which was hamster-strain and virus-strain dependent. With WE-inocula, MHA and PD4 inbred hamsters were all susceptible to lethal-disease and failed to completely eliminate infection. All LSH and CB inbred hamsters resisted lethal-disease and totally cleared WE-infection. Random colony-bred LVG hamsters and inbred LHC hamsters were intermediate in WE-susceptibility; some died with wasting, while others survived with minimal to no illness. ARM was avirulent for all hamsters and infections were totally cleared. By immunosuppressive treatment, all hamsters were rendered completely susceptible to lethaldisease by WE, and had unresolved infections and diminished serum LCMV-antibody titers. Immunosuppression also rendered all hamster strains partially susceptible to lethal infection by ARM. The hamster immune response was thus shown to suppress LCMV-infection and protect against lethal illness.E.V.G. was funded by the Resident Research Associateship program of the National Research Council.     

A Murine Model for Infection with Burkholderia cepacia with Sustained Persistence in the Spleen

Burkholderia cepacia is an opportunistic pathogen that causes severe systemic infections in patients with chronic granulomatous disease (CGD) or with cystic fibrosis (CF), but its mechanisms of virulence are poorly understood. We developed a murine model of systemic infection in wild-type (WT) and gamma interferon knockout (GKO) BALB/c mice to facilitate dissection of components of pathogenicity and host defense. Both WT and GKO mice were susceptible to chronic splenic infection with B. cepacia, but not with Pseudomonas aeruginosa. B. cepacia strains from patients with CGD persisted longer than those from CF patients. C57BL/6 mice were the most susceptible murine strain; bacteria persisted in the spleen for 2 months. DBA/2, BALB/c, and A/J strains of mice were relatively resistant to infection. Certain strains of B. cepacia complex can persist in the murine spleen after systemic infection; this may provide clues to its virulence in compromised hosts, such as those with CGD and CF.     

Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A

The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied. The i.n. inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death. The H10407 strain was found to be consistently more virulent than the B7A strain. Following i.n. challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period. Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A. In mice i.n. challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens. In mice i.n. challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS. The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice. The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response). These studies indicate that the i.n. challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants. This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.     

Susceptibility of Blastomyces dermatitidis strains to products of oxidative metabolism

Three strains of Blastomyces dermatitidis which differ in their virulence for mice were exposed in their yeast form to various components of the peroxidase-hydrogen peroxide-halide system. Susceptibility to H2O2 alone correlated with virulence, with the most virulent strain (ATCC 26199) least susceptible (50% lethal dose, greater than 50 mM) and an avirulent strain (ATCC 26197) most susceptible (50% lethal dose less than 3.3 mM). A strain of intermediate virulence (ATCC 26198) was of intermediate susceptibility (50% lethal dose, 11.5 mM). The addition of a nontoxic concentration of KI (5 X 10(-4) M) did not increase H2O2 toxicity. However, the addition of either myeloperoxidase or horseradish peroxidase and KI markedly decreased the amount of H2O2 required to kill the organisms, with 100 +/- 0% of all strains killed at 5 X 10(-5) M H2O2 and 97 +/- 4, 100 +/- 0, and 94 +/- 8% of ATCC 26199, ATCC 26198, and ATCC 26197 killed, respectively, at 5 X 10(-6) M H2O2. Kinetic studies with H2O2 alone revealed a delayed onset of killing, but virtually 100% of organisms were killed by 120 min of exposure in all strains. By comparison, the peroxidase-hydrogen peroxide-halide system was 100% lethal for all strains at 1 min. The relatively high concentrations of H2O2 required to kill the yeast phase of B. dermatitidis suggest that H2O2 alone does not account for host resistance to the organism. However, the rapidly lethal effect of the peroxidase-hydrogen peroxide-halide system at physiologically relevant concentrations suggests that this may be one mechanism of host defense to B. dermatitidis.     

Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice.

The virulence mechanisms of Vibrio vulnificus biotype 2 have been studied and compared with those of biotype 1 in mice as the experimental animals. Biotype 2 isolates from European eels were as virulent for mice as biotype 1 strains (50% lethal dose, about 10(5) CFU per mouse); a septicemic infection developed in less than 24 h. These strains had several properties in common with biotype 1 organisms including capsule expression, uptake of various iron sources, and production of exoproteins, whose role in mouse virulence has been demonstrated. We also discuss the implication of biotype 2 strains in human infections.     

Mixed vaginal infections of Balb/c mice with low virulent herpes simplex type 1 strains result in restoration of virulence properties: vaginitis/vulvitis and neuroinvasiveness

Vaginal infections of BALB/c Ann mice with herpes simplex virus type 1 (HSV-1) were studied. Mice were inoculated with virulent strains ANG path and 17 syn⁺ or low-virulent recombinant strains 27/III and 17-syn3 that differ from parental strains in their glycoprotein B (gB) gene sequences. When low-virulent strains were inoculated separately, no vaginitis/vulvitis was produced despite replication in the vagina. In contrast, after coinfection of mice with the two low-virulent strains, vaginitis/vulvitis was produced and virus could be recovered from the central nervous system (CNS). Two of the CNS isolates produced vaginitis/vulvitis, neuroinvasiveness and death of mice after vaginal infection. Restriction fragment analysis and sequencing were used to assess recombination events in the gB gene sequence of the CNS isolates. After mixed vaginal infection recombination between non-virulent HSV strains occurs, resulting in vaginitis/vulvitis and neuroinvasiveness. No correlation was detected between the syncytial phenotype and local vaginal virulence. Virulence of HSV is not solely dependent on gB function; it seems to be more probable that several genes act in concert to induce virulence and neuroivasiveness. Received: 28 May 1996     

Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection

The course of mouse cytomegalovirus (MCMV) infection was compared between wild-type and mutant C57BL / 6 (B6) mice deficient in either RAG-2, perforin, granzyme A, granzyme B or combinations thereof at two time points post infection (p. i.). At day 15 p. i., virus titers were similarly elevated in salivary glands of all mutant, but not wild-type B6 mice and undetectable in lung and spleen tissues of any of the mouse strains. Significant pathological alterations were only seen in salivary glands and spleen from RAG2(- / -), but not in those from other mice whereas few inflammatory foci were observed in lung tissues of all mice except B6. At day 30 p. i., elevated virus titers were observed only in salivary glands, lung and spleen from RAG2(- / -), but in none of the other mice, and were accompanied by extended pathological alterations in all three organs. The data extend previous reports on the critical role of NK / CD8(+) T cells in the early control of MCMV infection by showing that both perforin and granzymes A / B contribute to viral elimination in salivary glands; however, neither of the three molecules alone seem to be indispensable for the final control of infection.     

Experimental infection of suckling mice with a host range mutant of Junin virus.

Experimental infection of three mouse strains with a non-pathogenic mutant of Junin virus named Cl67 was compared with respect to the parental XJCl3 strain. After intracerebral (ic) or intraperitoneal inoculation, XJCl3 was highly virulent for 2 day-old C3H/HeJ, OF1, and BALB/cJ mouse strains, whereas its derivative Cl67 was attenuated. Survival of the Cl67-infected mouse was associated with a restricted replication at the site of inoculation which would impair spread of virus. Thus, the reduced virulence of Cl67 for suckling mice is independent of the mouse strain and the route of viral entry. When Cl67 was preinoculated ic 10 days before the challenge inoculation with XJCl3 by the same route, mice were partially protected from lethal infection. Since neutralizing antibodies were first detected at 30 days post-infection, an interference mechanism is postulated as a mechanism of protection of the mice.