VIROLOGY 233, 118–129 (1997) ARTICLE NO. VY978556

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Attenuation of B5R Mutants of Rabbitpox in Vivo Is Related to Impaired Growth provided by Elsevier - Publisher Connector and Not an Enhanced Host Inflammatory Response

Richard J. Stern,* James P. Thompson,† and R. W. Moyer*,1

*Department of Molecular Genetics and Microbiology University of Florida College of Medicine, Gainesville, Florida 32610-0266; and †Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Gainesville, Florida 32610-0216

Received December 23, 1996; accepted March 21, 1997

The rabbitpox virus (RPV) B5R protein, synthesized late in infection, is found as a 45-kDa membrane-associated protein of the envelope of infectious extracellular enveloped virus (EEV) and as a 38-kDa protein secreted from the cell by a process independent of morphogenesis. The protein is not found associated with intracellular mature virus (IMV). Deletion of the gene attenuates the virus (RPVDB5R) in animals (mice and rabbits), has relatively little effect on formation of IMV, prevents EEV formation in some but not all cells, and leads to a reduced host range. Analysis of the sequence of the protein suggests relatedness to factor H of the complement cascade. Collectively, these observations suggest that attenuation of the virus in vivo could be linked to an inhibition of the inflammatory response, a deficiency in growth, or both. In this report we have analyzed the behavior of RPVDB5R in infected mice and rabbits and conclude that attenuation of the mutant virus likely results from simple failure to grow within the infected animal and that the inflammatory response probably contributes little to the observed attenuation. ᭧ 1997 Academic Press

INTRODUCTION that endow these with the ability to replicate in a wide variety of cell types. The poxviruses comprise an extensive family of large, In recent years, it has come to be appreciated that the cytoplasmic DNA viruses capable of infecting a broad poxviruses also produce several proteins which, in vitro, range of animal species. Several of these viruses are are capable of interacting with and neutralizing critical known to cause a severe and often fatal illness in certain components of the host immune system. Indeed, in some hosts. One dramatic example is variola major, an ortho- cases, it has been demonstrated that expression of these poxvirus which is the causative agent of in man. viral proteins is important for full viral virulence. Many A second member of the genus is rabbit of these viral-encoded proteins either share sequence poxvirus (RPV), which was originally isolated at the homology and/or react directly with regulatory compo- Rockefeller Institute in 1934, during a spontaneous, lethal nents of the host immune system. Examples of such viral- outbreak of infection in rabbits. RPV in rabbits is similar encoded homologues include a secreted interleukin-1b to variola in virulence, in that infection of the host animal receptor (Alcami and Smith, 1992; Spriggs et al., 1992), an rapidly leads to a generalized, disseminated infection interferon-g receptor (Alcami and Smith, 1995; Seregin et which results in death in the majority of cases (Bedson al., 1996; Mossman et al., 1995b, 1995a), TNF receptor and Duckworth, 1963). The production of a lethal infec- (Smith et al., 1991; Hu et al., 1994; Schreiber and McFad- tion, combined with the availability of an animal model, den, 1994), a complement C4b-binding protein (Kotwal makes RPV an attractive candidate for studying the ef- et al., 1990; Kotwal and Moss, 1988), and a serine protein- fects of specific genes on poxvirus virulence. ase inhibitor, designated as SPI-2 or crmA (Palumbo et In the laboratory, the infection of rabbits with RPV is al., 1989; Pickup et al., 1986). initiated by intradermal inoculation. In order to develop Interest in the crmA gene was heightened when it was the subsequent lethal infection, the virus must initially shown that inactivation of the gene in cowpox virus (CPV) replicate within a variety of cell types at the localized resulted in the production of white, inflammatory pocks site of inoculation. Then, the virus must efficiently spread on the chorioallantoic membrane (CAM) of embryonated to secondary sites (tissues and organs) and continue chicken eggs, instead of the red, hemorrhagic, nonin- replication as the host attempts to mount a defensive flammatory pocks typical of wild-type CPV (Pickup et al., response. It has been known for quite some time that 1986). Subsequently, it was shown that the crmA protein many poxviruses possess a variety of ancillary genes is a powerful inhibitor of the interleukin-1b convertase (ICE), a key enzyme in the production of the proinflamma- tory cytokine, interleukin-1b (IL-1b) (Ray et al., 1992). 1 To whom reprint requests should be addressed. The evaluation of pock color on the CAM is a well-

0042-6822/97 $25.00 118 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

AID VY 8556 / 6a37$$$121 06-02-97 23:24:22 vira AP: Virology RPV GROWTH AND INFLAMMATION 119 established system which has proven invaluable for the with the result that the virus fails to plaque on this cell study of the immune modulating effects of the crmA pro- line (Martinez Pomares et al., 1993). Similar results in tein. RPV, like CPV, also produces red, hemorrhagic other cell lines have been reported for B5R mutants of pocks on the CAM and mutants of the RPV crmA gene , where mutations in the B5R gene inhibit extra- likewise produce white, inflammatory lesions. Those le- cellular virus production (Engelstad and Smith, 1993; sions, like those of CPVDcrmA(SPI-2), are impacted with Wolffe et al., 1993). In addition, we showed that mutations heterophils (Fredrickson et al., 1992; Palumbo et al., 1989; in the B5R gene resulted in the production of nonhemor- R. J. Stern and R. W. Moyer, unpublished results). The rhagic, white-pocks on the CAM, similar in appearance heterophilia and resultant white pock phenotype are pre- to those produced by crmA mutants of CPV or RPV (Marti- sumed to result directly from the inability of the mutant nez Pomares et al., 1993). This information, combined virus to block the host inflammatory response as demon- with the homology to Factor H, suggested a possible strated by the phenotypic reversion of this white-pock role for the B5R protein in the regulation of the host phenotype back to red pocks when infections of the CAM inflammatory response. Since it was suspected that viral are done in the presence of anti-inflammatory agents suppression of the host inflammatory response is im- such as dexamethasone (Fredrickson et al., 1992). portant in allowing full manifestation of viral virulence, The B5R protein homolog encoded by RPV is virtually we suggested that the observed attenuation of virus in identical to that encoded by the B5R gene of vaccinia animals resulting from deletion of the B5R gene might virus. The B5R protein is yet another poxvirus protein well be related to an enhanced inflammatory response that shares sequence homology with a component of the in accordance with what was believed to be the explana- immune system (Factor H) (Takahashi Nishimaki et al., tion for white pocks on the CAM. 1991; Engelstad et al., 1992). The 45-kDa glycoprotein In this paper, we extend our previous results and show encoded by this gene is produced late during the viral that mutations in the B5R protein of RPV result in major infection and is found as a component of extracellular attenuation of RPV virulence in rabbits as well as mice. enveloped virus particles (EEV) released from the cell The attenuation could, however, be a manifestation of (Isaacs et al., 1992; Wolffe et al., 1993; Engelstad et al., poor growth in the animal, related to a failure to contain 1992; Engelstad and Smith, 1993; Takahashi Nishimaki the inflammatory response of the host or both. In the et al., 1991; Martinez-Pomares et al., 1995). The B5R pro- studies presented here, we attempt to resolve these pos- tein is not found in intracellular mature virus (IMV) (Isaacs sibilities and specifically address the possible involve- et al., 1992; Wolffe et al., 1993; Engelstad and Smith, ment of the host inflammatory response on RPV B5R 1993; Martinez Pomares et al., 1993), an infectious form mutant infections in both the CAM and animal systems. of the virus which is further processed to form EEV. In We conclude that attenuation results most likely from an addition, the B5R protein is found in the supernatant of impaired ability of the B5R mutant virus to grow effec- infected cells as a smaller, secreted 38-kDa protein de- tively in animal hosts. rived by cleavage of the 45-kDa form, a process which is believed to occur on the cell surface independent of MATERIALS AND METHODS EEV formation (Martinez Pomares et al., 1993). Cells, viruses, and eggs Examination of the amino acid sequence of the B5R protein reveals four short, conserved repeat (SCR) re- RPV Utrecht strain was obtained from the American gions of approximately 60 amino acids each, flanked by Tissue Type Culture collection. CPV Brighton Red strain cysteine residues (Engelstad and Smith, 1993; Takahashi was a generous gift from Dr. David Pickup (Duke Univer- Nishimaki et al., 1991). These SCR regions are typically sity). All virus stocks were grown on RK-13 cells (ATCC). found in members of a protein family involved in the Virus used for animal infections was purified as follows: regulation of the complement system (Hourcade et al., Infected cells were harvested, lysed by dounce homoge- 1989). Comparison of the amino acid sequence with the nization, and spun at 500 g to remove nuclei. The super- protein databases using the BLAST program (National natants were then layered onto 36% sucrose and spun Center for Biotechnology Information (NCBI), Bethesda, at 65,000 g for 90 min. Viral pellets were resuspended MD), revealed that the B5R protein shares regions of in PBS and stored at 070ЊC. Plaque assays were done on homology with Factor H, a regulatory component of the RK-13 cells except where mycophenolic acid selection of complement cascade. Factor H is a component of the recombinant virus was performed, in which case, assays alternative pathway of the complement cascade which were performed on CV-1 cells (ATCC). acts to destabilize the formation of C3b complex and RK-13 and CV-1 cells were cultured in Gibco minimal can therefore be viewed as a negative regulator of the essential medium (MEM) containing streptomycin (50 IU/ complement system. ml), penicillin (50 mcg/ml), glutamine (2 mM), sodium In previous work, we have shown that interruption of pyruvate (1 mM), MEM nonessential amino acids (0.1 the RPV B5R open reading frame leads to low production mM), and 5% fetal bovine serum (GIBCO). Chicken em- of IMV and virtually no EEV in chick embryo fibroblasts, bryo fibroblasts were prepared and maintained for eight

AID VY 8556 / 6a37$$$122 06-02-97 23:24:22 vira AP: Virology 120 STERN, THOMPSON, AND MOYER passages in medium 199 (Gibco) with 5% fetal bovine serum. Embryonated White Leghorn chicken eggs were ob- tained from SPAFAS, Inc. (Roanoke, IL) and incubated at 37.5ЊC in a Kuhl incubator (Flemington, NJ) according to the manufacturers instructions. At 11 days postfertiliza- tion, the CAM was inoculated with virus suspended in 100 ml of sterile PBS. In CAM experiments involving dexa- methasone, the virus was resuspended in PBS con- taining 8 mM dexamethasone (Sigma Chemical Co.), streptomycin (50 IU/ml), and penicillin (50 mcg/liter) (Fredrickson et al., 1992). The infected eggs were incu- FIG. 1. Rectal temperatures of intradermally infected rabbits. Rabbits bated for an additional 72 hr after which, the CAMs were were infected intradermally with either 500 PFU (᭺, ᮀ) or 50000 PFU removed for examination. Staining of CAMs with NBT (᭿, ࡗ) of wtRPV (A) or RPVDB5R (B). Temperatures were recorded (Fredrickson et al., 1992) was accomplished by incuba- every 24 hr following infection. D, died; E, euthanized. tion of PBS rinsed membranes for 30 min at 37ЊC in PBS containing 0.1% nitro-blue tetrazolium (Kodak). where (Martinez Pomares et al., 1993). Briefly, the open reading frame for the Escherichia coli guanyl-ribosylpho- Animals and animal infections sphotransferase (gpt) gene under the control of the pox- Adult (approximately 3 kg) female rabbits were ob- viral p7.5 promoter was cloned into a unique EcoRV re- tained from M & P’s rabbitry (Tampa, FL) and were striction site within the cloned RPV B5R gene. A plasmid housed in individual cages. To initiate the infection in containing this interrupted gene was then used to con- these animals, virus, resuspended in 100 ml of PBS, was struct an RPVDB5R mutant following transfection of this injected intradermally into the shaved flank of each ani- plasmid into wtRPV-infected cells followed by selection mal. Rectal temperatures were recorded using a digital of the recombinant virus based on resistance to myco- thermometer (Becton –Dickinson, Franklin, NJ). phenolic acid (MPA). Six- to 8-week-old (15–20 g) female Balb/c mice were obtained from either Harlan–Sprague–Dawley Inc. (Indi- RESULTS anapolis, IN) or the Jackson Laboratories (Bar Harbor, Attenuation of RPVDB5R in rabbits ME). Intranasal infections of mice were performed as follows: Mice were first anesthetized by intraperitoneal In order to expand our knowledge of the effects of injection of 100 ml of sodium pentobarbital (0.05–0.1 mg/ eliminating the RPV B5R gene on virulence, we compared g body weight) following which the mice were inoculated, the ability of wtRPV and RPVDB5R to cause disease in 10 ml per nostril, with virus diluted in PBS, using a micro- rabbits. Rabbits may be considered a ‘‘more natural host’’ capillary pipet. In experiments involving the use of dexa- for RPV and are exquisitely sensitive to infection. A lethal methasone, mice were given twice-daily intraperitoneal infection can frequently be initiated by intradermal inocu- injections of 100 ml of sterile PBS containing dexametha- lation of a single plaque forming unit (PFU) of RPV. The sone-21 phosphate (Sigma) (500 mg/ml). Ear swelling progress of the infection in these animals can easily be was induced in mice by the application to one ear of 25 followed by monitoring the increase in body temperature ml of croton oil (5% in ethanol) (Sigma). Ear thickness as well as by noting the secondary signs of infection was measured using an engineers micrometer (Starret, which develop such as nasal discharge, dyspnea, and Athol, MA) (Trottier and Ogolla, 1990). secondary/satellite lesions which are indicative of vire- mia. The data of Fig. 1 compares the febrile response Lung collection and virus titration resulting from intradermal injection of rabbits with either RPV or RPVDB5R, whereas, the data of Table 1 compares Mice were sacrificed by cervical dislocation. Lungs overt clinical symptoms. were removed, weighed, and stored at 070ЊC until fur- Rabbits infected with wild-type RPV (Fig. 1A) showed ther processing. Lungs were thawed, the tissue was dis- a sharp increase in body temperature (pyrexia) beginning rupted in 2.0 ml of sterile PBS using a Stomacher lab approximately 2 days postinoculation and persisting until blender 80 (Tekmar Co., Cincinnati, OH), and the homog- death or, in the 25 –50% of the animals which survived, enate was frozen and thawed twice more. Samples were until approximately 10 days postinoculation. Presumably, then briefly sonicated before titration. this pyrexia results in part from the fact that RPV, unlike many fails to synthesize a secreted IL- Construction of RPVDB5R 1b receptor (Alcami and Smith, 1992), which is important The construction of RPVDB5R, a virus containing a in preventing infection-induced fever (Alcami and Smith, distruption in the B5R gene has been described else- 1992, 1996).

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TABLE 1

Development of Clinical Symptoms in Rabbits Infected with Two Doses of either RPV or RPVDB5R

Nasal Secondary Virus and Dose discharge Dyspnea Hemorrhage lesions Fever Death

RPV 50 pfu A 00 0 /// RPV 50 pfu B // 0 /// RPV 5 1 103 pfu A 0/ 0 /// RPV 5 1 103 pfu B /0 0 //0

RPVDB5R 50 pfu A 00 0 000 RPVDB5R 50 pfu B 00 0 000 RPVDB5R 5 1 103 pfu A 00 0 000 RPVDB5R 5 1 103 pfu B 00 0 000

Note. Individual rabbits were inoculated intradermally with 50 or 5 1 103 PFU of either RPV or RPVDB5R and examined each day thereafter as described in the legend to Fig. 1. Two rabbits (designated A and B) were used for each dose of virus. The chart tabulates individual symptoms observed during the course of the infection. / Indicates the animal was positive for that symptom while 0 indicates the animal was negative.

All animals infected with wild-type RPV exhibited at also showed that many exhibited a neutrophilic infiltrate least some of the classical signs of illness (Table 1) and within the dermis. It is noteworthy that many more inflam- three of the four infected animals failed to survive the matory cells were observed from RPV-generated lesions infection. By contrast, rabbits infected with RPVDB5R than those generated by RPVDB5R. One explanation for (Fig. 1B) showed no significant increase in body tempera- these observations could be a reduced growth of ture at any time during the infection. There was no vire- RPVDB5R within the lesion. We observed similar results mia as evidenced by the complete absence of secondary for animals infected with 5 1 104 PFU of virus (data not lesions, little if any symptoms of clinical disease, and all shown). of the animals survived (Table 1). Animals infected with wild-type RPV developed signifi- Attenuation of RPVDB5R in mice cant induration at the site of inoculation beginning 2 days postinoculation, which remained throughout the infec- The extreme sensitivity of rabbits to lethal infection tion. Secondary lesions, resulting from viremia, were fre- with RPV, makes the determination of the LD50 for this quently observed by 6 days postinfection. Examination virus impractical. To more accurately quantitate the ex- of the RPVDB5R primary lesions indicated that there was tent of RPVDB5R attenuation, the LD50 for this mutant some induration present in these animals at the site of was reexamined in a mouse intranasal infection model inoculation, although the indurated area was much less in which weight loss and mortality are used to gauge the significant than that seen in the RPV-infected animals virulence and lethality of the virus (Bloom et al., 1991). (data not shown). Unlike RPV-infected animals, where The results of this experiment are illustrated in Fig. 3. the induration increased throughout the experiment, the Mice inoculated with wild-type RPV lost weight in a dose- induration generated by RPVDB5R infections began de- dependent manner as expected and death, resulting from creasing on Day 6 postinoculation and had usually re- the viral infection, occurred in animals infected with 5 solved by Day 12. doses greater than 10 PFU yielding an LD50 (Reed and A histological examination of the primary lesions was Meunch, 1938) of 105.6 PFU (Fig. 3A). In contrast, inocula- performed 4 days after infection from RPV and RPVDB5R tion of mice with identical doses of RPVDB5R resulted animals inoculated with 5 1 102 PFU of virus (Fig. 2). in either minimal or no weight loss (Fig. 3B). No mortality The results indicated that lesions from RPVDB5R-in- ever resulted from infection of mice with RPVDB5R, even fected animals exhibited no necrosis, no hemorrhage, following inoculation of the animals with doses as high 8 few if any inflammatory cells, and basically resembled as 10 PFU (data not shown). The LD50 for RPVDB5R can those of animals inoculated with PBS (Fig. 2a). This was therefore be estimated to be about a 1000-fold greater in sharp contrast to the histological picture of animals than the LD50 for wild-type RPV (Reed and Meunch, 1938). infected with RPV where extensive tissue destruction and This result is in agreement with our earlier results (Marti- hemorrhage was evident (Fig. 2b). The epidermis of RPV- nez Pomares et al., 1993) and with similar studies in infected animals showed severe vacuolization together vaccinia (Engelstad and Smith, 1993). with necrosis that extended through the dermis and into The observation that RPVDB5R was severely attenu- the skeletal muscle bundles at the deep border of the ated in mice was consistent with the results obtained sections. While not particularly evident in Fig. 2b, an ex- with this mutant in rabbits. To evaluate inflammation and amination of thin sections from RPV-infected animals necrosis, a histological examination of the lungs from

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FIG. 2. Histological examination of primary lesions from infected rabbit skin. PBS (a) or PBS containing 5 1 102 PFU of either RPV (b) or RPVDB5R (c) was injected intradermally into a single rabbit. A skin sample including the site of inoculation was removed 4 days postinfection, sectioned, and the sections were stained with hematoxylin and eosin prior to examination. Skin sections are shown at 2001 magnification. FIG. 4. Lung sections from intranasally infected mice. 20 ml of PBS (A) or PBS containing 104.5 PFU of RPV (B), RPVDB5R (C), or RPVDSPI-2 (D) was administered intranasally to BALB/c mice. Four days following infection, the lungs were removed, sectioned, and the sections were stained with hematoxylin and eosin for examination. Sections are shown at 1001 magnification.

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FIG. 7. Histopathology of pocks formed on infected CAMs. The chorioallantoic membrane of 11-day-old chicken embryos were inoculated with PBS (uninfected), wild-type RPV, or RPVDB5R and incubated as described under Materials and Methods. After 72 hr, the CAMs were removed and individual pocks were excised, sectioned, and stained with hematoxylin/eosin for analysis. Sections are shown at both 2001 and 4001 magnification. infected mice were performed 4 days after intranasal Infection with either RPV or RPVDSPI-2 resulted in the infection with either PBS, RPV, RPVDB5R, or RPVDSPI- development of periarteriolar, peribronchiolar, and alveo- 2. Lungs from RPVDSPI-2(crmA)-infected mice were lar edema. Both RPV- and RPVDSPI-2-infected lungs con- included as a positive control as the presence of the SPI- tained a mixed (neutrophilic and lymphocytic) inflamma- 2(crmA) protein is believed to supress the host inflamma- tory cell infiltrate; however, the response was consis- tory response. The results are summarized in Fig. 4. tently more severe in RPVDSPI-2-infected lungs. In contrast, lungs from RPVDB5R-infected mice showed no evidence of either edema or necrosis. However, a low level of mixed inflammatory cell infiltrate, similar to that observed in the RPV-infected lungs, was noted. The level of inflammatory cell infiltrate present in these lungs was somewhat increased in comparison to RPV although it was not as severe as the infiltration observed in RPVDSPI-2-infected tissue.

Growth of RPVDB5R in the mouse lung We and others have previously reported that defects in the B5R gene of RPV or vaccinia resulted in a cell- FIG. 3. Weight change in mice following intranasal infection. Mice line-dependent defect in extracellular-enveloped virus 3 4 5 6 were infected intranasally with 10 (l), 10 (᭿), 10 (ࡗ), or 10 (᭡) PFU production, resulting in an altered host range (Takahashi of RPV or RPVDB5R. Eight animals were examined at each dose. Weights of the animals were determined daily for up to 10 days follow- Nishimaki et al., 1991; Engelstad and Smith, 1993; Eng- ing inoculation. At the highest dose of RPV (106 PFU) three animals elstad et al., 1992; Isaacs et al., 1992; Wolffe et al., 1993; failed to survive the infection (D). Martinez Pomares et al., 1993). If a similar host range

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the simple inability to grow could also explain the de- creased titer of RPVDB5R and the resulting attenuation of this mutant observed in infected animals. To determine if the decreased yield of RPVDB5R observed in the lung was due to an increased host inflammatory response in the RPVDB5R-infected animal, we measured the amount of virus produced in the lungs of infected mice under conditions where the inflammatory response has been suppressed. Suppression of the inflammatory response was achieved by treating the mice prior to infection, twice-daily, with dexamethasone, a known and potent, anti-inflammatory agent (see Materials and Methods). Suppression of the inflammatory response in the dexa- methasone-treated mice was confirmed by measuring the reduction in phorbol-ester-induced ear swelling (Fig. 6A). The level of virus present in the lungs of control and

FIG. 5. Growth of RPV and RPVDB5R in mouse lung. Mice were infected intranasally with 106 PFU of either wtRPV (l) or RPVDB5R (᭺). At the times indicated, lungs were removed and the amount of virus present was measured by determination of titers on RK-13 cells. Four mice were analyzed at each time point. restriction occurs in the infected animal, then impaired viral growth alone could explain the severe attenuation observed. To measure the ability of RPVDB5R to grow in the animal, within a localized environment, a growth experiment was performed using the mouse (lung) as a growth chamber. Mice were infected intranasally with a lethal dose of virus and the ability of the virus to grow was measured by monitoring viral titers in the lung at various times throughout the course of the infection. As shown in Fig. 5, the titers of virus in the lungs of wild- type RPV-infected animals rapidly increases to yield a titer of 108 PFU/lung on Day 5 postinfection. In contrast, the yield of virus in RPVDB5R-infected animals is consid- erably (at least 100-fold) lower when compared to the wild-type RPV group, correlating with the diminished weight loss and lack of morbidity observed in these ani- mals. Although the level of RPVDB5R produced is much less than that of wild-type RPV, comparison of the final titer of RPVDB5R with that seen at zero time in RPVDB5R-infected animals shows an overall small but reproducible increase in titer, demonstrating that the mu- tant is able to grow, to some extent, in the mouse lung.

Effect of dexamethasone on virus replication in mice FIG. 6. Effect of dexamethasone treatment on subsequent viral yields in mice. (A) To confirm the anti-inflammatory effects of dexamethasone, We have previously shown that RPVDB5R produces mice were either untreated (black bar) or treated twice-daily with dexa- white pocks on the CAM. The white pocks produced by methasone as described under Materials and Methods (gray bar). On RPVDB5R are similar in appearance to those produced the 4th day of treatment, ear swelling was induced by the application by SPI-2 deletion mutants of either RPV and CPV which of croton oil to one ear. Ear thickness was measured 4 hr postapplica- have been shown to be the result of an enhanced host tion. (B) Groups of mice, either untreated (open symbols) or dexametha- sone treated (closed symbols) were infected with 104.5 PFU of RPV (᭺/ inflammatory response (Palumbo et al., 1989, 1993; Fred- l) or RPVDB5R (᭝/᭡). At the specified times, lungs were removed from rickson et al., 1992). Loss of the ability of the virus to four animals and the amount of virus was measured by determination of suppress the host inflammatory response, rather than titers on RK-13 cells. Error bars, when significant, are shown.

AID VY 8556 / 6a37$$$123 06-02-97 23:24:22 vira AP: Virology RPV GROWTH AND INFLAMMATION 125 dexamethasone-treated, infected animals was deter- TABLE 2 mined, 4, 6, and 8 days postinoculation, by plaque assay. Virus Titers from Lungs of Infected Mice The results of this experiment are shown in Fig. 6B. The total amount of virus present in the lungs of infected, Day 4 P.I. Day 6 P.I. untreated animals was similar to that seen in the previ- ous experiment, with wild-type RPV-infected animals pro- (0)MPA (/)MPA (0)MPA (/)MPA ducing 2–3 orders of magnitude more virus than RPV 7.17a 0 7.3 0 RPVDB5R-infected animals. Dexamethasone-induced RPVDB5R 4.52 4.48 4.72 4.72 suppression of the host inflammatory response did not RPV / alter the level of virus seen in either wtRPV- or RPVDB5R- RPVDB5R 7.3 4.57 7.5 4.74 infected animals at Day 4 following infection although Note. Mice (four per group) were infected with 104.5 of RPV, the virus titers appeared to decrease at a slower rate RPVDB5R, or both of these viruses. Lungs were removed at the times through Day 8 in the dexamethasone-treated mice, per- indicated and the total amount of virus present was quantified by deter- haps indicating some inhibition in the clearance of the mination of the titer on CV-1 cells in the absence (0) of mycophenolic virus. acid (MPA). The level of recombinant virus present in each sample was estimated by determination of the titer in the presence (/) of MPA. a The log of the titer is presented. Measurement of virus growth in mice coinfected with wtRPV and RPVDB5R was similar to that seen in animals infected with wtRPV Although the results from the previous experiment sug- alone. Titering of the samples in the presence of MPA, gested that the dexamethasone-sensitive steps of the to determine the level of RPVDB5R virus in coinfected inflammatory response are not responsible for the de- lungs, revealed that coinfection with RPV had no effect creased viral yields observed in RPVDB5R-infected ani- on RPVDB5R levels. In all cases the level of mutant virus mals, it remained formally possible that the B5R protein was identical to that seen in the lungs of animals infected acts on a component of the inflammatory system not with RPVDB5R alone. This result suggests that the B5R effected by dexamethasone. To address this possibility, protein cannot produce an environment in the lung which we asked whether the B5R protein present in the mouse leads to enhanced growth of the RPVDB5R mutant. lung could, in some way, alter the environment of the lung and allow for increased production of RPVDB5R; in The nature of the white pocks produced by RPVDB5R other words, could the B5R protein produced by wtRPV achieve a physiological, localized, environmental com- We have no evidence, based on animal experiments, plementation of the B5R mutation. This was tested by to suggest that infection with RPVDB5R leads to an en- infecting mice intranasally with either RPVDB5R, wild- hanced inflammatory response which could account for type RPV, or an equal mixture of the two viruses, and the poor growth of this virus. None of the data obtained measuring the resulting yields of each virus at 4 and from infected rabbits would suggest that infection with 6 days following infection. Since the RPVDB5R mutant RPVDB5R leads to enhancement of the host inflamma- contains the gene for the E. coli guanyl-ribosylphospho- tory response. In addition, treatment of mice with dexa- transferase (gpt), the levels of this virus in the coinfected methasone prior to infection to inhibit the inflammatory lung can be distinguished from that of wild-type RPV by response, did not lead to increased growth of RPVDB5R. performing plaque assays in the presence or absence These results would seem to contradict our earlier re- of mycophenolic acid. The results of this experiment are sults showing that RPVDB5R produces white pocks on shown in Table 2. Lungs removed from animals infected the CAM, which by analogy with CPVDSPI-2 would be with either RPV or RPVDB5R alone contained levels of expected to produce an enhanced inflammatory re- virus similar to those seen in previous experiments at sponse (Fredrickson et al., 1992; Palumbo et al., 1993, both 4 and 6 days following infection with either virus 1989; Pickup et al., 1986). If the formation of white pocks alone, when the titers were determined in the absence by RPVDB5R is the result of enhanced inflammation, then of mycophenolic acid. Determination of the level of virus the pocks, like those of CPV or RPVD SPI-2 (crmA) mu- present in the lungs of both of these singly infected tants should contain activated heterophils (Fredrickson groups in the presence of MPA gave the expected result; et al., 1992). i.e., lung samples from RPV-infected animals produced We therefore performed a histological analysis of no plaques in the presence of MPA, whereas samples CAMs infected with wild-type RPV and RPVDB5R (Fig. from RPVDB5R-infected animals produced the same 7). Uninfected membranes show no pathology and were numbers of plaques in the presence of MPA as seen in free of heterophils. Infection with wild-type RPV results the absence of the drug. in a marked proliferation of both the chorionic epithelial When lung samples from the mixedly infected mice cells and the fibroblast cells of the stroma. The epithe- were titered in the absence of MPA, the level of virus lium in the lesion showed ballooning degeneration as

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AID VY 8556 / 6a37$$8556 06-02-97 23:24:22 vira AP: Virology RPV GROWTH AND INFLAMMATION 127 well as necrosis and exfoliation of the epithelial layer. hibited by dexamethasone, the white pocks produced by A mild heterophilic cell infiltrate was observed in the CPVDcrmA mutants phenotypically revert to red pocks epithelium as well as in the stromal layer. Blood vessels (Fredrickson et al., 1992). CAMs were infected with CPV, within the stroma appeared dilated and hemorrhage was RPV, RPVDcrmA, CPVDcrmA, or RPVDB5R in the ab- evident. These last features were more easily observed sence or presence of 8 mM dexamethasone and exam- under low power magnification of intact pocks (data not ined 72 hr after infection (Fig. 9). All mutant pocks were shown). Surprisingly, infection of the CAM with RPVDB5R white in the absence of dexamethasone, whereas RPV produces pocks containing many of the same features and CPV generate the expected red pocks. In the pres- as noted for wild-type RPV (Fig. 7), although there were ence of dexamethasone, pocks produced by both several noticeable differences. RPVDB5R pocks, like RPVDcrmA and CPVDcrmA pocks were red, consistent those of RPV were also defined by extensive proliferation with a dexamethasone-mediated inhibition of inflamma- of the epithelial and stromal layers. The epithelium tion. However, dexamethasone had no effect on the white showed similar ballooning degeneration, necrosis, and pocks produced by RPVDB5R. The resulting pocks re- exfoliation as noted for RPV. However, the degree of mained white and appeared similar to those produced in exfoliation was less severe in the RPVDB5R pocks. Un- the absence of the drug. Therefore, despite the increased like the RPV pocks, the pocks generated by RPVDB5R presence of heterophils in the lesions of RPVDB5R appear to contain large numbers of heterophils, most of pocks, there is no other indication that this represents a which were contained within the stromal layer. In addi- typical inflammatory response. tion, the innervating blood vessels of the RPVDB5R le- sions were not dilated and there was a noticeable lack DISCUSSION of hemorrhage within these pocks in comparison to the pocks generated by wild-type RPV. Many of the membrane-associated poxvirus proteins The presence of heterophils in the lesions on the CAM found in the extracellular enveloped virus particle (EEV) produced by RPVDB5R is surprising in view of our results have been studied to gain a better understanding of their in infected animals and would suggest that the white contribution to infection and the morphogenesis of the pocks produced by the B5R mutant are inflammatory and virus (Schmutz et al., 1991; Blasco and Moss, 1991, 1992; therefore quite similar to those produced by CrmA (SPI- Blasco et al., 1993; McIntosh and Smith, 1996; Parkinson 2) mutants of CPV (Palumbo et al., 1989). A simple but et al., 1995; Engelstad and Smith, 1993; Duncan and indirect test for the presence of activated heterophils Smith, 1992; Engelstad et al., 1992; Payne, 1992, 1980; involves the staining of the pocks with nitroblue tetrazo- Payne and Kristensson, 1982). A common feature shared lium (NBT), which is reduced to formazan by free-oxygen by many of these proteins is that deletion of the relevant radicals produced by the activated heterophils within in- ORF often interrupts the terminal steps of viral morpho- flammatory lesions. Production of formazan is indicated genesis, resulting in inhibition in the formation of EEV, by an intense blue color. Indeed, the assay has been the extracellular form of infectious virus (Blasco and used as an indicator of the presence of activated hetero- Moss, 1991; Schmutz et al., 1991; Hirt et al., 1986; Duncan phils within these pocks (Palumbo et al., 1994). and Smith, 1992; Parkinson and Smith, 1994; McIntosh We have reconfirmed those results and shown that and Smith, 1996; Rodriguez and Smith, 1990; Blasco and RPV or CPV mutants defective in the SPI-2 gene (but not Moss, 1992; Isaacs et al., 1992; Gong et al., 1989). Since wild-type virus) produce white pocks on the CAM which it is believed that it is the enveloped form of the virus turn dark blue in the presence of NBT. However, the that is responsible for virus spread, both in tissue culture white pocks produced by RPVDB5R do not stain blue and in the infected animal (Payne, 1980; Blasco and even upon prolonged exposure to NBT (data not shown), Moss, 1992; Payne and Kristensson, 1982) it is not sur- suggesting that despite the presence of increased num- prising that several of these EEV protein mutants have bers of heterophils, these white pocks are of a fundamen- been shown to fail to plaque and/or are attenuated in tally different nature than those produced by the SPI-2 vivo (Schmutz et al., 1991; Blasco and Moss, 1991; McIn- mutants (Fig. 8). tosh and Smith, 1996). What is particularly interesting Another method of evaluating the white pocks pro- about the B5R ORF in this context, is that deletion of the duced on the CAM is the response of the developing gene confers a host range (Takahashi Nishimaki et al., pock to dexamethasone, a commonly used suppressor 1991; Martinez Pomares et al., 1993; Wolffe et al., 1993; of inflammation. If the host inflammatory response is in- Engelstad and Smith, 1993), implying that the protein is

FIG. 8. NBT staining of infected CAMs. CAMs were infected as described under Materials and Methods. The upper panel shows CAMs after removal from the eggs at 72 hr postinfection. The lower panel shows the same membranes following a 30-min incubation at 37Њ in PBS containing 0.1% NBT (wv.). FIG. 9. Effect of dexamethasone on pock color in eggs. The CAMS were infected and the eggs were incubated with the indicated virus resuspended in either PBS (top panel) or PBS containing 8 mM dexamethasone (lower panel). Membranes were removed and examined 72 hr postinfection.

AID VY 8556 / 6a37$$$124 06-02-97 23:24:22 vira AP: Virology 128 STERN, THOMPSON, AND MOYER essential for EEV formation in some cells and not in of infected animals suggest that despite the ability of others. The B5R protein is also rather unique among the RPVDB5R to grow in certain cells in culture, the data EEV proteins in that in addition to being a membrane is most consistent with the notion that attenuation of component of EEV, the protein is also secreted from the RPVDB5R mutants in animals is linked to a simple failure cell in a morphogenesis-independent manner; i.e., in the to grow and that the inflammatory process is likely not absence of virion assembly (Martinez Pomares et al., affected by the B5R gene product. Therefore, we reinves- 1993). The B5R protein has homology to Factor H, a com- tigated the nature of the white pocks produced by ponent of the complement cascade (Takahashi Nishi- RPVDB5R. The RPVDB5R white pocks, like the white maki et al., 1991; Engelstad and Smith, 1993), suggesting pocks produced by crmA mutants have an enhanced that it might belong to the family of proteins known to be influx of heterophils but unlike the crmA-induced white host response modifiers and might somehow function to pocks failed to reduce NBT to formazan and were not inhibit inflammation. Hence it was not surprising when phenotypically reverted to red pocks by application of it was discovered that a mutant of RPV, lacking the B5R dexamethasone to the infected CAM. These results sug- ORF, produced white pocks on the CAM, similar to those gest that the white pocks produced by B5R mutants are produced by the well-studied virus mutants in the crmA noninflammatory, and therefore intrinsically different than protein. One known function of the crmA protein is to those produced by crmA mutants, implying that there is inhibit the interleukin-1b convertase (ICE). Virus mutants more than one type of white pock. Preliminary experi- lacking the crmA (SPI-2) gene allow an influx of inflamma- ments suggest that the RPVDB5R white pocks might in- tory cells into the lesion which will ultimately develop stead be a consequence of hyperplasia of the CAM at into a white pock. We have attempted to address a basic the site of infection, a feature easily observed under low- question in this communication; namely, whether the at- power magnification. It may also be that the failure of tenuation of the RPVDB5R mutant in vivo is the result these pocks to turn red is due to a lack of vasodilation of an enhanced inflammatory response which serves to and/or vascular damage within the pock, which may re- better contain the infection or simply a consequence of sult from an impaired production of virus. failure of the RPVDB5R mutant to effectively grow or spread. ACKNOWLEDGMENTS The RPVDB5R mutant is severely attenuated in both mice (Martinez Pomares et al., 1993; Engelstad and This work was supported by Grant AI157322 from the National Insti- tutes of Health. R.J.S. acknowledges support from Training Grant T32 Smith, 1993; Wolffe et al., 1993), (Fig. 2) and rabbits (Fig. AI07110. 1). This effect is particularly dramatic in rabbits, where infection of a rabbit with very few infectious wtRPV parti- REFERENCES cles, frequently results in death. Infection of rabbits with 5 1 104 RPVDB5R virions is not only not lethal, but fails Alcami, A., and Smith, G. L. (1992). A soluble receptor for interleukin-1b encoded by vaccinia virus: A novel mechanism of virus modulation of to cause fever or illness. However, in mice, where the the host response to infection. Cell 71, 153–167. mutant is also severly attenuated, some growth is ob- Alcami, A., and Smith, G. L. (1995). Vaccinia, cowpox, and camelpox served (Fig. 3). 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