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Myxoma virus is a leporipoxvirus of New World rabbits (Sylvilagus sp.) that induces a rapidly lethal infection known as myxomatosis in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, myxoma virus encodes a plethora of proteins to circumvent or inhibit a variety of host antiviral immune mechanisms. M-T7, the most abundantly secreted protein of myxoma virus-infected cells, was originally identified as an interferon-gamma receptor homolog (Upton, Mossman, and McFadden, Science 258, 1369-1372, 1992). Here, we demonstrate that M-T7 is dispensable for virus replication in cultured cells but is a critical virulence factor for virus pathogenesis in European rabbits. Disruption of both copies of the M-T7 gene in myxoma virus was achieved by the deletion of 372 bp of M-T7 coding sequences, replacement with a selectable marker, p7.5Ecogpt, and selection of a recombinant virus (vMyxlac-T7gpt) resistant to mycophenolic acid. vMyxlac-T7gpt expressed no detectable M-T7 protein and infected cells supernatants were devoid of any detectable interferon-gamma binding activities. Immunohistochemical staining with anti-beta-galactosidase and anti-CD43 antibodies demonstrated that in vMyxlac-T7gpt-infected rabbits the loss of M-T7 not only caused a dramatic reduction in disease symptoms and viral dissemination to secondary sites, but also dramatically influenced host leukocyte behavior. Notably, primary lesions in wild-type virus infections were generally underlayed by large masses of inflammatory cells that did not effectively migrate into the dermal sites of viral replication, whereas in vMyxlac-T7gpt infections this apparent block to leukocyte influx was relieved. A second major phenotypic distinction noted for the M-T7 knockout virus was the extensive activation of lymphocytes in secondary immune organs, particularly the spleen and lymph nodes, by Day 4 of the infection. This is in stark contrast to infection by wild-type myxoma virus, which results in relatively little, if any, cellular activation of germinal centers of spleen and lymph node by Day 4. We conclude that M-T7 functions early in infection to (1) retard inflammatory cell migration into infected tissues and (2) disrupt the communication between sentinel immune cells at the site of primary virus infection in the subdermis and lymphocytes in the secondary lymphoid organs, thereby disabling the host from mounting an effective cellular immune response. To summarize, in addition to neutralizing host interferon-gamma at infected sites, we propose that M-T7 protein also modifies leukocyte traffic in the vicinity of virus lesions, thus effectively severing the link between antigen presenting cells of the infected tissue and the effector lymphocytes of the peripheral immune organs.     

Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition

The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T2 protein were constructed. One mutant, deltaL113, containing intact copies of only two cysteine-rich domains, was not secreted and was incapable of binding rabbit TNF-alpha yet retained full ability to inhibit virus-induced apoptosis of RL-5 cells. Thus, the minimal domain of intracellular M-T2 protein required to inhibit apoptosis is distinct from that required by the extracellular M-T2 for functional TNF-alpha binding and inhibition. This is the first report of a virus-encoded immunomodular protein with two distinct antiimmune properties.     

Expression of the myxoma virus tumor necrosis factor receptor homologue and M11L genes is required to prevent virus-induced apoptosis in infected rabbit T lymphocytes

Myxoma virus is a leporipoxvirus that causes a highly lethal virulent disease known as myxomatosis in the European rabbit. An important aspect of myxoma virus pathogenesis is the ability of the virus to productively infect lymphocytes and spread to secondary sites via lymphatic channels. We investigated the infection of the CD4+ T lymphoma cell line RL-5 with myxoma virus and Shope fibroma virus, a related but benign leporipoxvirus, and observed that myxoma virus, but not Shope fibroma virus, was able to productively infect RL-5 cells. We also discovered that infection of RL-5 cells with Shope fibroma virus or attenuated myxoma virus mutants containing disruptions in either the T2 or the M11L gene resulted in the rapid induction of DNA fragmentation, followed by morphological changes and loss in cell integrity characteristic of cell death by apoptosis. Purified exogenous T2 protein was unable to prevent apoptosis, suggesting that T2 functions intracellularly. Thus, myxoma virus T2, originally described as a secreted homologue of the tumor necrosis factor receptor, and M11L, a novel transmembrane species with no known cellular homologue, function to extend virus host range for replication in rabbit T lymphocytes through the inhibition of apoptosis in infected T lymphocytes.     

Myxoma virus encodes an alpha2,3-sialyltransferase that enhances virulence

A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion with N-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that alpha2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host's defenses against infection are discussed.     

Interruption of cytokine networks by poxviruses: lessons from myxoma virus

Myxoma virus is an infectious poxvirus pathogen that induces a virulent systemic disease called myxomatosis in European rabbits. The disease is rapidly and uniformly fatal to susceptible rabbits and is characterized by generalized dysfunction of cellular immunity and multiple interruptions of the host cytokine network. A number of virus genes are classified as virulence factors because virus constructs bearing targeted gene disruptions induce attenuated disease symptoms. Many of these genes encode proteins that interact directly with effector elements of the host immune system. Included among these immunosubversive viral proteins are secreted mimics of host ligands or regulators (virokines) and homologues of cellular cytokine receptors (viroceptors). Five examples of these immune modulator proteins encoded by myxoma virus are reviewed: (1) myxoma growth factor, a member of the epidermal growth factor ligand superfamily; (2) SERP-1, a secreted serine proteinase inhibitor; (3) M11L, a receptor-like surface protein; (4) T2, a tumor necrosis factor receptor homologue; and (5) T7, an interferon-gamma receptor homologue. The origin of viral strategies designed to subvert immune regulation by host cytokines is considered in the context of the biology of myxoma virus within immunocompetent hosts.     

Secreted poxvirus chemokine binding proteins

Poxviruses encode a variety of immunomodulatory proteins that subvert the cytokine networks of infected hosts. Myxoma virus, a poxvirus pathogen of rabbits, expresses two distinct 35- to 40-kDa secreted glycoproteins that bind a broad spectrum of chemokines. The first of these, designated M-T7, is encoded by the T7 gene and is the first example of what is here referred to as type-I chemokine binding protein (CBP-I). M-T7 was initially discovered as a secreted viral homologue of cellular interferon-gamma receptor but binding studies indicate that purified M-T7 protein also interacts with members of the CXC, CC, and C chemokine families through the conserved heparin-binding domains. The second myxoma protein, M-T1, also called CBP-II, is a member of a larger superfamily of poxvirus proteins that includes related secreted 35-kDa proteins encoded by a wide variety of orthopoxviruses. Deletion analysis of either CBP-I or -II genes within recombinant poxvirus constructs revealed profound alterations in the trafficking of infiltrating leukocytes into virus-infected lesions. It is proposed that the interaction of CBP-I with the conserved heparin-binding domains found on most chemokines represents a novel mechanism for altering multiple chemokine functions in vivo. In summary, CBP-I and CBP-II are the first examples of secreted virus proteins that bind to multiple chemokine family members as part of a strategy to prevent the early phase of inflammatory cell migration into virus-infected tissues.     

Developmental and tissue-specific expression of human CD4 in transgenic rabbits

A major obstacle to understanding AIDS is the lack of a suitable small animal model for studying HIV-1 infection and the subsequent development of AIDS, and for testing diagnostic, therapeutic, and preventive modalities. Our goal is to produce a rabbit model for the study of AIDS. Here we report on the generation of transgenic rabbits that express the human CD4 (hCD4) gene. The transgene, which contains the coding region for hCD4 and approximately 23 kb of sequence upstream of the translation start site, was used previously to direct hcD4 expression on the surface of CD4+ T cells of transgenic mice (Gillespie et al., 1993: Mol Cell Biol 13:2952-2958). The hCD4 transgene was detected in five males and two females derived from the microinjection in five males and two females derived from the microinjection of 271 rabbit embryos. Both hCD4 RNA and protein were expressed in peripheral blood lymphocytes (PBLs) from all five males but neither of the females. Human CD4 was expressed on PBLs from F1 offspring of all founder males. T-cell subset analysis revealed that hCD4 expression was restricted to rabbit CD4 (rCD4) expressing lymphocytes; mature rCD4- rCD8+ lymphocytes did not express hCD4. In preliminary studies, PBLs from hCD4 transgenic rabbits produced greater amounts of HIV-1 p24 core protein following HIV-1 infection in vitro than HIV-1 p24 antigen in nontransgenic rabbit infected cultures. These results extend to rabbits our previous observation that this transgene contains the sequence elements required for high-level expression in the appropriate cells of transgenic mice. Furthermore, these and previous studies demonstrating that expression of hCD4 protein enhances HIV-1 infection of rabbit T cells in vitro, coupled with reports that normal, nontransgenic rabbits are susceptible to HIV-1 infection, suggests that the hCD4 transgenic rabbits described herein will have an increased susceptibility to HIV-1 infection. In vivo HIV-1 infection studies with these rabbits are under way.     

Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation

Non-isotopic in situ hybridisation was used at the electron microscope level to determine the localisation of viral RNA in dengue-2 infected mosquito cells at 14, 24, 48 and 72 h post-infection. In situ hybridisation was carried out on sections of dengue-2 infected mosquito cells using a digoxigenin-labelled DNA probe to the envelope protein gene sequence of the virus. Viral RNA was consistently localised over the rough endoplasmic reticulum and the virus-induced smooth membrane structures which form within the endoplasmic reticulum. During the later stages of infection electron-dense areas were observed to develop in close proximity to the smooth membrane structures. Electron microscopic in situ hybridisation showed that these denser areas contained both viral RNA and virus particles. Our results show that in dengue-2 infected mosquito cells the smooth membrane structures are an important site for the concentration of dengue viral RNA and its possible subsequent encapsidation into virus particles.     

Rotavirus disease, but not infection and development of intestinal histopathological lesions, is age restricted in rabbits

The rabbit model of rotavirus infection has proved to be useful for assessing active immunity and protection after infection or vaccination with virus or virus-like particles. One limitation of the rabbit model is that after experimental infection of rabbits, clinical diarrhea is not routinely induced. Lack of diarrhea in the rabbit model has been proposed to be due to the fluid absorptive capability of the cecum or attenuation of virus strains through tissue culture adaptation. To test whether a wild-type lapine rotavirus strain BAP (BAPwt) isolated from diarrheic rabbits would cause disease on passage in rabbits, 1-, 2-, 10-, and 16-week-old rabbits were orally inoculated with BAPwt, its tissue culture-adapted counterpart strain (BAP-2), tissue culture-adapted lapine strain ALA, or PBS. Lapine rotavirus infection in 1-week-old, but not >/=2-week-old, rabbits resulted in the development of disease characterized by soft, wet, yellow-to-brownish-green partially formed-to-liquid stools observed only at the time of virus antigen shedding. The level and duration of virus shedding after infection were prolonged in 1-week-old rabbits compared with rabbits >/=2 weeks of age. Although diarrhea was not observed beyond the first 2 weeks of life, histopathological changes, including villus shortening and fusion, increased vacuolation of epithelial cells, and mononuclear infiltration of the lamina propria, were observed throughout the small intestine between 12 and 120 h after ALA infection in 1-week-old, 1- to 2-month-old, and 11-month-old rabbits. In 11-month-old rabbits, onset of intestinal damage appeared to be slightly delayed, was less severe, and was not observed in the duodenum. There were no differences in the immune responses to rotavirus infection in rabbits of different age groups (1 week to 5 years of age). All lapine rotavirus-inoculated rabbits seroconverted and were protected from virus challenge at 28 days postinoculation. Like in mice, rotavirus disease is age restricted in rabbits.     

Myxoma virus in rabbits

Myxoma virus in European rabbits (Oryctolagus cuniculus) is one of the best documented examples of host-virus co-evolution. In the natural hosts (Sylvilagus brasiliensis or S. bachmani rabbits in the Americas), myxoma virus causes a benign cutaneous fibroma. In European rabbits, however, myxoma virus causes the fulminant disease, myxomatosis. When introduced into wild European rabbit populations in Australia, Europe and Great Britain, the virus was initially highly lethal, killing in excess of 99% of infected rabbits. Development of resistance was encouraged by the emergence of attenuated virus strains which allowed the survival of moderately resistant rabbits. This may have occurred more rapidly in hot climates, as high ambient temperatures increase the survival rate of infected rabbits. Resistant rabbits are less effective transmitters of the virus and this may encourage the emergence of more virulent virus strains. Little is known of the mechanism of resistance. There have been suggestions of non-genetic resistance. However, these are yet to be confirmed experimentally.     

Immunogenic properties of rabbit haemorrhagic disease virus structural protein VP60 expressed by a recombinant baculovirus: an efficient vaccine

We have constructed a recombinant baculovirus containing the gene encoding the structural protein VP60 from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). Infection of cultured Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of high yields of VP60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. A VP60-dose study showed that the recombinant protein was able to elicit a protective response in rabbits against a nasal challenge with 100 LD50 of RHDV. The effective dose able to protect 50% of the animals in the absence of adjuvant was found to be 10-25 micrograms of recombinant VP60.     

Resistance to alpha/beta interferons correlates with the epizootic and virulence potential of Venezuelan equine encephalitis viruses and is determined by the 5' noncoding region and glycoproteins

We compared the alpha/beta interferon (IFN-alpha/beta) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-alpha/beta resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5' noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.     

Role of gamma interferon in natural clearance of Bordetella pertussis infection

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.     

An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the salmonid disease furunculosis

Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. The disease is responsible for severe economic losses in intensively cultured salmon and trout. Bacterin vaccines provide inadequate protection against infection. We have constructed an aromatic-dependent mutant of A. salmonicida in order to investigate the possibility of an effective live-attenuated vaccine. The aroA gene of A. salmonicida was cloned in Escherichia coli, and the nucleotide sequence was determined. The codon usage pattern of aroA was found to be quite distinct from that of the vapA gene coding for the surface array protein layer (A layer). The aroA gene was inactivated by inserting a fragment expressing kanamycin resistance within the coding sequence. The aroA::Kar mutation was introduced into the chromosome of virulent A. salmonicida 644Rb and 640V2 by allele replacement by using a suicide plasmid delivery system. The aroA mutation did not revert at a detectable frequency (< 10(-11). The mutation resulted in attenuation when bacteria were injected intramuscularly into Atlantic salmon (Salmo salar L.). Introduction of the wild-type aroA gene into the A. salmonicida mutants on a broad-host-range plasmid restored virulence. A. salmonicida mutant 644Rb aroA::Kar persisted in the kidney of brown trout (Salmo trutta L.) for 12 days at 10 degrees C. Vaccination of brown trout with 10(7) CFU of A. salmonicida 644Rb aroA by intraperitoneal injection resulted in a 253-fold increase in the 50% lethal dose (LD50) compared with unvaccinated controls challenged with a virulent clinical isolate 9 weeks later. A second vaccination after 6 weeks increased the LD50 by a further 16-fold.     

Expression of myb-related genes in the moss, Physcomitrella patens

Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged < 5% of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30% of the cell volume in 14-21-DPN animals, and approximated adult levels (> 40%) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from > 10% of the cell volume at 27 DGA to < 5% in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells. Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70% of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75% of adult levels by 28 DPN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of rabbit renal mixed-function oxidases by phenobarbital: cell specific ultrastructural changes in the proximal tubule

Administration of phenobarbital (60 mg/kg) daily for 4 days to male rabbits resulted in induction of renal cytochrome P-450 (3.5-fold) and a corresponding increase in ethoxycoumarin-O-deethylase and benzphetamine-N-demethylase activity (17- and 4-fold, respectively). Kidney weight to body weight ratio and renal ethoxyresorufin-O-deethylase were not affected by phenobarbital pretreatment. Numerous focal areas of proliferation of smooth endoplasmic reticulum (SER) were evident in proximal tubule cells from phenobarbital treated rabbits while proximal tubular cells from control rabbits had only small and sparcely located aggregates of SER. Phenobarbital-induced SER proliferation was specifically localized to the S3 segment of the proximal tubule. Proliferation was not observed in S2 cells of the proximal tubule, cells of Henle's loop, distal tubules, or collecting tubules in rabbits pretreated with phenobarbital. These data demonstrate the biochemical heterogeneity of cell types within the proximal tubules of rabbits. Furthermore, induction of mixed-function oxidases specifically in S3 cells of the proximal tubule may be of toxicological significance in the metabolic activation of certain nephrotoxicants.     

Recombinant respiratory syncytial virus (RSV) bearing a set of mutations from cold-passaged RSV is attenuated in chimpanzees

A set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold-passaged (cp) respiratory syncytial virus (RSV) has been introduced into a recombinant wild-type strain of RSV. This recombinant virus, designated rA2cp, appears to replicate less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than either biologically derived or recombinant wild-type RSV. Infection with rA2cp also resulted in significantly less rhinorrhea and cough than infection with wild-type RSV. These findings confirm the role of the cp mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates.     

Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to the human immunodeficiency virus type 1 Vpu protein

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.