Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice

Neuroadapted Sindbis virus (NSV) causes acute encephalitis and paralyzes and kills adult mice unless they are treated with primary immune serum after infection. To study the nature and specificity of curative antibodies, we gave mice 30 different monoclonal antibodies (MAbs) against Sindbis virus (SV) 24 h after lethal intracerebral inoculation of NSV. By the time of MAb treatment, NSV replication in the brain had been well established (7.5 X 10(7) PFU/g). Seventeen MAbs directed against multiple biological domains on the NSV E1 and E2 envelope glycoproteins prevented paralysis and death. Anticapsid MAbs failed to protect. Altogether, 15 of 17 curative MAbs either neutralized NSV infectivity or lysed NSV-infected cells with complement, but neither ability was necessary or sufficient to guarantee recovery. All 5 protective anti-E2 MAbs neutralized NSV infectivity; 6 of 10 protective anti-E1 MAbs neutralized NSV; 4 did not. Plaque assay or immunohistochemical staining showed that neutralizing and nonneutralizing curative MAbs decreased NSV in the brain, brainstem, and spinal cord. Despite high neutralization titers, hyperimmune anti-SV and anti-NSV mouse sera prevented only 6 and 30% of deaths, respectively, while primary immune sera prevented 50 (SV) and 90% (NSV) of deaths. Secondary intravenous immunization with a live virus apparently diminished, obscured, or failed to boost a class of protective antibodies. When separate mouse groups were given these 30 MAbs 24 h before lethal intracerebral inoculation of NSV, a slightly different set of 17 neutralizing or nonneutralizing anti-E1 and anti-E2 antibodies protected. Two nonneutralizing MAbs and hyperimmune anti-SV serum, which had failed to promote recovery, prophylactically protected 100% of the mice. The antibody requirements or mechanisms of prophylaxis and recovery may differ.     

Expression of antigens in virulent and avirulent Indian strains ofLeishmania donovani

Indo-Gen mediated surface labelling with¹²⁵I demonstrated differences in surface oriented antigens between virulent and virulent promastigote ofLeishniania donovani, In case of virulent strains, surface polypeptides with molecular masses of 63, 53, 42 and 38 kDa were found to be labelled with¹²⁵I whereas in the case of aviralent stains 68, 55, 50, 46, 42 and 33 Da, components were iodinated. Further studies by immunoblot assay using different subcellular fractions of virulent and avirulent parasites demonstrated that antibody raised against gp63 cross-reacted with the 63 and 60 kDa antigen of the virulent and avirulentLeishmania donovani strains of Indian origin respectively. It indicates that these two polypeptides are antigenically similar. When virulent and avirulent cells were grown in the presence of varying concentration of tunicarnycin and immunoblot with anti gp63, it was observed that with increasing concentration of tunicamycin the 63 kDa polypeptide of the virulent cells shifted to approximately 58–57 kDa and the 60 kDa polypoptide of the aviruleni cells shifted to 57 kDa. This suggests that glycosylation may play an important role in antigenic variation between virulent and avirulent parasites.     

Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis

We used Porphyromonas gingivalis gene microarrays to compare the total gene contents of the virulent strain W83 and the avirulent type strain, ATCC 33277. Signal ratios and scatter plots indicated that the chromosomes were very similar, with approximately 93% of the predicted genes in common, while at least 7% of them showed very low or no signals in ATCC 33277. Verification of the array results by PCR indicated that several of the disparate genes were either absent from or variant in ATCC 33277. Divergent features included already reported insertion sequences and ragB, as well as additional hypothetical and functionally assigned genes. Several of the latter were organized in a putative operon in W83 and encoded enzymes involved in capsular polysaccharide synthesis. Another cluster was associated with two paralogous regions of the chromosome with a low G+C content, at 41%, compared to that of the whole genome, at 48%. These regions also contained conserved and species-specific hypothetical genes, transposons, insertion sequences, and integrases and were located adjacent to tRNA genes; thus, they had several characteristics of pathogenicity islands. While this global comparative analysis showed the close relationship between W83 and ATCC 33277, the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277 is suggestive of chromosomal islands that may have been acquired by lateral gene transfer.     

Signals involved in Arabidopsis resistance to Trichoplusia ni caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringae pathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1, pad4, eds5, and sid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance to T. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv. maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 or avrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with Psm ES4326(avrRpt2) or Psm ES4326(avrB), nahG transgenic and npr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahG and npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.     

A comparative study of virulent and avirulent strains of Chromobacterium violaceum

A clinical isolate and a soil isolate of Chromobacterium violaceum were compared to determine differences in virulence-related characteristics. Purified lipopolysaccharide (endotoxin) from the virulent, clinical strain was more reactive than that from the avirulent soil strain as determined by the Limulus amebocyte lysate assay. There were no differences in hemolysin or cyanide production between the two strains. The virulent strain was more resistant to phagocytosis and intracellular killing by human polymorphonucleocytes. The clinical strain showed a superoxide dismutase activity 30% higher and a catalase activity fivefold higher than the activities of the soil-isolated strain. The clinical strain also was capable of producing approximately twice as much hydrogen peroxide during growth as compared with the soil isolate. This study suggests that virulence of C. violaceum may be, at least in part, associated with endotoxin, and some protection of the virulent, clinical strain from phagocytic attack is afforded by elevated levels of superoxide dismutase and catalase.     

Adherence as a method of differentiating virulent and avirulent strains of Vibrio parahaemolyticus.

Usually only Kanagawa-positive strains of Vibrio parahaemolyticus are considered virulent; yet, a significant portion of V. parahaemolyticus food poisonings appear to be caused by Kanagawa-negative strains. Therefore, additional and more accurate measurements of a strain's food-poisoning potential are needed. Adherence of V. parahaemolyticus to human fetal intestinal (HFI) cells in vitro seems to offer this information. All strains of V. parahaemolyticus adhered to the HFI cells, but the degree of adherence was related to a number of factors. These included the age of the culture, the strain's Kanagawa reaction and source, the length of time the bacteria were exposed to the HFI cells, and the composition of the growth medium. Cells harvested during the late log phase of growth adhered more intensely than those harvested from the late stationary phase. Virulent strains, i.e., those involved in food poisoning, were observed to have a high adherence ability regardless of their Kanagawa reaction, whereas Kanagawa-negative strains isolated from seafood exhibited weak adherence intensities. Kanagawa-positive strains isolated from seafood adhered strongly to the HFI cells. The difference between the virulent and avirulent strains was quantitative in nature, and the greatest differences in adherence intensities were observed after short (10 to 15 min) exposure times. The presence of ferric iron in the growth medium was found to increase the adherence intensities of the virulent strains.     

Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene.

We developed a model system to study the signal transduction pathways leading to the activation of Arabidopsis thaliana genes involved in the defense against pathogen attack. Here we describe the identification and characterization of virulent and avirulent Pseudomonas syringae strains that elicit disease or resistance symptoms when infiltrated into Arabidopsis leaves. The virulent and avirulent strains were characterized by determining growth of the pathogen in Arabidopsis leaves and by measuring accumulation of mRNA corresponding to Arabidopsis phenylalanine ammonia-lyase (PAL), beta-1,3-glucanase (BG), and chalcone synthase (CHS) genes in infected leaves. The virulent strain, P. syringae pv maculicola ES4326, multiplied 10(5)-fold in Arabidopsis leaves and strongly elicited BG1, BG2, and BG3 mRNA accumulation but had only a modest effect on PAL mRNA accumulation. In contrast, the avirulent strain, P. syringae pv tomato MM1065, multiplied less than 10-fold in leaves and had only a minimal effect on BG1, BG2, and BG3 mRNA accumulation, but it induced PAL mRNA accumulation. No accumulation of CHS mRNA was found with either ES4326 or MM1065. We also describe the cloning of a putative avirulence (avr) gene from the avirulent strain MM1065 that caused the virulent strain ES4326 to grow less well in leaves and to strongly elicit PAL but not BG1 and BG3 mRNA accumulation. These results suggest that the Arabidopsis PAL and BG genes may be activated by distinct signal transduction pathways and show that differences in plant gene induction by virulent and avirulent strains can be attributed to a cloned presumptive avr gene.     

Pathogenesis of street rabies virus infections in resistant and susceptible strains of mice.

Seven strains of mice were examined to determine why susceptibility differences and variations in clinical central nervous system (CNS) disease occurred among these animals after intraperitoneal inoculation of street rabies virus (SRV). Trace experiments for infectious virus indicated that these differences were associated with restriction of virus replication within the CNS. Limitation of viral replication appeared to correlate with the antibody response in that prominent serum anti-SRV neutralizing antibody titers were detected in resistant strains, whereas susceptible strains produced minimal amounts of antibody until their death. The importance of the immune response was reaffirmed with cyclophosphamide studies in that all resistant SJL/J mice died after immunosuppressive treatment. In contrast, cyclophosphamide-treated SJL/J mice whose immune systems were reconstituted with either unfractionated immune spleen cells or with sera 24 h after SRV inoculation survived a lethal dose of SRV. More importantly, immunosuppressed SJL/J and immunodeficient athymic mice were protected when reconstituted with immune serum 72 h after SRV inoculation, a time in which infectious virus was detected in the spinal cords of some mice but was not present in the peritoneal cavity. Additional studies showed that antibody in the cerebrospinal fluid was unimportant in the resistance of mouse strains which remained clinically asymptomatic, but it appeared to be associated with the survival of mice which developed clinical CNS disease. Furthermore, CNS resistance to intranasal or intracerebral inoculation with challenge virus standard rabies virus developed as early as 5 days post-intraperitoneal inoculation of SRV.     

Haemocyte changes in resistant and susceptible strains of D. melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi

Two strains of Drosophila melanogaster (resistant and susceptible) were parasitized by a virulent or avirulent strain of the parasitoid wasp Leptopilina boulardi. The success of encapsulation depends on both the genetic status of the host strain and the genetic status of the parasitoid strain: the immune cellular reaction (capsule) is observed only with the resistant strain-avirulent strain combination. The total numbers of host haemocytes increased in all 4 combinations, suggesting that an immune reaction was triggered in all hosts. Resistant host larvae infected with the virulent or avirulent strains of parasitoid wasp had slightly more haemocytes per mm(3) than did susceptible host larvae at the beginning of the reaction (less than 15 h post-parasitization). This difference disappeared later. Only the virulent parasitoid strain caused the production of a high percentage of altered lamellocytes (from a discoid shape to a bipolar shape), half the total number of lamellocytes are altered. This suggests that the alteration of lamellocyte shape alone is not sufficient to explain the lack of capsule formation seen in resistant hosts parasitized by the virulent strain. Lastly, there were very few altered lamellocytes in resistant or susceptible hosts parasitized by the avirulent parasitoid strain, two combinations in which no capsule was formed. As is now established for Drosophila-parasitoid interactions, virus-like particles contained in the long gland of the female wasp affect the morphology of the lamellocytes. The results presented here are further proof of the action (direct or indirect) of virus like particles of the virulent strain on lamellocytes.     

Molecular basis of Sindbis virus neurovirulence in mice.

We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.