Acute Virulence Research Articles

Rapid Membrane Disruption by a Perforin-Like Protein Facilitates Parasite Exit from Host Cells

Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe Toxoplasma perforin-like protein 1 (TgPLP1), a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore formation. After intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to rapidly permeabilize the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture but resulted in a reduction greater than five orders of magnitude of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.

Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase

Chronic lung infections caused by Pseudomonas aeruginosa are the leading cause of morbidity and mortality for cystic fibrosis (CF) patients. Adaptation of P. aeruginosa to the CF lung results in the loss of acute virulence determinants and appears to activate chronic virulence strategies in this pathogen. In order to identify such strategies, a random transposon mutagenesis was performed and 18 genes that were required for optimal infection of alfalfa seedlings by FRD1, a CF isolate of P. aeruginosa, were recognized. The largest subset of genes (seven of the 18), were associated with central carbon metabolism, including the gene that encodes isocitrate lyase (ICL), aceA. Because FRD1 is avirulent in animal infection models, we constructed an ICL mutant in P. aeruginosa strain PAO1 in order to assess the requirement of ICL in mammalian infection. The PAO1 ICL mutant was less virulent in the rat lung infection model, indicating that ICL is required for the pathogenesis of P. aeruginosa in mammals. Furthermore, FRD1 showed increased ICL activity and expression of an aceA : : lacZ fusion compared to PAO1. We suggest that upregulation of ICL occurred during adaptation of FRD1 to the CF lung and that some of the novel virulence mechanisms employed by FRD1 to infect alfalfa seedlings may be the same mechanisms P. aeruginosa relies upon to persist within human niches.

Relationship between Escherichia coli Strains Causing Acute Cystitis in Women and the Fecal E. coli Population of the Host

Previous epidemiological assessments of the prevalence versus special-pathogenicity hypothesis for urinary tract infection (UTI) pathogenesis in women may have been confounded by underlying host population differences between women with UTI and healthy controls and have not considered the clonal complexity of the fecal Escherichia coli population of the host. In the present study, 42 women with acute uncomplicated cystitis served as their own controls for an analysis of the causative E. coli strain and the concurrent intestinal E. coli population. Clonality among the urine isolate and 30 fecal colonies per subject was assessed by repetitive-element PCR and macrorestriction analysis. Each unique clone underwent PCR-based phylotyping and virulence genotyping. Molecular analysis resolved 109 unique clones (4 urine-only, 38 urine-fecal, and 67 fecal-only clones). Urine clones exhibited a significantly higher prevalence of group B2 than fecal-only clones (69% versus 10%; P < 0.001) and higher aggregate virulence scores (mean, 6.2 versus 2.9; P < 0.001). In multilevel regression models for predicting urine clone status, significant positive predictors included group B2, 10 individual virulence traits, the aggregate virulence score, fecal dominance, relative fecal abundance, and (unique to the present study) a pauciclonal fecal sample. In summary, within the fecal E. coli populations of women with acute cystitis, pauciclonality, clonal dominance, virulence, and group B2 status are closely intertwined. Phylogenetic group B2 status and/or associated virulence factors may promote fecal abundance and pauciclonality, thereby contributing to upstream steps in UTI pathogenesis. This relationship suggests a possible reconciliation of the prevalence and special-pathogenicity hypotheses.

Impact of variation in acute virulence of BVDV1 strains on design of better vaccine efficacy challenge models

Due to antigenic differences between BVDV1 and BVDV2 strains, both pestivirus species are included in U.S. vaccines. The efficacy of these vaccines in preventing acute infections is evaluated based on reduction of clinical disease. While high virulence BVDV2 strains are used in U.S. vaccine efficacy studies, the BVDV1 strain used (NY-1) produces very little in the way of clinical disease. In order to identify a BVDV1 strain that generates a more pronounced clinical presentation, three field strains were compared to NY-1. Infection with two of the field strains resulted in significantly more pronounced clinical disease compared to NY-1. Decreasing the inoculation of a field strain by two logs did not significantly change clinical presentation.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in São Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits.

Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients

The opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4-hydroxy-2-heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss-of-function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased beta-lactamase activity that increased tolerance to ceftazidime, a widely used beta-lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.

Elimination of ie 1 Significantly Attenuates Murine Cytomegalovirus Virulence but Does Not Alter Replicative Capacity in Cell Culture

The major immediate-early (MIE) genes of cytomegaloviruses (CMV) are broadly thought to be decisive regulators of lytic replication and reactivation from latency. To directly assess the role of the MIE protein IE1 during the infection of murine CMV (MCMV), we constructed an MCMV with exon 4 of the ie1 gene deleted. We found that, independent of the multiplicity of infection, the resulting recombinant virus, MCMVdie1, which fails to express the IE1 protein, was fully competent for early gene expression and replicated in different cultured cell types with identical kinetics to those of parental or revertant virus. Immunofluorescence microscopy studies revealed that MCMVdie1 was greatly impaired in its capacity to disrupt promyelocytic leukemia bodies in NIH 3T3 cells early after infection, a process that has been proposed to increase viral transcription efficiency. We examined MCMVdie1 in the murine model using both immunocompetent BALB/c and severe combined immunodeficient (SCID) mice. When MCMVdie1 was inoculated into these two types of mice, significantly lower viral titers were detected in infected organs than in those of the wild-type virus-infected animals. Moreover, the ie1-deficient MCMV exhibited a markedly reduced virulence. While all animals infected with 5 x 10(4) PFU of parental virus died by 30 days postinfection, SCID mice infected with a similar dose of MCMVdie1 did not succumb before 60 days postinfection. The in vivo defective growth phenotype of MCMVdie1 was abrogated upon rescue of ie1. These results demonstrate the significance of the ie1 gene for promoting an acute MCMV infection and virulence yet indicate that MCMV is able to grow in vivo, although impaired, in the absence of the ie1 gene.

Virus persistence in an animal model of multiple sclerosis requires virion attachment to sialic acid coreceptors.

Persistent Theiler's virus infection in the central nervous system (CNS) of mice provides a highly relevant animal model for multiple sclerosis. The low-neurovirulence DA strain uses sialic acid as a coreceptor for cell binding before establishing infection. During adaptation of DA virus to growth in sialic acid-deficient cells, three amino acid substitutions (G1100D, T1081I, and T3182A) in the capsid arose, and the virus no longer used sialic acid as a coreceptor. The adapted virus retained acute CNS virulence, but its persistence in the CNS, white matter inflammation, and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. Substitution of G1100D in an infectious DA virus cDNA clone demonstrated a major role for this mutation in loss of sialic acid binding and CNS persistence. These data indicate a direct role for sialic acid binding in Theiler's murine encephalomyelitis virus persistence and chronic demyelinating disease.

Identification of quantitative trait loci controlling acute virulence in Toxoplasma gondii.

Strains of Toxoplasma gondii can be grouped into three predominant clonal lineages with members of the type I group being uniformly lethal in mice. To elucidate the basis of this extreme virulence, a genetic cross was performed between a highly virulent type I strain (GT-1) and a less-virulent type III strain (CTG), and the phenotypes of resulting progeny were analyzed by genetic linkage mapping. Analysis of independent recombinant progeny identified several quantitative trait loci that contributed to acute virulence. A major quantitative trait locus located on chromosome VII accounted for approximately 50% of the virulence phenotype, whereas a minor locus on chromosome IV, linked to the ROP1 gene, accounted for approximately 10%. These loci are conserved in other type I strains, indicating that acute virulence is controlled by discrete genes common to the type I lineage.

Genetic approaches to studying virulence and pathogenesis in Toxoplasma gondii.

Toxoplasma gondii is a common protozoan parasite that causes disease in immunocompromised humans. Equipped with a wide array of experimental tools, T. gondii has rapidly developed as a model parasite for genetic studies. The population structure of T. gondii is highly clonal, consisting of three distinct lineages that differ dramatically in virulence. Acute virulence is probably mediated by the genetic differences that distinguish strain types. We have utilized a combination of genetic approaches to investigate the acute virulence of toxoplasmosis using the mouse model. These studies reveal the surprising finding that pathogenicity is due to the over-stimulation of normally protective immune responses. Classical genetic linkage mapping studies indicate that genes that mediate acute virulence are linked to chromosome VII in the parasite. To increase the resolution of genetic mapping studies, single-nucleotide polymorphisms are being developed based on an extensive database of expressed sequence tags (ESTs) from T. gondii. Separately, DNA microarray studies are being used to examine the expression of parasite and host genes during infection. Collectively, these approaches should improve current understanding of virulence and pathogenicity in toxoplasmosis.

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines.

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.

Toxoplasma gondii induction of interleukin-12 is associated with acute virulence in mice and depends on the host genotype

The intracellular parasite Toxoplasma gondii can influence host resistance by modulating immune functions in various cell types. The stimulation of interleukin (IL)-12 production in macrophages, dendritic cells and neutrophils by T. gondii has been implicated to be important for skewing anti-parasite immunity early after infection as well as in mediating the pathologic effects induced by the parasite. The present study demonstrates secretion of IL-12 p40 and the bioactive p70 heterodimer by inflammatory macrophages following exposure to live Toxoplasma or tachyzoite lysate. Parasite induction of IL-12 occurred in a dose-dependent manner. Predigestion of T. gondii lysate with proteinase K abrogated its IL-12 inducing activity, thus indicating that a parasite protein(s) triggers this response. Macrophages from various mouse inbred strains showed a differential responsiveness: cells from T. gondii-susceptible mice released more IL-12 upon toxoplasmic challenge than those from resistant mice, although the infection rate and intracellular parasite growth were similar. In triggering macrophage production of IL-12, tachyzoites proved superior to bradyzoites prepared from the same T. gondii isolate. Furthermore, parasites of a mouse-virulent isolate became less efficient inducers of IL-12 following attenuation. The parallel loss in macrophage stimulation in vitro and acute virulence in vivo suggests a linkage of both parasite capacities. Together with the correlation on host side between the genotype-dependent mouse susceptibility to infection and cellular responsiveness to the parasite trigger, these findings indicate that an overproduction of parasite-induced IL-12 might represent a basic mechanism of T. gondii pathogenicity.

Attenuation of mouse-virulent Toxoplasma gondii parasites is associated with a decrease in interleukin-12-inducing tachyzoite activity and reduced expression of actin, catalase and excretory proteins

Determinants of Toxoplasma gondii virulence are still unknown, although genetic markers associated with T. gondii pathogenicity or host susceptibility to infection have been identified. To define indicator proteins of mouse virulence, type I strain parasites were attenuated by continuous passage in fibroblast culture and compared with the parental strain passaged in mice. The loss of acute virulence, evident by a 1 000-fold higher pathogen dose causing 100% lethality in mice correlated with a less efficient infection of inflammatory cells at the site of inoculation, while parasite proliferation and invasiveness in vitro proved unimpaired. Infection with the attenuated parasites elicited earlier local interleukin-12 and strong interferon-γ responses in vivo, although the activity that triggers interleukin-12 secretion in macrophages is reduced in the attenuated compared to the virulent strain variant. The interleukin-12-inducing T. gondii stimulus was identified as a protein(s) present in tachyzoite excretory products. Comparative proteome analysis combined with immunodetection and quantitation of a variety of T. gondii antigens indicated that the steady-state levels of actin, catalase, microneme protein 5, as well as dense granule proteins 1, 2, 3, 4, 5, 7, 8 and nucleoside triphosphate hydrolase 1 are decreased in the attenuated phenotype, whereas the surface antigen 1 and rhoptry protein 1 are produced at a similar level by virulent and attenuated parasites. In conclusion, these findings reveal a correlation between the efficient establishment of T. gondii infection in vivo and parasite synthesis of actin, catalase and several excretory proteins, and thus postulate a role for these molecules in acute virulence.

Experimental Approaches to Understanding Virulence in Toxoplasmosis

Toxoplasma gondii is a widespread protozoan parasite that causes severe disease only in immunocompromised individuals. Equipped with excellent animal models and relatively advanced systems for genetics, T. gondii provides an excellent system for understanding pathogenesis. Resistance to toxoplasmosis is governed by rapid innate and adaptive immunity that is characterized by a Thl type profile of cytokines. Despite this effective response, acute infections can cause considerable damage and the parasite effectively establishes a long-term chronic infection that predisposes the host to reactivation and provides a means of eventual transmission. This complex interaction is brought about by the differentiation of the parasite from a rapidly replicating, lytic form (known as the tachyzoite) to a slow-growing form (known as the bradyzoite) that gives rise to chronic infection. The population structure of T. gondii is remarkably clonal, consisting of just three predominant lineages that are geographically widespread and found in a variety of hosts including humans. Acute virulence is strongly associated with the type I genotype which exhibits an enhanced replication rate in vitro and higher tissue burdens in vivo relative to non-virulent lineages. The pathology associated with acute infection appears to be due to excessive production of acute inflammatory mediators, suggesting that disease is partly due to over-response of the host immune system. A combination of refined animal models and newly developed genetic tools for establishing the relative contribution of genes to pathogenesis will enable a comprehensive analysis of the molecular basis of virulence in toxoplasmosis.

Targeted disruption of the GRA2 locus in Toxoplasma gondii decreases acute virulence in mice.

Following invasion into the host cell, the protozoan Toxoplasma gondii secretes a variety of proteins that modify the parasitophorous vacuole. Within the vacuole, the 28-kDa dense granule protein known as GRA2 is specifically targeted to the tubulovesicular network which forms connections with the vacuolar membrane. To investigate the importance of GRA2, we derived from strain RH a mutant T. gondii line in which GRA2 was disrupted by replacement with the marker Ble (selecting for phleomycin resistance). The Deltagra2 mutant invaded and grew normally in both fibroblasts and macrophages in vitro; however, it was less virulent during acute infection in mice. The survival rate of mice inoculated with Deltagra2 was significantly higher; some infected mice survived the acute infection, whereas all mice infected with the wild-type strain RH succumbed to early death. Chronic infection by Deltagra2 was detected by positive serology, immunohistochemical detection of parasites and cysts in the brain, and reisolation of parasites by bioassay at 6 weeks postinfection. Thus, absence of GRA2 partially attenuates the virulence of T. gondii during the acute phase of infection and allows for establishment of chronic infection by the otherwise highly virulent RH strain. These results establish that GRA2 plays an important role during in vivo infection and provide a potential model for examining acute pathogenesis by T. gondii.

Targeted Disruption of the GRA2 Locus inToxoplasma gondii Decreases Acute Virulence in Mice

Following invasion into the host cell, the protozoanToxoplasma gondii secretes a variety of proteins that modify the parasitophorous vacuole. Within the vacuole, the 28-kDa dense granule protein known as GRA2 is specifically targeted to the tubulovesicular network which forms connections with the vacuolar membrane. To investigate the importance of GRA2, we derived from strain RH a mutant T. gondii line in which GRA2 was disrupted by replacement with the marker Ble (selecting for phleomycin resistance). The Δgra2 mutant invaded and grew normally in both fibroblasts and macrophages in vitro; however, it was less virulent during acute infection in mice. The survival rate of mice inoculated with Δgra2 was significantly higher; some infected mice survived the acute infection, whereas all mice infected with the wild-type strain RH succumbed to early death. Chronic infection by Δgra2 was detected by positive serology, immunohistochemical detection of parasites and cysts in the brain, and reisolation of parasites by bioassay at 6 weeks postinfection. Thus, absence of GRA2 partially attenuates the virulence of T. gondii during the acute phase of infection and allows for establishment of chronic infection by the otherwise highly virulent RH strain. These results establish that GRA2 plays an important role during in vivo infection and provide a potential model for examining acute pathogenesis by T. gondii.

Escherichia coli virulence factors and serotypes in acute bacterial prostatitis.

Escherichia coli is the most frequent pathogen in both acute bacterial prostatitis and acute uncomplicated urinary infections. To assess the virulence profiles of E. coli in acute prostatitis, the serotypes and virulence factor (VF) genotypes were determined. We studied 107 E. coli isolates from cases of acute bacterial prostatitis, 76 isolates from acute pyelonephritis, 194 isolates from acute Cystitis and 80 fecal isolates from healthy people. All pyelonephritis and cystitis isolates were from women. Seven urovirulence determinants were analyzed by DNA colony hybridization, including the genes for type 1 fimbria (pil), P fimbria (pap), S fimbria (sfa), afimbrial adhesin AFA-I (afaI), alpha-hemolysin (hly), cytotoxic necrotizing factor 1 (cnf1) and aerobactin (aer). O:H:K serotypes were also determined. With the exception of pil and afaI, all VFs were significantly more often associated with prostatitis, pyelonephritis and cystitis isolates than with the fecal isolates. The prevalence of sfa, hly and cnf1 was higher in prostatitis isolates than in pyelonephritis and cystitis isolates, and the pap+sfa+hly+cnf1+ genotype was dominant among prostatitis isolates (48.8%). Nine O serotype (O1, O2, O4, O6, O16, O18, O22, O25 and O75) accounted for 79.4%, 73.7% and 78.4% of the prostatitis, pyelonephritis and cystitis strains, respectively. There was an apparent correlation between serotype and genotype in uropathogenic E. coli. The predominance of O serotypes in female urinary tract infections and a high percentage of multiple VFs among the prostatitis isolates suggested that VFs play important roles in the pathogenesis of acute bacterial prostatitis.

Acute virulence in mice is associated with markers on chromosome VIII in Toxoplasma gondii

Toxoplasma gondii has an unusual population structure consisting of three widely distributed clonal lineages. Acute virulence in mice is strictly observed in type I strains, indicating that a genetic determinant(s) unique to this lineage controls acute pathogenesis. We have analyzed several naturally occurring recombinant strains of T. gondii that carry allele 1 at the SAG1 locus; this allele is characteristic of the type I strains and was previously found to be 100% correlated with the acute virulence phenotype. Recombinant strains G622-M and ROD both had a predominantly type III genotype, with the significant exception of allele 1 at the SAG1 locus. Although these two strains had virtually identical multilocus genotypes, they differed in their virulence in mice. Strain ROD was virulent whereas strain G622-M was nonvirulent, thus demonstrating that the presence of allele 1 at SAG1 is not alone sufficient to confer acute virulence. Several sequence polymorphisms upstream of SAG1 were found to be highly correlated with the acutely virulent lineages. Collectively, these results suggest that acute virulence is regulated by a region linked to the SAG1 locus on chromosome VIII in T. gondii.

Persistence of Virus-Specific IgM and Clinical Recovery after Japanese Encephalitis *

We have searched for evidence of a chronic Japanese encephalitis virus (JEV) infection in six Thai patients convalescing from acute Japanese encephalitis (JE) in whom JEV-specific IgM antibody was last detected 116 to 350 days after their acute illness. These six patients were compared with 94 other JE patients matched for age, sex and serological response and in whom JEV-specific IgM was either short-lived (less than 90 days) or not tested. All patients were evaluated for the presence or absence of seven abnormal neurological signs over a 1- to 2-year period. During the first 30 days of illness the mean numbers (+/- S.E.M.) of abnormal signs per patients for the IgM and control groups were 3.8 +/- 0.3 and 2.3 +/- 0.1, respectively (P less than 0.01). After 1 year the six IgM patients still had significantly more abnormal neurological signs than controls (1.3 +/- 0.3 and 0.6 +/- 0.1, respectively [P less than 0.01]). By 2 years, the IgM group showed no neurological impairment; examination of cerebrospinal fluids revealed no evidence of subclinical viral infections. The recovery of the six IgM patients between 1 and 2 years after their relatively severe acute illness suggests that IgM antibody persistence was related to acute virulence rather than chronicity of the JEV infection.