Investigation and management of serum sodium after subarachnoid haemorrhage (SaSH): a survey of practice in the United Kingdom and Republic of Ireland.

BACKGROUND: Hyponatraemia is a common complication of aneurysmal subarachnoid haemorrhage (SAH). We aimed to determine current neurosurgical practice for the identification, investigation and management of hyponatraemia after SAH. METHODS: An online questionnaire was completed by UK and Irish neurosurgical trainees and consultant collaborators in the Sodium after Subarachnoid Haemorrhage (SaSH) audit. RESULTS: Between August 2019 and June 2020, 43 responses were received from 31 of 32 UK and Ireland adult neurosurgical units (NSUs). All units reported routine measurement of serum sodium either daily or every other day. Most NSUs reported routine investigation of hyponatraemia after SAH with paired serum and urinary osmolalities (94%), urinary sodium (84%), daily fluid balance (84%), but few measured glucose (19%), morning cortisol (13%), or performed a short Synacthen test (3%). Management of hyponatraemia was variable, with units reporting use of oral sodium supplementation (77%), fluid restriction (58%), hypertonic saline (55%), and fludrocortisone (19%). CONCLUSIONS: Reported assessment of serum sodium after SAH was consistent between units, whereas management of hyponatraemia varied. This may reflect the lack of a specific evidence-base to inform practice.

Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells.

Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

How does discharge against medical advice affect risk of mortality and unplanned readmission? A retrospective cohort study set in a large UK medical admissions unit.

OBJECTIVES: To assess the frequency of discharge against medical advice (DAMA) in a large UK teaching hospital, explore factors which increase the risk of DAMA and identify how DAMA impacts patient risk of mortality and readmission. DESIGN: Retrospective cohort study. SETTING: Large acute teaching hospital in the UK. PATIENTS: 36 683 patients discharged from the acute medical unit of a large UK teaching hospital between 1 January 2012 and 31 December 2016. MEASUREMENTS: Patients were censored on 1 January 2021. Mortality and 30-day unplanned readmission rates were assessed. Deprivation, age and sex were taken as covariates. RESULTS: 3% of patients discharged against medical advice. These patients were younger (median age (years) (IQR)): planned discharge (PD) 59 (40-77); DAMA 39 (28-51), predominantly of male sex (PD 48%; DAMA 66%) and were of greater social deprivation (in three most deprived quintiles PD 69%; DAMA 84%). DAMA was associated with increased risk of death in patients under the age of 33.3 years (adjusted HR 2.6 (1.2-5.8)) and increased incidence of 30-day readmission (standardised incidence ratio 1.9 (1.5-2.2)). LIMITATIONS: Readmission to acute hospitals outside of the local health board may have been missed. We were unable to include information regarding comorbidity or severity of presentation. CONCLUSIONS: These data highlight the vulnerability of younger patients who DAMA, even in a free-at-the-point-of-delivery healthcare setting.

Prospective, multicentre study of screening, investigation and management of hyponatraemia after subarachnoid haemorrhage in the UK and Ireland.

BACKGROUND: Hyponatraemia often occurs after subarachnoid haemorrhage (SAH). However, its clinical significance and optimal management are uncertain. We audited the screening, investigation and management of hyponatraemia after SAH. METHODS: We prospectively identified consecutive patients with spontaneous SAH admitted to neurosurgical units in the United Kingdom or Ireland. We reviewed medical records daily from admission to discharge, 21 days or death and extracted all measurements of serum sodium to identify hyponatraemia (<135 mmol/L). Main outcomes were death/dependency at discharge or 21 days and admission duration >10 days. Associations of hyponatraemia with outcome were assessed using logistic regression with adjustment for predictors of outcome after SAH and admission duration. We assessed hyponatraemia-free survival using multivariable Cox regression. RESULTS: 175/407 (43%) patients admitted to 24 neurosurgical units developed hyponatraemia. 5976 serum sodium measurements were made. Serum osmolality, urine osmolality and urine sodium were measured in 30/166 (18%) hyponatraemic patients with complete data. The most frequently target daily fluid intake was >3 L and this did not differ during hyponatraemic or non-hyponatraemic episodes. 26% (n/N=42/164) patients with hyponatraemia received sodium supplementation. 133 (35%) patients were dead or dependent within the study period and 240 (68%) patients had hospital admission for over 10 days. In the multivariable analyses, hyponatraemia was associated with less dependency (adjusted OR (aOR)=0.35 (95% CI 0.17 to 0.69)) but longer admissions (aOR=3.2 (1.8 to 5.7)). World Federation of Neurosurgical Societies grade I-III, modified Fisher 2-4 and posterior circulation aneurysms were associated with greater hazards of hyponatraemia. CONCLUSIONS: In this comprehensive multicentre prospective-adjusted analysis of patients with SAH, hyponatraemia was investigated inconsistently and, for most patients, was not associated with changes in management or clinical outcome. This work establishes a basis for the development of evidence-based SAH-specific guidance for targeted screening, investigation and management of high-risk patients to minimise the impact of hyponatraemia on admission duration and to improve consistency of patient care.

Corticotroph isolation from Pomc-eGFP mice reveals sustained transcriptional dysregulation characterising a mouse model of glucocorticoid-induced suppression of the hypothalamus-pituitary-adrenal axis.

Glucocorticoids (GC) are prescribed for periods > 3 months to 1%-3% of the UK population; 10%-50% of these patients develop hypothalamus-pituitary-adrenal (HPA) axis suppression, which may last over 6 months and is associated with morbidity and mortality. Recovery of the pituitary and hypothalamus is necessary for recovery of adrenal function. We developed a mouse model of dexamethasone (DEX)-induced HPA axis dysfunction aiming to further explore recovery in the pituitary. Adult male wild-type C57BL6/J or Pomc-eGFP transgenic mice were randomly assigned to receive DEX (approximately 0.4 mg kg-1 bodyweight day-1 ) or vehicle via drinking water for 4 weeks following which treatment was withdrawn and tissues were harvested after another 0, 1, and 4 weeks. Corticotrophs were isolated from Pomc-eGFP pituitaries using fluorescence-activated cell sorting, and RNA extracted for RNA-sequencing. DEX treatment suppressed corticosterone production, which remained partially suppressed at least 1 week following DEX withdrawal. In the adrenal, Hsd3b2, Cyp11a1, and Mc2r mRNA levels were significantly reduced at time 0, with Mc2r and Cyp11a1 remaining reduced 1 week following DEX withdrawal. The corticotroph transcriptome was modified by DEX treatment, with some differences between groups persisting 4 weeks following withdrawal. No genes supressed by DEX exhibited ongoing attenuation 1 and 4 weeks following withdrawal, whereas only two genes were upregulated and remained so following withdrawal. A pattern of rebound at 1 and 4 weeks was observed in 14 genes that increased following suppression, and in six genes that were reduced by DEX and then increased. Chronic GC treatment may induce persistent changes in the pituitary that may influence future response to GC treatment or stress.

Neuroadapted yellow fever virus strain 17D: a charged locus in domain III of the E protein governs heparin binding activity and neuroinvasiveness in the SCID mouse model.

A molecular clone of yellow fever virus (YFV) strain 17D was used to identify critical determinants of mouse neuroinvasiveness previously localized to domain III of the neuroadapted SPYF-MN virus envelope protein. Three candidate virulence substitutions (305F-->V, 326K-->E, and 380R-->T) were individually evaluated for their roles in this phenotype in a SCID mouse model. The virus containing a glutamic acid residue at position 326 of the envelope protein (326E) caused rapidly lethal encephalitis, with a mortality rate and average survival time resembling those of the parental SPYF-MN virus. Determinants at positions 380 (380T) and 305 (305V) did not independently affect neuroinvasiveness. Testing a panel of viruses with various amino acid substitutions at position 326 revealed that attenuation of neuroinvasiveness required a positively charged residue (lysine or arginine) at this position. Molecular-modeling studies suggest that residues 326 and 380 contribute to charge clusters on the lateral surface of domain III that constitute putative heparin binding sites, as confirmed by studies of heparin inhibition of plaque formation. The neuroinvasiveness of YFVs in the SCID model correlated inversely with sensitivity to heparin. These findings establish that residue 326 in domain III of the E protein is a critical determinant of YFV neuroinvasiveness in the SCID mouse model. Together with modeling of domain III from virulent YFV strains, the data suggest that heparin binding activity involving lysine at position 326 may be a modulator of YFV virulence phenotypes.

The impact of obesity on male fertility.

Obesity in men of reproductive age is globally on the increase. There is clear evidence from epidemiological studies that obesity impacts negatively on male fertility; it is associated with hypogonadism, although it is less consistently linked to impaired spermatogenesis and tests of sperm function, including DNA fragmentation. Sperm from obese men used for in vitro fertilisation/intra cytoplasmic sperm injection is associated with a greater number of pregnancy losses and is less likely to result in live births. There are also increasing data from animal studies that paternal obesity may impact negatively on the reproductive and metabolic health of offspring and grand-offspring. It has been suggested that high-fat dietary exposures could affect the epigenetic content of sperm or the endocrine content of seminal fluid and thus impact early fetal development. Experimental and epidemiological data show that male fertility, and offspring health, can be improved by weight loss in obese and overweight males.

West Nile 25A virus infection of B-cell-deficient ((micro)MT) mice: characterization of neuroinvasiveness and pseudoreversion of the viral envelope protein.

The attenuated West Nile virus 25A strain (WN25A) was investigated for its neuroinvasive properties in B-cell-deficient (microMT) mice. After peripheral inoculation, WN25A caused fatal encephalitis in the majority of 6-8-week-old mice, characterized by a systemic infection with viraemia, moderate virus burdens in peripheral tissues and a high titre of brain-associated virus. Mice generally succumbed to infection within a few weeks of infection. However, others survived for as long as 10 weeks, and some for even longer. Normal age-matched C57BL/6 mice showed no signs of illness after inoculation with WN25A virus. Nucleotide sequencing of WN25A viruses recovered from the brains of B-cell-deficient mice revealed that the conserved N-linked glycosylation site in the viral envelope protein was abolished by substitution of a serine residue at position 155. This was found to be a pseudoreversion relative to the wild-type WN-Israel strain, based on virulence testing of one such brain-associated virus in both B-cell-deficient and normal C57BL/6 mice. This study provides further characterization of the mouse virulence properties of the attenuated WN25A virus in the context of B-cell deficiency. Replication in these mice does not involve rapid neuroadaptation or reversion of WN25A virus to a neuroinvasive phenotype. Molecular modelling studies suggest a difference in local structure of the E protein associated with either an asparagine or serine residue at position 155 compared with the tyrosine found in the virulent parental WN-Israel virus.

JE Nakayama/JE SA14-14-2 virus structural region intertypic viruses: biological properties in the mouse model of neuroinvasive disease.

A molecular clone of Japanese encephalitis (JE) virus Nakayama strain was used to create intertypic viruses containing either the 5'-C-prM-E or the prM-E region of the attenuated JE SA14-14-2 virus in the JE Nakayama background. These two intertypic JE viruses, JE-X/5'CprME(S) and JE-X/prME(S), respectively, generally resembled the parental JE virus in cell culture properties. Similar to virus derived from the JE Nakayama molecular clone (JE-XJN), JE-X/prME(S) was highly neuroinvasive and neurovirulent for young adult mice, whereas JE-X/5'CprME(S) was attenuated for neuroinvasiveness and only partially attenuated for neurovirulence. Immunization of young mice with JE-X/5'CprME(S) virus elicited neutralizing antibodies against JE Nakayama virus and conferred protection against encephalitis following challenge with JE Nakayama virus. The sequence of the JE-X/5'CprME(S) virus differed from that of JE-X/prME(S) virus at two nucleotides in the 5' UTR, 3 amino acid positions in the capsid protein, 4 positions in the prM protein and 1 in the envelope protein. For JE-X/prME(S) virus, the 4 differences in prM and the single substitution in the envelope represented reversions to the sequence of JE Nakayama virus. Overall, this study reveals that molecular determinants associated with the prM-E region of the attenuated JE SA14-14-2 virus are insufficient by themselves to confer an attenuation phenotype upon JE Nakayama virus. This suggests a role for determinants in the 5' UTR and/or the capsid protein of the JE SA 14-14-2 virus genome in influencing the virulence properties of the JE Nakayama virus in the mouse model.

Chimeric Japanese encephalitis virus/dengue 2 virus infectious clone: biological properties, immunogenicity and protection against dengue encephalitis in mice.

A molecular clone of Japanese encephalitis virus (JE virus) was derived from the JE virus Nakayama strain and used to produce infectious JE virus in cell culture. The engineered JE virus resembled the parental JE virus in cell-culture properties and was related closely to other JE virus strains based on nucleotide sequence analysis. The JE virus clone was used as a genetic background for construction of a chimeric virus containing the structural proteins prM and E of Dengue virus, serotype 2. The chimeric JE/dengue 2 virus generated authentic dengue 2 structural proteins as assessed by immunoassays for the dengue E protein. It exhibited a small plaque size and less efficient growth in various cell lines than the parental JE virus. JE/dengue 2 virus was non-neuroinvasive for young adult mice, but displayed partial neurovirulence at doses up to 4 log p.f.u. given intracerebrally. Immunization of 3-week-old mice with JE/dengue 2 virus yielded neutralizing-antibody titres against dengue 2 virus and conferred protection against dengue encephalitis caused by neuroadapted dengue 2 virus. A rise in post-challenge neutralizing-antibody titres against dengue 2 virus in surviving mice suggests that immunization is associated with establishment of a memory antibody response in this model. This study demonstrates the capacity of JE virus to serve as a vector for expression of heterologous flavivirus structural proteins. Similar to previous studies with other chimeric flaviviruses, this approach may be useful as a genetic system for engineering experimental vaccines against Dengue virus and other medically important flaviviruses.

Neuroblastoma cell-adapted yellow fever virus: mutagenesis of the E protein locus involved in persistent infection and its effects on virus penetration and spread.

Persistent infection of mouse neuroblastoma NB41A3 cells with yellow fever 17D virus generates viral variants which exhibit defective cell penetration, poor cell-to-cell spread, small plaque size and reduced growth efficiency, caused by substitution of glycine for aspartic acid or glutamic acid at positions 360 and 362 in the envelope protein. These positions occur within a charge cluster, Asp360-Asp361-Glu362, located in domain III, near its interface with domain I. To characterize further the molecular basis for the variant phenotype, a series of mutant viruses containing substitutions at position 360, 361 and 362, were studied for effects on the cell culture properties typical of the neuroblastoma-adapted variant. Most substitutions at position 360 gave rise to viruses that were very defective in cell penetration, growth efficiency and cell-to-cell spread, whereas substitution with glutamic acid yielded a virus indistinguishable from parental yellow fever 17D. Substitution with lysine was not tolerated and substitution with asparagine resulted in frequent wild-type revertants. A glycine residue was not tolerated at position 361, but substitution at 362 yielded a small plaque virus, similar to the effect of substitution at position 360. These data indicate that the yellow fever virus E protein contains a locus within domain III where a negative-charge cluster is important for optimal function of this domain in virus-cell interactions beyond the stage of virus attachment. Modelling predictions suggest that the mutations alter the local properties of the loop within domain III, and may compromise interactions of this domain with an adjacent region of domain I during conformational changes that occur in the E protein in association with virus entry.

Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities.

A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B-NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 degrees C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B-NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.

Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection.

A series of 29 patients undergoing treatment for chronic hepatitis C virus (HCV) genotype 1 infection with pegylated alpha-2a interferon plus ribavirin were studied for patterns of response to antiviral therapy and viral quasispecies evolution. All patients were treatment naive and had chronic inflammation and fibrosis on biopsy. As part of an analysis of pretreatment variables that might affect the outcome of treatment, genetic heterogeneity within the viral E1-E2 glycoprotein region (nucleotides 851 to 2280) was assessed by sequencing 10 to 15 quasispecies clones per patient from serum-derived PCR products. Genetic parameters were examined with respect to response to therapy based on serum viral RNA loads at 12 weeks (early viral response) and at 24 weeks posttreatment (sustained viral response). Nucleotide and amino acid quasispecies complexities of the hypervariable region 1 (HVR-1) were less in the responder group in comparison to the nonresponder group at 12 weeks, and genetic diversity was also less both within and outside of the HVR-1, with the difference being most pronounced for the non-HVR-1 region of E2. However, these genetic parameters did not distinguish responders from nonresponders for sustained viral responses. Follow-up studies of genetic heterogeneity based on the HVR-1 in selected responders and nonresponders while on therapy revealed greater evolutionary drift in the responder subgroup. The pretreatment population sequences for the NS5A interferon sensitivity determinant region were also analyzed for all patients, but no correlations were found between treatment response and any distinct genetic markers. These findings support previous studies indicating a high level of genetic heterogeneity among chronically infected HCV patients. One interpretation of these data is that early viral responses are governed to some extent by viral factors, whereas sustained responses may be more influenced by host factors, in addition to effects of viral complexity and diversity.

Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C.

Chronic infection with the hepatitis C virus (HCV) is associated with failures of T-cell-mediated immune clearance and with abnormal B-cell growth and activation. We examined the levels of chemokines that bind to CXC chemokine receptor 3 (CXCR3) to determine whether such chemokines might play a role in the failure of the immune system to clear HCV infection. Elevations in CXC ligand 9 (CXCL9), CXCL10, and CXCL11 were observed in all patients with HCV. CXCR3 expression was increased significantly on peripheral blood B lymphocytes, but not T lymphocytes, from individuals with HCV infection. Chemokine levels were measured in samples collected before, during, and after antiviral therapy from a group of 29 patients infected with HCV genotypes 1a (24 patients) and 1b (5 patients). Levels of CXCL10 and CXCL9 decreased following successful antiviral therapy; CXCL11 did not decline significantly during or in the first 6 months after therapy. The baseline level of CXCL10 (measured before the start of antiviral treatment) was greatest in patients with HCV who subsequently became nonresponders to therapy. These results suggest that plasma concentrations of immunoreactive CXCL10 may be a predictor of responsiveness or nonresponsiveness to antiviral therapy with pegylated interferon (IFN) with or without ribavirin. This observation has implications for understanding the pathogenesis of HCV infection.

Neuroblastoma cell-adapted yellow fever 17D virus: characterization of a viral variant associated with persistent infection and decreased virus spread.

Serial passage of yellow fever 17D virus (YF5.2iv, derived from an infectious molecular clone) on mouse neuroblastoma (NB41A3) cells established a persistent noncytopathic infection associated with a variant virus. This virus (NB15a) was dramatically reduced in plaque formation and exhibited impaired replication kinetics on all cell lines examined compared to the parental virus. Nucleotide sequence analysis of NB15a revealed a substitution in domain III of the envelope (E) protein at residue 360, where an aspartic acid residue was replaced by glycine. Single mutations were also found within the NS2A and NS3 proteins. Engineering of YF5.2iv virus to contain the E(360) substitution yielded a virus (G360 mutant) whose plaque size and growth efficiency in cell culture resembled those of NB15a. Compared with YF5.2iv, both NB15a and G360 were markedly restricted for spread through Vero cell monolayers and mildly restricted in C6/36 cells. On NB41A3 cells, spread of the viruses was similar, but all three were generally inefficient compared with spread in other cell lines. Compared to YF5.2iv virus, NB15a was uniformly impaired in its ability to penetrate different cell lines, but a difference in cell surface binding was detected only on NB41A3 cells, where NB15a appeared less efficient. Despite its small plaque size, impaired growth, and decreased penetration efficiency, NB15a did not differ from YF5.2iv in mouse neurovirulence testing, based on mortality rates and average survival times after intracerebral inoculation of young adult mice. The data indicate that persistence of yellow fever virus in NB41A3 cells is associated with a mutation in the receptor binding domain of the E protein that impairs the virus entry process in cell culture. However, the phenotypic changes which occur in the virus as a result of the persistent infection in vitro do not correlate with attenuation during pathogenesis in the mouse central nervous system.

Neuroadapted yellow fever virus 17D: determinants in the envelope protein govern neuroinvasiveness for SCID mice.

A molecular clone of mouse-neuroadapted yellow fever 17D virus (SPYF-MN) was used to identify critical determinants of viral neuroinvasiveness in a SCID mouse model. Virus derived from this clone differs from nonneuroinvasive YF5.2iv virus at 29 nucleotide positions, encoding 13 predicted amino acid substitutions and 2 substitutions in the 3' untranslated region (UTR). The virulence determinants of SPYF-MN for SCID mice were identified by constructing and characterizing intratypic viruses in which the E protein of SPYF-MN was expressed in the YF5.2iv background (SPYF-E) or the E protein of YF5.2iv was expressed in the SPYF-MN background (YF5.2-E). SPYF-E caused lethal encephalitis in young adult SCID mice after intraperitoneal inoculation, with average survival times and tissue virus burdens resembling those of mice inoculated with the parental SPYF-MN virus. To define which domains of the E protein are involved in neuroinvasiveness, two viruses were tested in which the amino acid substitutions in domains I-II and III were segregated. This revealed that substitutions in domain III (residues 305, 326, and 380) were critical for the neuroinvasive phenotype, based on average survival times and tissue burdens of infectious virus. Comparison of growth properties of the various intratypic viruses in cell culture indicated that no inherent defects in replication efficiency were likely to account for the biological differences observed in these experiments. These findings demonstrate that the E protein is a critical factor for yellow fever virus neuropathogenesis in the SCID mouse model and that the neuroinvasive properties depend principally on functions contributed by domain III of this protein. To assess whether critical determinants for neuroinvasion of normal ICR mice by SPYF virus were also in the E protein, sequences of viruses recovered from brains of ICR mice succumbing to encephalitis with the parental SPYF virus were derived. No differences were found in the E protein; however, two substitutions were present in the 3' UTR compared to that of SPYF-MN, one of which is predicted to alter RNA secondary structure in this region. These findings suggest that the 3' UTR may also affect neuroinvasiveness of SPYF virus in the mouse model.

Yellow fever 17D virus: pseudo-revertant suppression of defective virus penetration and spread by mutations in domains II and III of the E protein.

A yellow fever (YFV) 17D virus variant, which causes persistent infection of mouse neuroblastoma cells associated with defective cell penetration and small plaque size, yielded plaque-revertant viruses from cells transfected with viral transcripts encoding the adaptive mutation (Gly360 in the E protein). Reconstruction of a plaque-purified revertant which contained Gly360 and additional substitutions (Asn for Lys303 and Val for Ala261) yielded a virus whose infectious center size, growth efficiency, and cell penetration rate similar to the parental YF5.2iv virus, whereas viruses with Asn303 or Val261 alone with Gly360 yielded either a small-plaque virus or a parental revertant. These data indicate that the YFV E protein is subject to suppression of mutations in domain III that are deleterious for viral entry and spread by a second-site mutation in domain II. Position 261 lies within the hydrophobic ligand-binding pocket at the domain I-II interface, a site believed to be involved in the hinge-like conformational change of domain II during activation of membrane fusion-activity. Results of this study provide genetic data consistent with findings on flavivirus structure and implicate domain III in functions beyond simply cell surface attachment.

HCV E2 glycoprotein: mutagenesis of N-linked glycosylation sites and its effects on E2 expression and processing.

An expression system for analysis of the synthesis and processing of the E2 glycoprotein of a hepatitis C virus (HCV) genotype 1a strain was developed in transiently transfected cells. E2 proteins representing the entire length of the protein, including the transmembrane segment (E2) as well as two truncated versions (E2(660) and E2(715)), were characterized for acquisition of N-linked glycans and transport to the media of transfected cells. To investigate the utilization of the 10 potential N-linked glycosylation sites on this E2 protein, a series of mutations consisting of single or multiple (two, three, four or eight) ablations of asparagine residues in the background of the E2(660) construct were analyzed. E2(660) proteins harboring single or multiple site mutations were produced at levels similar to that of wild-type protein, but secretion of the single mutants was mildly diminished, and elimination of two or more sites dramatically reduced delivery of the protein to the media. Similar results were obtained in Huh-7 cells with respect to intracellular synthesis and secretion of the mutant proteins. Analysis of oligosaccharide composition using endoglycosidase digestion revealed that all of the glycan residues on the intracellular forms of E2(660), E2(715), and E2 contained N-linked glycans modified into high-mannose carbohydrates, in contrast to the secreted forms, which were endo H resistant. The parental E2(660) protein could be readily detected in Huh-7 cells using anti-polyhistidine or antibody to recombinant E2. In contrast, E2(660) lacking the eight N-linked glycans was expressed but not detectable with anti-E2 antibody, and proteins lacking four glycans exhibited reduced reactivity. These experiments provide direct evidence that the presence of multiple N-linked glycans is required for the proper folding of the E2 protein in the ER and secretory pathway as well as for formation of its antigenic structure.

Accumulation of B lymphocytes with a naive, resting phenotype in a subset of hepatitis C patients.

Chronic infection with hepatitis C virus (HCV) is associated with disturbances of B lymphocyte activation and function: autoantibody production, mixed cryoglobulinemia, and B cell lymphomas. It has been proposed that these abnormalities reflect chronic antigenic stimulation or aberrant signaling through the B cell coreceptor, the latter mediated by binding of the HCV E2 glycoprotein to CD81. To test this hypothesis, we measured expression of activation and differentiation markers on peripheral blood B cells from patients with chronic HCV infection. Thirty-six HCV patients with and without mixed cryoglobulinemia were compared with 18 healthy control volunteers and 17 sustained virologic responders who had cleared HCV infection. Ten of the 36 HCV patient samples showed increased B cell frequencies; B cell frequency was higher in patients with more severe hepatic fibrosis. However, these samples lacked evidence of Ag-driven activation or proliferation. The expanded cells were low in the activation markers CD25, CD69, CD71, CD80, and CD86. Proliferation of circulating B cells was unchanged in HCV patients. These cells did not express the differentiation marker CD27, suggesting that they were not enriched in memory B cells. Furthermore, the expanded B cells expressed both IgD and IgM, suggesting that they were antigenically naive. Together, these results indicate that B cell expansion in the peripheral blood of HCV patients is not associated with Ag-mediated activation and differentiation. Instead, factors other than antigenic stimulation may promote the accumulation of peripheral blood B cells with a naive phenotype in a subset of HCV patients.

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model.

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.