The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell.

Flaviviruses elicit a humoral immune response to two virus-encoded, membrane-associated glycoproteins. One is the major virion surface envelope protein (E), which is recognized by antibody, whereas the other is a secreted, heavily glycosylated non-structural protein (NS1). Inoculation with either protein can give rise to a protective immune response, as can the passive transfer of E and NS1 monospecific monoclonal antibodies. Experiments reported here demonstrate that the secreted form of NS1, whether from cells infected with tick-borne encephalitis virus (TBEV) or from cells infected with a defective recombinant adenovirus containing the NS1 gene, occurs chiefly as a pentamer or hexamer and occasionally as a decamer or dodecamer. Intracellular forms of this protein however occur only as dimers. The higher M(r) forms secreted from the cell are exquisitely sensitive to detergent, suggesting they are held together by hydrophobic bonds. Both intracellular and extracellular forms of the dimer can be dissociated by heat, but at different temperatures. Unlike similar proteins from mosquito-borne viruses. NS1 from TBEV-infected cells cannot be dissociated at ambient temperatures by extremes of pH. Studies on the antigenic structure of this protein show it to have several highly conserved epitopes, confirming similar earlier conclusions from amino acid sequence analyses.

X-ray solution scattering of Sindbis virus. Changes in conformation induced at low pH.

Alphaviruses, like many enveloped animal viruses, enter the cell by fusing with the cell membrane. This fusion occurs only in coated vesicles at a low pH. By using X-ray solution scattering of highly purified virus particles we have gained direct evidence that a drop in pH does not alter the structure of the virus core but does cause a significant change in the structure of the virus envelope. Thus these experiments give direct evidence to support the hypothesis that a reduction in pH causes a conformational change in the virus E protein, which enables it to promote fusion with the cell envelope and trigger virus infection.

Purification and analysis of infectious virions and native non-structural antigens from cells infected with tick-borne encephalitis virus.

By employing the techniques of column chromatography and membrane filtration, we have succeeded in purifying flavivirus particles with low particle to infectivity ratios and free from contamination with cellular proteins. Virus particles purified by this method have an average diameter of 53 nm, a particle to infectivity ratio of less than 10, and a KD (partition coefficient) consistent with a molecular weight of 2.63 x 10(7). In addition it has been possible to purify the extracellular form of non-structural protein 1 (NS1), which in its native form appears to be a hexamer. It is also apparent from these studies that the slowly sedimenting haemagglutinin particle (or SHA) is an artifact of purification methods using gradient centrifugation. This technology should not only prove useful in the laboratory for studying the detailed structure of these viruses and the proteins encoded by them, but should also prove useful in industrial vaccine manufacture where large volumes of highly pathogenic material are handled.

The synthesis and maturation of a non-structural extracellular antigen from tick-borne encephalitis virus and its relationship to the intracellular NS1 protein.

The replication of flaviviruses results in the secretion of four virus-coded proteins into the extracellular environment. Three of these proteins, E, C and M (or pre-M), are found in purified virions. A fourth virus-specified extracellular protein which was not present in either the slowly sedimenting haemagglutinin particles or in virions is described. The relationship of this protein to the intracellular NS1 polypeptide was investigated along with its similarity to the soluble complement-fixing antigen (SCF) reported for mosquito-borne flaviviruses. The difference in the Mr of NS1 and SCF is the result of additional glycosylation of SCF, mostly by the addition of fucose molecules. The synthesis of E and NS1 is sequential but their secretion is simultaneous, suggesting a role for NS1 in virion protein transport or virion release.

The synthesis of immunogenic polypeptides encoded by tick-borne encephalitis virus.

Tick-borne encephalitis virus codes for two major immunogenic polypeptides, one of which is the major virion envelope protein E, and the other, NV3, does not have a designated function at present. The intracellular forms of both the E and NV3 polypeptides contain at least four types of sugar residues, i.e. galactose, glucosamine, fucose and mannose. The only glycoprotein in the extracellular virion particles is E. Experiments performed in the presence of tunicamycin have demonstrated that most of these sugars are N-linked. The kinetics of synthesis of E and NV3 have been studied and both show a distinct lag period between initiation of protein synthesis and the appearance of either protein. The kinetics of synthesis of these proteins are consistent with the hypothesis that initiation of protein synthesis starts at the 5' end of a polycistronic genome but the synthesis of the E and NV3 proteins only occurs after translocation of the polyribosome complex to specific areas in the infected cell. No precursors to either the E or NV3 glycoproteins were detected. Synthesis of both glycoproteins can be detected as early as 6 h after infection and rises to a maximum at 15 h after infection.

Treatment of uncomplicated gonorrhoea with single dose aztreonam.

Infection with Neisseria gonorrhoeae was cleared in 61 men and 26 women at all sites (except in the pharynx of one male bisexual patient with urethral and pharyngeal gonorrhoea) after treatment with aztreonam as a single 1 g intramuscular injection. Aztreonam was well tolerated with no adverse effects. This monobactam antibiotic was effective against both penicillin sensitive and resistant strains.

The purification of alphavirus virions and subviral particles using ultrafiltration and gel exclusion chromatography.

Conventional methods of virus purification using ultracentrifugation frequently result in distorted particles with low levels of biological activity, and are thus unsuitable for preparing samples for high-resolution techniques such as neutron scattering, X-ray scattering in solution, and X-ray crystallography. Moreover, in the event of an instrument failure, ultracentrifugation can also pose a significant hazard when preparing pathogenic viruses or subunits derived from them. By employing exclusively ultrafiltration and gel exclusion chromatography, a method has been developed to prepare highly purified, intact alphavirus particles retaining high levels of biological activity. These procedures have also been adapted to prepare aggregates of viral envelope protein with a defined immunogenic content.

Ultrasonic appearances associated with prostatic inflammation: a preliminary study.

Per-rectal ultrasonography was performed on 40 patients in whom a diagnosis of prostatitis had been made on the basis of symptoms and signs of prostatic inflammation confirmed by bacteriology, microscopy or pH changes of expressed prostatic secretion. Certain ultrasonic features were present in all patients to a variable degree. A change in volume and weight of the prostate could be an indicator of treatment response.

A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers.

A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-wellpolystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays using peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods.