Efficacy of a whole inactivated EI vaccine against a recent EIV outbreak isolate and comparative detection of virus shedding.

An outbreak of H3N8 Equine Influenza virus (EIV) that occurred in vaccinated horses in Japan was caused by a genetically divergent EIV isolate of the Florida clade 1 sub-lineage. This virus subsequently entered Australia where it infected thousands of immunologically naïve horses. The objective of this study was to evaluate the ability of a non-updated whole inactivated equine influenza (EI) vaccine to protect if used in the face of an outbreak induced by a virus similar to the ones circulating in Japan and Australia in 2007. Seven naïve Welsh mountain ponies were immunised twice with the commercially available vaccine Duvaxyn IE-T Plus and experimentally infected with A/eq2/Sydney/2888-8/07. Five ponies remained unvaccinated as controls. The ponies were challenged in an ACDP (Advisory Committee on Dangerous Pathogens) Category III containment facility by exposure to a nebulised aerosol of A/eq2/Sydney/2888-8/07 two weeks after the second vaccination. Clinical signs and virus shedding were monitored for 14 days post-challenge infection. After challenge infection, all control ponies developed clinical signs of disease with coughing being particularly noteworthy when compared with vaccinated ponies. Only 3 out of 5 controls developed pyrexia for up to 3 days, and 1 out of 7 vaccinates was pyretic for 1 day. Nasal discharge was evident in both control and vaccinated ponies with no significant difference between groups. Three different methods were used to measure virus shedding in nasal secretions (i.e. titration in embryonated hens' eggs, EIV NP ELISA and EIV NP qRT-PCR). The intensity and duration of EIV shedding significantly decreased in the vaccinated group when compared with the control ponies. All control ponies seroconverted after experimental infection with A/eq2/Sydney/2888-8/07 whereas only 1 out of 7 vaccinated ponies had a significant increase in antibody. Duvaxyn IE-T Plus therefore reduced clinical signs and virus shedding when ponies were challenged with A/eq2/Sydney/2888-8/07 (H3N8), 2 weeks after a second dose of vaccine.

The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.

Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements.

The RNA genome of influenza virus is encapsidated by the virus nucleoprotein (NP) to form ribonucleoprotein (RNP) structures of defined morphology. These structures result from the ability of NP to oligomerise and to bind single-strand RNA. To characterise NP oligomerization, we developed a binding assay using immobilised NP fusion proteins and in vitro translated NP. This system was used to estimate a dissociation constant for NP-NP contacts of 2 x 10 (-7)M. Analysis of NP deletion mutants identified three sequence elements important for oligomerization. Two regions corresponding to the middle and C-terminal thirds of the polypeptide were identified as the minimal sequences capable of promoting NP-NP contacts. However, the C-terminal 23 amino-acids of NP inhibited oligomerization, as their removal increased self-association 10-fold. Single codon changes identified amino acids important for the function of these regions. Alanine substitution of R199 decreased binding affinity threefold, whereas alteration of R416 had a more drastic effect, reducing binding >10-fold. In contrast, mutation of F479 increased self-association fivefold. Mutations altering NP oligomerization affected the ability of the polypeptides to support influenza virus gene expression in an in vivo assay. Decreased oligomerization activity correlated with decreased transcriptional function. However, mutations that increased self-association also decreased transcription competence. This indicates that NP contains both positive and negative sequence elements involved in oligomerization and is consistent with the importance of NP-NP contacts for the formation of a transcriptionally active RNP.

Modulation of nuclear localization of the influenza virus nucleoprotein through interaction with actin filaments.

The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.

Analysis of HPV1 E4 complexes and their association with keratins in vivo.

The HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (>95%) exists as complexes which do not contain keratins. The identification of discrete species of 34K, 58K, 70K, 88K, and 105K suggests that these are simple multimers of the 17K monomer, with very little of the soluble E4 being present in complexes larger than 140K. The truncated 10/11K E4 species, which comprise the C-terminal domain of E4, exist as trimers and dimers in vivo. Less than 5% of the E4 was present as complexes greater than 140K, and these were found to be insoluble. The 34K (dimer) and 58K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105K E4 complex was abolished (and formation of the 58K species enhanced) when keratins were present during E4 synthesis in vitro. We conclude that while E1-E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 E4 protein in established tumors in vivo is neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers.

Use of urine albumin measurement as a replacement for total protein.

We have investigated the replacement of urine total protein estimations for the assessment of glomerular permeability, by the measurement of urine albumin excretion using a latex particle enhanced immunoturbidimetric assay. An initial screen was performed using Albustix to assess the sample pre-dilution necessary for immunoanalysis. A total of 167 24-hour urine samples were analysed and urine albumin concentration correlated well with that of urine total protein (r = 0.93) over the range 0-16,800 mg/l. This protocol provides a more cost effective and analytically valid assessment of glomerular permeability.

Targeting and degradation of p53 by E6 of human papillomavirus type 16 is preferential for the 1620+ p53 conformation.

E6-mediated degradation of p53 is believed to play a role in the transformation of cells by high-risk types of human papillomavirus. In order to explore the structural requirements for targeting of p53 we have compared E6-mediated degradation of variant p53 forms expressed in vitro. Complete degradation was observed in samples containing monomers, dimers and higher molecular weight structures of wild-type p53, indicating that E6 targets all quaternary forms of wild-type p53. Wild-type human and murine p53s reactive with PAb 1620 (which recognizes a conformation-dependent epitope) were degraded when incubated with E6. Mutant p53 proteins were variably resistant to E6-mediated degradation, and this correlated with PAb 1620 reactivity. Thus, mutants hp53Val-154, hp53Val-266 and hp53Pro-273 (1620 degrees) were completely resistant to degradation, whereas hp53Ile-247 and hp53Trp-248 (1620+) were degraded. Mutants hp53Leu-273 and mp53Val-135, which are temperature sensitive for conformation, were completely degraded in the 1620+ form but degradation resistant in the 1620 degrees form. Although the PAb 1620+ conformation appeared important for recognition of p53 by E6, the epitope itself is unlikely to be the actual recognition target since the PAb 1620 monoclonal antibody failed to protect against E6-mediated degradation.

Partially transformed T3T3 cells express high levels of mutant p53 in the 'wild-type' immunoreactive form with defective oligomerization.

High levels of wild-type p53 suppress transformed growth of many cell lines and yet murine T3T3 cells shown partially transformed growth despite high endogenous levels of phenotypically 'wild-type' p53. On sequencing T3T3 p53 was found to encode missense mutations at codons 230 and 287 and, although endogenous T3T3 p53 is 'wild type', the protein adopted the mutant phenotype when expressed in vitro. Size fractionation of T3T3 cell lysate indicated monomeric p53 possibly in complex with a low molecular weight protein. When expressed in vitro T3T3 p53 formed dimers and higher order structures. Thus T3T3 cells appear (i) to drive endogenous mutant p53 to adopt conformational epitopes characteristic of the 'wild-type' protein, and (ii) to interfere with normal assembly of p53 quaternary structure. Phosphopeptide mapping of p53 from 3T3x cells, T3T3 cells and SV3T3 cells indicated reduced amino terminal phosphorylation of the mutant p53 phenotype. Alternative splicing of p53 was also detected in 3T3x cells; similar splicing occurs in wild-type p53 (Han & Kulesz-Martin, 1992; Nucl. Acids Res., 20, 1979-1981) and a possible regulatory function is discussed.

Temperature-sensitive mutants of p53 associated with human carcinoma of the lung.

We have compared the effects of specific point mutations on the tertiary and quaternary structure of the human p53 protein. Eight mutants, each derived from primary resected tissues of lung carcinomas, were expressed in vitro under strictly defined conditions, such that the only known variant was the point mutation present in each p53 mRNA. All the mutations were located in highly conserved domains. The tertiary structure of each mutant protein was investigated by reactivity with anti-p53 monoclonal antibodies directed against conformation-dependent epitopes. Quaternary structure was examined by gel filtration. Although all the mutant proteins exhibited abnormal tertiary structures, their quaternary structures appeared similar to wild type, the one exception being p53-tyr135, which contains tyrosine in place of cysteine at residue 135. The conformational phenotype of mutant human p53 was found to be dependent upon (i) the locus of the mutation and (ii) the nature of the amino acid substitution: two different substitutions at residue 273 yielded two mutants with differing structural properties. We have discovered three mutants of human p53 that are temperature sensitive for conformation; one is mutated at codon 273, a 'hotspot' for p53 mutation in human cancer.

Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles.

Turbidimetric immunoassay is commonly used to quantify serum proteins. Latex-particle enhancement of this type of assay has been primarily associated with increasing assay sensitivity. However, covalent coupling of an antibody to a latex particle can offer other advantages that are also pertinent in measurement of high concentrations of analytes. By using a common antibody with IgG as a model analyte, we describe the development of a nonenhanced and a latex-particle-enhanced turbidimetric assay for measuring serum IgG. Both assays show adequate analytical recovery and parallelism, and results compare well with those by rate nephelometry. The latex-enhanced assay has equivalent sensitivity, working range, and interassay precision, but much greater signal change and calibration stability than the nonenhanced assay. In addition, with latex particles, less antiserum is needed. Coupling antibodies to latex particles offers considerable advantages, even when an improved assay detection limit is not required.

Development of a rapid latex enhanced turbidimetric assay for retinol binding protein in urine.

We describe a simple, rapid and sensitive homogeneous immunoassay for urinary retinol-binding protein (RBP) using latex particle-enhanced turbidimetric immunoassay. Rabbit anti-human RBP is covalently coupled to 40 nm latex particles and the assay performed on the IL Monarch 2000 centrifugal analyser, with a 20 microL sample volume and the reaction monitored at 340 nm over an 8 min period. The assay range is 0-6 mg/L with a detection limit of 25 micrograms/L. The within and between assay coefficients of variation are less than 1.5% and less than 2.5%, respectively. Comparison with radioimmunoassay for RBP showed good agreement.

Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation.

Activating mutations of p53 promote tumor progression. The mutant protein adopts a characteristic conformation, which lacks the growth suppressor function of wild-type p53. We show that mutant p53 can drive cotranslated wild-type p53 into the mutant conformation: a similar effect in vivo would block wild-type suppressor function with dominant negative effect. The cotranslational effect of mutant p53 on wild-type conformation depends upon interaction between nascent polypeptides and oligomerization of the full-length proteins. We also show that oligomers of p53 proteins can be induced to change conformation in a cooperative manner. Cell growth stimulation induces a similar conformational change in p53, and our present results indicate that this may involve allosteric regulation.

Particle-enhanced turbidimetric immunoassay of sex-hormone-binding globulin in serum.

A particle-enhanced turbidimetric immunoassay (PETIA) for human sex-hormone-binding globulin (SHBG) is described. The method involves use of antibody covalently coupled to latex particles and is almost fully automated, with sample processing being complete in less than 20 min. The working reagents are stable for at least three months, and full calibration of the assay each day is not essential. A particular advantage is that pretreatment of samples is rarely required because the working range of the assay is from 2.0 to 320 nmol/L for nondiluted serum. Intra- and interassay CVs were less than 4.5% and 8.5%, respectively, and mean analytical recovery was 101.5%. SHBG concentrations of 129 serum samples determined by this method and by a commercially available immunoradiometric assay correlated highly.

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135.

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.

A rapid and robust particle-enhanced turbidimetric immunoassay for serum beta 2 microglobulin.

A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum beta 2-microglobulin is described. The method has a working range of 0.2-40 mg/l, with good precision and a correlation coefficient of 0.97 when compared with an established radioimmunoassay method. One of the major advantages of this assay is the stability of the calibration curve (up to at least 20 months). This, and the fact that no pretreatment of serum samples is necessary, makes the assay ideally suited for all types of routine determination.

Rapid, robust method for measuring low concentrations of albumin in urine.

We describe a rapid particle-enhanced turbidimetric immunoassay for albumin in urine. Intra- and interassay CVs were less than 5% and less than 10%, respectively, the detection limit is 2 mg/L, and the working range extends to 200 mg/L. Mean analytical recovery of albumin added to centrifuged urines was 100% (SD 10.6%), and, when results were compared with those by the Pharmacia RIA, the correlation coefficient was 0.99. The working reagents are stable for at least six months; thus this assay is suited for both batch and urgent analysis.