Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell.

Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses' proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD) and varicella zoster glycoprotein E (VZV gE). Recombinant plasmids were generated, transfected into insect cells (SF9), and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

Pan-human coronavirus and human bocavirus SYBR Green and TaqMan PCR assays; use in studying influenza A viruses co-infection and risk of hospitalization.

PURPOSE: Influenza A viruses, human coronaviruses (hCoV) and human bocavirus (hBoV) are emerging respiratory viruses. This study investigated the association between influenza A viruses co-infection with hBoV and hCoV and severity and the sensitivity of a real-time polymerase chain reaction (RT-PCR) assay for identification of 15 coronaviruses. METHODOLOGY: Published sequences for the 15 human coronaviruses were used to design a consensus PCR targeting the replicase open reading frame 1b. A previously published PCR targeting the NS1 Gene of all known human bocavirus strains was also utilized. A series of 217 samples from patients aged 37.7 (SD ± 30.4)] with seasonal influenza A viruses (SeasFluA) identified between 06/2011 and 06/2012 in NW England were tested for hCoV and hBoV using RT-PCR. Association between co-infection and disease outcome was assessed using logistic regression. RESULTS: The limit of detection of hCoV RT-PCR assay was 2 copies/µl of human coronavirus RNA template, a sensitivity comparable to a previously published SYBR green assay for human coronaviruses. A total of 12 hCoV and 17 hBoV were identified in the 217 influenza A positive samples. A higher proportion (61.5%; 8/13) of SeasFluA/hBoV co-infections were identified in patients that were admitted either to a general ward or the intensive care unit compared to 44.3% (66/149) of single SeasFlu A virus infections (OR 2.5 95% CI 0.67-9.34, p = 0.17). In a stratified analysis, there was a trend towards higher association between FluA, hCoV and hBoV with increasing age (especially in patients aged 24-45 years and >65 year old). CONCLUSION: Our hCoV RT-PCR protocol appeared to be of adequate analytical sensitivity for diagnosis. More and larger studies are needed to confirm the role of hCoV, hBoV in causing severe disease when they co-infect with influenza A viruses.

Single, dual and multiple respiratory virus infections and risk of hospitalization and mortality.

Respiratory virus infections cause a significant number of hospitalization and deaths globally. This study investigated the association between single and multiple respiratory virus infections and risk of admission to a general ward, intensive care unit or death in patients aged 0-105 years (mean ± s.d. = 24·4 ± 24·1 years), from North West England, that were tested for respiratory virus infections between January 2007 and June 2012. The majority of infections were in children aged ⩽5 years. Dual or multiple infections occurred in 10·4% (1214/11 715) of patients, whereas single infection occurred in 89·6% (10 501/11 715). Rhinovirus was the most common co-infecting virus (occurring in 69·5%; 844/1214 of co-infections). In a multivariate logistic regression model, multiple infections were associated with an increased risk of admission to a general ward [odds ratio (OR) 1·43, 95% confidence interval (CI) 1·2-1·7, P < 0·0001]. On the other hand, patients with respiratory syncytial virus (RSV) and human parainfluenza virus types 1-3 (hPIV1-3), as a single infection, had a higher risk of being admitted to a general ward (OR 1·49, 95% CI 1·28-1·73, P < 0·0001 and OR 1·34, 95% CI 1·003-1·8, P = 0·05, respectively); admitted to an intensive-care unit or dying (OR 1·5, 95% CI 1·20-2·0, P = 0·001 and OR 1·60, 95% CI 1·02-2·40, P = 0·04, respectively). This result emphasizes the importance of RSV, hPIV and mixed infections and calls for research on vaccines, drugs and diagnostic tests targeting these respiratory viruses.

Mutations associated with severity of the pandemic influenza A(H1N1)pdm09 in humans: a systematic review and meta-analysis of epidemiological evidence.

Mutations in the haemagglutinin (HA), non-structural protein 1 (NS1) and polymerase basic protein 2 (PB2) of influenza viruses have been associated with virulence. This study investigated the association between mutations in these genes in influenza A(H1N1)pdm09 virus and the risk of severe or fatal disease. Searches were conducted on the MEDLINE, EMBASE and Web of Science electronic databases and the reference lists of published studies. The PRISMA and STROBE guidelines were followed in assessing the quality of studies and writing-up. Eighteen (18) studies, from all continents, were included in the systematic review (recruiting patients 0 - 77 years old). The mutation D222G was associated with a significant increase in severe disease (pooled RD: 11 %, 95 % CI: 3.0 % - 18.0 %, p = 0.004) and the risk of fatality (RD: 23 %, 95 % CI: 14.0 %-31.0 %, p = < 0.0001). No association was observed between the mutations HA-D222N, D222E, PB2-E627K and NS1-T123V and severe/fatal disease. The results suggest that no virus quasispecies bearing virulence-conferring mutations in the HA, PB2 and NS1 predominated. However issues of sampling bias, and bias due to uncontrolled confounders such as comorbidities, and viral and bacterial coinfection, should be born in mind. Influenza A viruses should continue to be monitored for the occurrence of virulence-conferring mutations in HA, PB2 and NS1. There are suggestions that respiratory virus coinfections also affect virus virulence. Studies investigating the role of genetic mutations on disease outcome should make efforts to also investigate the role of respiratory virus coinfections.

Epstein-Barr virus infection in adult renal transplant recipients.

Epstein-Barr virus (EBV) DNAemia in the first year posttransplantation has been studied extensively. There is a paucity of information on prevalence and sequelae of EBV infection in adult renal transplantation beyond the first year. This single-center study examines the relationship between EBV DNAemia and demographic, immunosuppressive, hematologic and infection-related parameters in 499 renal transplant recipients between 1 month and 33 years posttransplant. Participants were tested repeatedly for EBV DNAemia detection over 12 months and clinical progress followed for 3 years. Prevalence of DNAemia at recruitment increased significantly with time from transplant. In multivariate adjusted analyses, variables associated with DNAemia included EBV seronegative status at transplant (p = 0.045), non-White ethnicity (p = 0.014) and previous posttransplant lymphoproliferative disease (PTLD) diagnosis (p = 0.006), while low DNAemia rates were associated with mycophenolate mofetil use (p < 0.0001) and EBV viral capsid antigen positive Epstein-Barr nuclear antigen-1 positive serostatus at transplant (p = 0.044). Patient and graft survival, rate of kidney function decline and patient reported symptoms were not significantly different between EBV DNAemia positive and negative groups. EBV DNAemia is common posttransplant and increases with time from transplantation, but EBV DNAemia detection in low-risk (seropositive) patients has poor specificity as a biomarker for future PTLD risk.

Comparative performance of a novel herpes simplex virus type 2-specific enzyme-linked immunosorbent assay using a targeted chain oligopeptide, peptide 55.

Herpes simplex virus (HSV) glycoprotein G (gG2) has been used as the basis of many serological assays for the detection of HSV type 2 (HSV-2)-specific antibodies. In the present study, an enzyme-linked immunosorbent assay (ELISA), the Pathozyme Viro HSV-2 immunoglobulin G (IgG) ELISA (Omega Diagnostics, Alva, United Kingdom), based on an immunodominant epitope of gG2 presented in a branched-chain format (peptide 55), was compared with two commercially available gG2-specific assays, the Bioelisa HSV-2 IgG assay (Biokit, S.A., Barcelona, Spain) and the HerpesSelect HSV-2 IgG assay (Focus Diagnostics, Cypress, CA). A panel of 218 well-characterized serum samples was tested. Thirty-one samples were determined to be HSV-2 IgG antibody positive and 164 samples were determined to be negative with all three kits. The levels of concordance between the tests were 95.9% between the Omega and HerpeSelect assays, 90.8% between the Omega and Bioelisa assays, and 94.5% between the HerpeSelect and Bioelisa assays. Twenty-three samples gave discordant results. Western blot results showed that of these, the results for 77% were correctly identified by the Omega assay, the results for 68% were correctly identified by the HerpeSelect assay, and the results for 13.6% were correctly identified by the Bioelisa assay. Although there was a high level of agreement between the results obtained by the three assays and no false-positive results were detected by any of the three kits, confirmation of the results for samples with discordant results by Western blotting suggested that the peptide 55-based Omega assay is the most sensitive and specific assay among the assays evaluated.

Age-related pattern of KI and WU polyomavirus infection.

BACKGROUND: The role of two recently identified polyomaviruses, KI and WU, in the causation of respiratory disease has not been established. OBJECTIVES: To determine the prevalence of KI and WU viruses (KIV and WUV) in 371 respiratory samples and evaluate their contribution to respiratory disease. STUDY DESIGN: Specimens were screened for KIV and WUV using single, multiplex or real time PCR; co-infection with other respiratory viruses was evaluated. RESULTS: Of the 371 samples analysed, 10 (2.70%) were positive for KIV and 4 (1.08%) were positive for WUV yielding an overall case prevalence of KIV and WUV infection of 3.77%. KIV and WUV were identified in patients aged<15 years (11 patients) with upper or lower respiratory tract infection and >45 years (3 patients) with upper respiratory tract infection. Co-infections were found in 5 (50%) and 3 (75%) of the KIV and WUV positive samples, respectively. CONCLUSIONS: This study supports previous conclusions that KIV and WUV detection in the respiratory tract may be coincidental and reflect reactivation of latent or persistent infection with these viruses. The age distribution of KIV and WUV infection in this study mirrors that found for the other human polyomaviruses, BK and JC.

Detection of BK virus and JC virus DNA in urine samples from immunocompromised (HIV-infected) and immunocompetent (HIV-non-infected) patients using polymerase chain reaction and microplate hybridisation.

BACKGROUND: The majority of the human population is infected with two human polyomaviruses BK virus (BKV) and JC virus (JCV) during childhood. After initial infection both viruses persist within renal system. Reactivation of both viruses may be linked with immunodeficiency or immunosuppressive therapy. OBJECTIVE: To evaluate the relationship between immunodeficiency and viruria, prevalence of BK and JC viruria over time was investigated in a cohort of HIV seropositive individuals at different stages of disease. The excretion in this group was compared with virus excretion in their HIV seronegative partners and in an unselected cohort of patients attending a Genito-Urinary Medicine (GUM) clinic. STUDY DESIGN: The excretion of BKV and JCV DNA in multiple urine samples from HIV-infected patients at different stages of disease and their HIV-negative partners, and in single samples from a cohort of patients at a GUM clinic was investigated. A microplate hybridisation method was developed to increase both the sensitivity and specificity of detection of the PCR product. The method was also applied to estimate the DNA copy numbers of BKV and JCV in urine samples. RESULTS: Within the HIV group, the level of immunosuppression (CD4+ category) was not associated with JCV viruria. By contrast, there was a modest correlation between immunodeficiency as indicated by a decline in CD4+ count and BKV viruria. Shedding of both BKV and JCV DNA together in urine samples of HIV-infected patients was much higher than in control groups (P = 0.02), indicating that HIV infection may associate with polyomavirus reactivation. The incidence of flu-like syndrome was much higher in HIV-infected asymptomatic individuals than acquired immunodeficiency syndrome (AIDS)-related complex (ARC)/AIDS patients. In general, the concentration of BKV DNA viruria (DNA copy number) was dependent to CD4+ counts (P = 0.008) while concentration of JCV DNA was independent to CD4+ cell count (P = 0.54). The prevalence of BKV and JCV DNA in patients who were infected with C. trachomatis was 9/50 (18%) and 11/50 (22%), respectively. BKV and JCV DNA was detected in 3/19 (15%) and 2/19 (10%) of patients who were infected with N. gonorrhoea. Results suggested that persons infected with C. trachomatis were more likely to show BKV and JCV viruria. CONCLUSION: These results confirm that shedding of BK and JC viruses in urine is not exclusively found in immunosupression, it may also occur in healthy individuals. The frequency of virus excretion is however, apparently increased in HIV-infected patients, although no firm statistical difference could be established. One of the interesting aspects of these findings was the relatively high incidence of BKV and JCV viruria in both control groups, i.e. HIV-negative partners of HIV-infected patients and patients attending a GUM clinic.

Association of acute parvovirus B19 infection with new onset of acute lymphoblastic and myeloblastic leukaemia.

AIMS: To investigate the association of acute parvovirus B19 infection with new onset of acute lymphoblastic and myeloblastic leukaemia. METHODS: Cerebrospinal fluid (CSF) samples from patients with acute myelogenous leukaemia (AML) at diagnosis (n = 2) and acute lymphoblastic leukaemia (ALL) at diagnosis (n = 14) were analysed for parvovirus B19 DNA by means of nested polymerase chain reaction. In addition, samples from patients with benign intracranial hypertension (BIH) (n = 10) and hydrocephalus (n = 13) were tested as controls. RESULTS: Four leukaemia cases were positive-common ALL (n = 2), null cell ALL (n =1), and M7 AML (n = 1)-whereas all controls were negative (Yates corrected chi(2) value, 3.97; p = 0.046; odds ratio, 16.92; confidence interval, 1.03 to 77.18). All four patients were significantly anaemic, but none was encephalitic or had evidence of central nervous system leukaemia. In three of these patients, serum tumour necrosis alpha, interferon gamma, interleukin 6, granulocyte-macrophage colony stimulating factor (range, 34.93-3800.06 pg/ml), and macrophage chemoattractant protein 1 were detectable. All of these four patients carried at least one of the HLA-DRB1 alleles, which have been associated with symptomatic parvovirus B19 infection. CONCLUSION: Erythroid suppression and immune cell proliferation are both associated with B19 infection and may also be important in the pathogenesis of acute leukaemia.

BK virus DNA in CSF of immunocompetent and immunocompromised patients.

AIM: To investigate the possible aetiological role of BK and JC viruses in immunocompetent and immunocompromised children with suspected encephalitis and meningoencephalitis. METHODS: The polymerase chain reaction and microplate hybridisation method was employed for the detection of polyomavirus DNA in 266 CSF specimens collected from immunocompetent and immunocompromised patients. RESULTS: BK virus DNA was detected in three (2.1%) CSF samples taken from patients aged 2-5 years; two were patients with acute lymphocytic leukaemia without overt neurological symptoms, the other was a patient with suspected encephalitis. BK virus DNA was also detected in two (1.6%) CSF samples taken from older children in the age range 10-16 years; both children had suspected encephalitis. JC virus DNA was not found in any CSF sample from either age group. CONCLUSIONS: Detection of BK virus in the CSF of immunocompromised and immunocompetent patients with suspected neurological disease suggests that this virus may have had a pathogenic role in the aetiology of this condition.

BKV-DNA and JCV-DNA in CSF of patients with suspected meningitis or encephalitis.

BACKGROUND: Few studies have looked for the polyoma viruses JC or BK virus in the central nervous system (CNS) of patients without neurological symptoms or with neurological symptoms other than progressive multifocal leukoencephalopathy (PML). PCR-microplate hybridization method was employed for the detection of BKV-DNA or JCV-DNA in cerebrospinal fluid (CSF) specimens from patients with suspected meningitis or encephalitis. MATERIALS AND METHODS: A total of 181 CSF specimens from 151 patients with suspected meningitis or encephalitis was examined for BKV or JCV using PCR-microplate hybridization method. None of the patients had (clinically diagnosed) PML. A control group consisting of 20 CSF specimens from normal subject was also included. RESULTS: BKV DNA was found in five out of 131 (3.8%) and JCV DNA in two out of 131 (1.5%) of the patients with suspected meningitis or encephalitis by PCR ELISA. BKV or JCV DNA was not detected in CSF samples of any of 19 HIV positive patients. BKV and JCV DNAs were detected respectively in two CSF samples in which Mycobacterium tuberculosis (TB) PCR was also positive. Another patient who was positive for JCV PCR died with a diagnosis of cerebral lymphoma. Among the BK virus infected patients there was a patient with a previous history of hemolytic uremia and acute renal failure. Neither BKV nor JCV DNA was found in any of the 20 CSF samples from normal patients undergoing lumbar puncture for myelography as a part of an investigation of lower back pain. CONCLUSION: These results suggest that BK virus may be associated with neurological diseases either in immunocompetent or immunocompromised patients. Detection of BKV and JCV DNA in the CSF of the patients suspected to have either meningitis or encephalitis suggests that these viruses may have an etiological role. Thus, diagnostic tests for BK and JC viruses should be included in the investigative program for meningitis or encephalitis patients.

Association of human parvovirus B19 infection with acute meningoencephalitis.

To find out the incidence and clinical presentation of parvovirus B19 meningoencephalitis, we tested samples of cerebrospinal fluid from 162 patients (one from each patient) with undiagnosed meningoencephalitis, who presented between March, 1997, and March, 1998 (an outbreak period) using nested PCR for B19 genes. Seven patients were positive; an incidence of 4.3%. Five additional cases of meningoencephalitis were detected from other years. Three patients with underlying disorders (haemophagocytic lymphohistiocytosis, Cockayne's syndrome, and Turner's syndrome) died. Neurological sequelae were observed in three surviving patients, all of whom had had striking abnormalities detected on brain scans done during the acute phase.

Needlestick injuries and hepatitis B virus vaccination in health care workers.

An analysis of 2646 needlestick injuries in hospitals in the Greater Manchester area between April 1992 and April 1999 was carried out. Ten per cent of members of staff injured in these incidents had never been vaccinated against hepatitis B virus (HBV) and 27% of those who had been vaccinated had no anti-HBs (< 10 IU/L). Although few health care workers were at risk of transmission of HBV through needlestick incidents in this study (0.6% (12/2084) of all source patients were HBsAg positive; 9 HBeAg positive, 7 anti-HBe positive), the large number of members of staff who were not protected from HBV infection indicates a need for occupational health departments to reinforce HBV vaccination policies.

Serological diagnosis of varicella-zoster virus in sera with antibody-capture enzyme-linked immunosorbent assay of IgM.

Evaluation was made of three enzyme-linked immunosorbent assay (ELISA) formats; varicella-zoster virus (VZV) indirect ELISA; VZV IgM capture using biotin and VZV IgM capture using peroxidase, for the detection of VZV-specific IgM antibodies in human sera. It was observed that there was no significant difference in sensitivity of detection using the three formats but there were important practical differences in the number of steps and hence time for assay completion between the three assay formats. All assays showed some cross-reactivity with sera containing anti-HSV1 antibodies.

Detection of BK virus in urine by polymerase chain reaction: a comparison of DNA extraction methods.

Using specimens spiked with BK virus, several DNA extraction methods were evaluated for their ability to remove polymerase chain reaction (PCR) inhibitors from urine samples. It was found that PCR inhibition could be completely overcome by extracting samples with 30% polyethylene glycol (PEG) in 3 M sodium chloride, and partially overcome by extracting samples with guanidine thiocyanate in the presence of high salt concentrations. The nature of the sample inhibition was investigated, leading to the conclusion that both urea and unidentified non-proteinaceous DNA associated substances inhibit BKV DNA amplification from urine.

Analysis of an HIV-infected cohort followed for as long as 15 years after seroconversion.

Data from a cohort of 62 HIV-positive individuals with hemophilia or von Willebrands disease infected for a maximum period of 15 years were analyzed. The relation between CD4+ and total lymphocyte counts and their rate of decline was analyzed with respect to age at seroconversion, time of seroconversion, and development of disease and subsequent death. As expected, the CD4+ and total lymphocyte population decline correlated with increased probability of disease and death. The patients fell into two distinct categories with respect to this decline: those whose cell count declined steadily (single slope) and those whose cell count remained steady or decreased very slowly for a variable period and then declined sharply (double slope). Within this cohort, the presence of a double slope appears to indicate a poorer prognosis, as 9 of 18 of the patients who have died showed this pattern, whereas only 6 of 42 of the remaining patients have this pattern even though more than half of this group have CD4+ lymphocyte counts < 0.2 x 10(9)/L. In addition, the ratio of CD4+ lymphocyte count to total lymphocyte count decreased with increasing cumulative frequency of the cumulative incidence of disease and death and the overall probability of death in this cohort was lower than expected, being 30% 12 years after seroconversion.

The identification of factors in seminal plasma responsible for suppression of natural killer cell activity.

We have identified two components of human seminal plasma which suppress natural killer (NK) cell activity in vitro. Dialysis and gel filtration experiments have shown both components to be of low molecular weight. The first will suppress NK cells following a short period of pretreatment, but this suppression is dependent upon the presence of bovine serum in the medium and is directly related to a loss of cell viability as measured by trypan blue dye exclusion. We suggest that this molecule is a polyamine. The second factor will not suppress NK activity following pretreatment of lymphocytes, but is a potent suppressor when added for the duration of the assay. This suppression is completely bovine serum independent, unrelated to toxicity and appears to be mediated by prostaglandin E2. The relevance of these results to a clinical situation is discussed.

Seminal plasma suppression of human lymphocyte responses in vitro requires the presence of bovine serum factors.

The effect of bovine sera on the ability of human seminal plasma (SP) to suppress lymphocyte responses was investigated. Marked suppression of natural cell-mediated cytotoxicity (NCMC) against K562 targets was observed when effectors were pretreated for 1 h with SP (1:50 dilution) in the presence of 10% newborn calf serum (NBCS). Some suppression of natural cytotoxicity was observed when the effectors were treated with SP in the presence of 10% fetal calf serum (FCS) and this suppression was greater if the length of pretreatment with SP was increased to 20 h. Suppression of NCMC did not occur, or was considerably less, when the effectors were treated with SP in the presence of 10% autologous human plasma or in HB103 serum-free medium. The effect of bovine sera on suppression of T lymphocyte response to mitogen was also examined. Pre-treatment of lymphocytes with SP (1:50 dilution) for 1 h in the presence of 10% NBCS was sufficient to abrogate completely the stimulatory effect of PHA. In the presence of 10% FCS it was necessary for SP to be present throughout the assay for suppression to occur. In HB103 medium, stimulation was only slightly decreased below control values when SP was present throughout the assay, but suppression was considerably less than that obtained upon addition of NBCS or FCS to the culture medium. These findings imply that suppression of lymphocyte activity by SP is dependent on the presence of exogenous serum co-factors and in the light of this finding, the clinical relevance of SP suppression may require re-examination.