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.

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.

We previously investigated EBV DNAemia in 500 renal transplant recipients and found 46% of patients positive, with chronic high viral loads (CHVL) in 7%. Risk factors for DNAemia included time from transplant and recipient seronegative status. Mycophenolate was associated with low rates of DNAemia. We now report a nested case control study recruiting 60 patients from the original 500, matched for age and time from transplant and stratified by viral load into; group 1, undetectable (n=19), group 2 low (<75% samples >1000copies/ml) (n=20) and group 3 CHVL ( ≥75% samples >1000copies/ml or ≥3 samples over >6 months with ≥10,000 copies/ml) (n=21). Methods We analysed serology, DNAemia in whole blood and plasma, lymphocyte subsets, ultrasound examination of cervical lymph nodes and immunosuppression burden. Results All participants were EBV seropositive. Anti-VCA levels were significantly higher in those with a CHVL. 7 patients had DNA detectable in plasma as well as whole blood (6/7 were in Group 3). This small sub-group had lower CD4:8 ratios than those with DNA in whole blood only (p=0.041), with higher CD8 counts (p=0.09). Group 3 had more individuals with ≥2 lymph nodes measuring >0.5mm than Group 1 (62% v 26%; OR 4.6; CI 1.2-17.5; p=0.03). A “heavy” immunosuppression burden was identified in 0% of Group 1 and 29% of Group 3 (p=0.012). No association between mycophenolate and lymphocyte counts was identified. PTLD has not been identified in any of the 3 patient groups to date. Conclusion Chronic immunosuppression and immune dysregulation may predispose to EBV DNAemia, with similarities to the HIV setting. There may be scope to optimise immunosuppression in the setting of high level EBV DNAemia. A planned 5 year follow-up of the study patients will provide outcome data in relation to immune function and lymph node findings.

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.

SUMMARY 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 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.

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.