In this study, we have sequenced the C-terminal part of the Epstein-Barr virus (EBV)-BNLF-1 gene encoding for the latent membrane protein-1 from tissues of EBV-positive Danish Hodgkin's disease (HD) and of Danish and Malaysian peripheral T-cell lymphomas (PTLs) and from tonsils of Danish infectious mononucleosis (IM). Our study showed that some of the 7 single-base mutations and the 30-bp deletion previously detected between codons of amino acid 322 and 366 in the BNLF-1 gene of the nasopharyngeal carcinoma cell line CAO were present in all Malaysian PTLs and in 60% of the Danish PTLs. In HD and the IM cases, the mutations were present in about 30%. The 30-bp deletion and the single base mutations occurred independently, and mutations were detectable in the majority of EBV type B-positive cases. These findings suggest that the 30-bp deletion and the 7 single-base mutations in the C-terminal part of the CAO-BNLF-1 gene do not characterize a new EBV type A substrain. Rather, some of the positions of single base mutations and the 30-bp deletion are hot spots that may have mutated independently through the evolution of EBV strains.
Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.
Nasopharyngeal carcinoma (NPC) is an epithelial cancer of the nasopharynx which is highly associated with Epstein-Barr virus (EBV). Worldwide, most of the top 20 countries with the highest incidence and mortality rates of NPC are low- and middle-income countries. Many studies had demonstrated that EBV could be detected in the tissue, serum and plasma of NPC patients. In this study, we explored the potential of assays based on non-invasive nasal washings (NW) as a diagnostic and prognostic tool for NPC. A total of 128 patients were evaluated for NW EBV DNA loads and a subset of these samples were also tested for 27 EBV and human miRNAs shortlisted from literature. EBV DNA and seven miRNAs showed area under the receiver operating characteristic curve (AUC) values of more than 0.7, suggestive of their potential utility to detect NPC. Logistic regression analyses suggested that combination of two NW assays that test for EBNA-1 and hsa-miR-21 had the best performance in detecting NPC. The trend of NW EBV DNA load matched with clinical outcome of 71.4% (10 out of 14) NPC patients being followed-up. In summary, the non-invasive NW testing panel may be particularly useful for NPC screening in remote areas where healthcare facilities and otolaryngologists are lacking, and may encourage frequent testing of individuals in the high risk groups who are reluctant to have their blood tested. However, further validation in an independent cohort is required to strengthen the utility of this testing panel as a non-invasive detection tool for NPC.
Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3'-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3' deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.