Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis

Diagnosing different stages of hepatitis B infection using a competitive polymerase chain reaction assay

Purpose: Different stages of hepatitis B virus (HBV) infection can be defined by serum HBV DNA levels. This study attempts to (1) investigate serum HBV DNA levels in inactive carriers and patients with chronic HBV (CHB) infection and (2) define cut-off value between inactive carriers and HBeAg (precore antigen of HBV) negative CHB patients in Indian population. Methods: One hundred and forty samples encompassing 42 inactive HBsAg carriers and 98 CHB patients (53 HBeAg-positive and 45 HBeAg-negative) were analysed. Serum HBV DNA levels were determined employing an in-house competitive polymerase chain reaction (cPCR) assay. Results: The HBeAg-positive patients were found to have the maximum median HBV DNA load, which was significantly higher than the HBeAg-negative ones (median; 1.25 x 10 8 vs. 2.30 x 10 5 copies/mL ; P < 0.05). Interestingly, the latter group has significantly higher HBV DNA levels than the inactive carriers (median; 2.30 x 10 5 vs. 4.28 x 10 3 copies/mL ; P < 0.05). The 2.5 x 10 4 copies/ml HBV DNA levels were optimal for discriminating CHB patients (HBeAg-negative) from inactive carriers with 75.6 and 78.6% sensitivity and specificity, respectively. Conclusions: Despite the extensive overlapping of HBV DNA levels in inactive carriers and HBeAg negative CHB patients, 2.5 x 10 4 copies/mL is the most favourable cut-off value to classify these individuals and would be imperative in the better management of this dreadful disease

Diagnosing different stages of hepatitis B infection using a competitive polymerase chain reaction assay

Purpose: Different stages of hepatitis B virus (HBV) infection can be defined by serum HBV DNA levels. This study attempts to (1) investigate serum HBV DNA levels in inactive carriers and patients with chronic HBV (CHB) infection and (2) define cut-off value between inactive carriers and HBeAg (precore antigen of HBV) negative CHB patients in Indian population. Methods: One hundred and forty samples encompassing 42 inactive HBsAg carriers and 98 CHB patients (53 HBeAg-positive and 45 HBeAg-negative) were analysed. Serum HBV DNA levels were determined employing an in-house competitive polymerase chain reaction (cPCR) assay. Results: The HBeAg-positive patients were found to have the maximum median HBV DNA load, which was significantly higher than the HBeAg-negative ones (median; 1.25 x 10 8 vs. 2.30 x 10 5 copies/mL ; P < 0.05). Interestingly, the latter group has significantly higher HBV DNA levels than the inactive carriers (median; 2.30 x 10 5 vs. 4.28 x 10 3 copies/mL ; P < 0.05). The 2.5 x 10 4 copies/ml HBV DNA levels were optimal for discriminating CHB patients (HBeAg-negative) from inactive carriers with 75.6 and 78.6% sensitivity and specificity, respectively. Conclusions: Despite the extensive overlapping of HBV DNA levels in inactive carriers and HBeAg negative CHB patients, 2.5 x 10 4 copies/mL is the most favourable cut-off value to classify these individuals and would be imperative in the better management of this dreadful disease