Timing Constraints of In Vivo Gag Mutations during Primary HIV-1 Subtype C Infection

Background: Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness

Genetic and epidemiological analysis of norovirus from children with gastroenteritis in Botswana, 2013–2015

Background: Norovirus is a leading cause of viral gastroenteritis worldwide with a peak of disease seen in children. The epidemiological analysis regarding the virus strains in Africa is limited. The first report of norovirus in Botswana was in 2010 and currently, the prevalence and circulating genotypes of norovirus are unknown, as the country has no systems to report the norovirus cases. This study investigated the prevalence, patterns and molecular characteristics of norovirus infections among children ≤5 years of age admitted with acute gastroenteritis at four hospitals in Botswana. Methods: A total of 484 faecal samples were collected from children who were admitted with acute gastroenteritis during the rotavirus vaccine impact survey between July 2013 and December 2015. Norovirus was detected using real-time RT-PCR. Positive samples were genotyped using conventional RT-PCR followed by partial sequencing of the capsid and RdRp genes. Norovirus strains were determined by nucleotide sequence analysis using the online Norovirus Genotyping Tool Version 1.0, and confirmed using maximum likelihood tree construction as implemented in MEGA 6.0. Results: The prevalence of norovirus was 9.3% (95% CI 6.7–11.9). The genotype diversity was dominated by the GII.4 strain at 69.7%. This was followed by GII.2, GII.12 each at 9.1%, GI.9 at 6.6% and GII.6, GII.10 each at 3.0%. The most common combined RdRp/Capsid genotype was the GII.Pe/GII.4 Sydney 2012. Norovirus was detected during most part of the year; however, there was a preponderance of cases in the wet season (December to March). Conclusion: The study showed a possible decline of norovirus infections in the last 10 years since the first report. An upward trend seen between 2013 and 2015 may be attributable to the success of rotavirus vaccine introductions in 2012. Knowledge of circulating genotypes, seasonal trends and overall prevalence is critical for prevention programming and possible future vaccine design implications.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacult

Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus

AbstractInfection of type II alveolar epithelial (ATII) cells by influenza A viruses (IAV) correlates with severe respiratory disease in humans and mice. To understand pathogenic mechanisms during IAV infection of ATII cells, murine ATII cells were cultured to maintain a differentiated phenotype, infected with IAV-PR8, which causes severe lung pathology in mice, and proteomics analyses were performed using liquid chromatography–mass spectrometry. PR8 infection increased levels of proteins involved in interferon signaling, antigen presentation, and cytoskeleton regulation. Proteins involved in mitochondrial membrane permeability, energy metabolism, and chromatin formation had reduced levels in PR8-infected cells. Phenotypic markers of ATII cells in vivo were identified, confirming the differentiation status of the cultures. Surfactant protein B had decreased levels in PR8-infected cells, which was confirmed by immunoblotting and immunofluorescence assays. Analysis of ATII cell protein profiles will elucidate cellular processes in IAV pathogenesis, which may provide insight into potential therapies to modulate disease severity