Background and AimsThe hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail.MethodsUsing a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy.ResultsUltra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents.ConclusionUse of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.

The current study proposes prediction formulas both for random wave runup and mean overtopping discharge at seawalls constructed on land or in very shallow water. Although several existing formulas for runup and overtopping use the incident wave characteristics at the toes of seawalls, this study adopts the equivalent deepwater wave characteristics and an imaginary seawall slope for easy application of the formulas, especially in relation to seawalls constructed on land. The prediction formulas for overtopping use the predicted runup values. For the wave runup prediction formulas two sets of experimental data are used; i.e., a new set of data and the data obtained in a previous study. For the wave overtopping prediction formulas, the experimental data measured in a previous study are used. Comparisons with measurements show good performances of both new prediction methods.

LDV/SOF therapy is highly effective and safe in elderly Japanese patients with HCV GT1, even in the presence of cirrhosis or NS5A RASs. Patients with SVR may have a lower risk of HCC.

To measure volume blood flow quantitatively in human abdominal arteries, we used an ultrasonic image-directed Doppler system and electromagnetic flowmeter to first measure volume flow in canine arteries. In dogs, there was a strong linear correlation (R = 0.98) between the product of the time average of the maximum blood flow velocity and the average cross-sectional area and the volume blood flow measured by an electromagnetic flowmeter. These results enabled measurement of volume blood flow in the human superior mesenteric (SMA), splenic (SPA), and common hepatic (CHA) arteries from the abdominal wall. Comparison of pulsatility index values indicated a larger vascular resistance in the SMA than in the SPA or CHA.

Backgrounds & AimsWe aimed to clarify the characteristics of resistance-associated substitutions (RASs) after treatment failure with NS5A inhibitor, daclatasvir (DCV) in combination with NS3/4A inhibitor, asunaprevir (ASV), in patients with chronic hepatitis C virus genotype 1b infection.MethodsThis is a nationwide multicenter study conducted by the Japanese Red Cross Liver Study Group. The sera were obtained from 68 patients with virological failure after 24 weeks of DCV/ASV treatment. RASs in NS5A and NS3 were determined by population sequencing.ResultsThe frequency of signature RASs at position D168 of NS3 was 68%, and at positions L31 and Y93 of NS5A was 79 and 76%, respectively. The frequency of dual signature RASs in NS5A (L31-RAS and Y93-RAS) was 63%. RASs at L28, R30, P32, Q54, P58, and A92 in addition to dual signature RAS were detected in 5, 5, 1, 22, 2, and 0 patients, respectively. In total, triple, quadruple, and quintuple RASs in combination with dual signature RAS were detected in 35, 10, and 1.5% patients, respectively. These RASs were detected in patients without baseline RASs or who prematurely discontinued therapy. Co-existence of D168 RAS in NS3 and L31 and/or Y93 RAS in NS5A was observed in 62% of patients.ConclusionTreatment-emergent RASs after failure with DCV/ASV combination therapy are highly complex in more than 50% of the patients. The identification of complex RAS patterns, which may indicate high levels of resistance to NS5A inhibitors, highlights the need for RAS sequencing when considering re-treatment with regimens including NS5A inhibitors.