Evolving Dynamics of Whole-Genome Influenza A/H3N2 Viruses Isolated in Cameroon

The number of amino acid (AA) mutations in the interferon sensitivity determining region (ISDR) of NS5A is reported to affect the response to interferon (IFN) therapy in patients with chronic hepatitis C (CHC). The aim of this study was to clarify whether host immunity is influenced by the number of AA mutations in the ISDR. Subjects included 44 patients with CHC infected with genotype 1b and high viral load. The number of AA mutations in the ISDR was retrospectively determined using stored serum samples taken immediately before starting therapy. All patients received IFN-alpha 2b or pegylated-IFN (PEG-IFN)-alpha 2b and ribavirin. When serum hepatitis C virus-ribonucleic acid (HCV-RNA) was negative at 4 or 12weeks after starting therapy, the patient was defined as having rapid viral response (RVR) or early viral response (EVR), respectively. CD4(+) T cell (Th1 or Th2) in peripheral blood (PB) before and until day 56 of treatment was analyzed. Rates of RVR and EVR were 0 (0/21) and 14% (3/21), respectively, in patients with one or fewer AA mutations in the ISDR (ISDR0-1), and 30 (7/23), and 74% (17/23), respectively, with two or more AA mutations in the ISDR (ISDR>2). Although the percentage of PB Th1 cells did not differ between the two groups during the study period, the percentage of PB Th2 cells was significantly lower in the ISDR0-1 group than in the ISDR>2 group at baseline and on days3, 7, 14, and 28 of treatment. The number of AA mutations in the ISDR influenced PB Th2 cells before and until day 28, and was associated with higher RVR and EVR rates.

Genetic characterization of influenza viruses from influenza-related hospital admissions in the St. Petersburg and Valencia sites of the Global Influenza Hospital Surveillance Network during the 2013/14 influenza season

Effects of Mutation Number in Interferon Sensitivity Determining Region on Peripheral Blood CD4-Positive T Cell Subsets (TH1, TH2) in Chronic Hepatitis C Patients With Hepatitis C Virus Genotype 1b a and High Viral Load

Mutations in the non-structural protein 5A gene in patients with chronic hepatitis C virus 1b infection during repeated interferon treatment

Objective To analyze the evolution characterization and resistance-associated mutations of the neuraminidase (NA) gene of influenza A (H1N1) pdm09 and H3N2 viruses. Methods A total of 12 influenza A (H1N1) pdm09 and 13 A (H3N2) strains in Tangshan were randomly selected from October 2013 to March 2016. Viral RNA was extracted and amplified by RT-PCR, NA genes from A (H1N1) pdm09 and A (H3N2) viruses were sequenced. A phylogenetic tree of the NA coding sequences was generated by Molecular Evolutionary Genetic Analysis (MEGA) version 6.0 software. The characteristics of nucleic acid sequence and amino acid variations were analyzed by BioEdit v7.2.5 software. Results During the 2013-2016 influenza seasons, the NA genes of 12 strains of influenza A (H1N1) pdm09 virus and 13 strains of influenza A (H3N2) virus in Tangshan shared the nucleotide identities of 98.5%-100.0% and 98.1%-100.0%, respectively. Phylogenetic analysis indicated that the two viruses belonged to clade 6B and 3C, respectively. The 6 isolates in 2013/2014 season was in clade 6B contained 9 amino acid substitutions comparing to the vaccine strain. The other 6 isolates from 2015/2016 season were further divided into different sub-clades, including three strains of subclade 6B.2 containing new L67I and T381I substitutions, two in subclade 6B.1 with substitutions V13I, P93S and I314M, one in clade 6B with I117M, I288V and S450G mutations. The influenza A (H3N2) virus strains from 2013/2014 season, 2014/2015 season and from 2015/2016 season were in clade 3C, subclade 3C.3a and 3C.2a1, respectively. Comparing to the vaccine strain A/Texas/50/2012(3C.1), strains in subclade 3C.3a contained V143M, E221D, P386S and I392T substitutions. Comparing to A/Switzerland/9715293/2013 (3C.3a), strains in subclade 3C.2a1 had point mutations of S245N (resulting in N-glycosylation motif: NAT245-247), S247T, T267K, P339N, I380V and P468H. None of Tangshan strains of both subtypes had the oseltamivir or zanamivir resistant mutation. Conclusions New genetic subgroups of NA gene of influenza A (H1N1) pdm09 and H3N2 viruses have emerged in Tangshan city. Key words: Influenza A virus; Neuraminidase; Genotype; Drug resistance

Amino acid mutations in the interferon sensitivity determining region and serum virus load in hepatitis C virus carriers with long-term normal ALT levels

Amino acid variations in several HCV genomic regions have been reported to be associated with response to interferon (IFN)-α plus ribavirin (RBV) combination therapy. However, the results remain controversial. In this study, we further investigated the amino acid variation of full-length HCV genome and its correlation to the response to pegylated interferon (PEG-IFN)-α2a and RBV combination therapy in patients with HCV genotype 1b (HCV-1b). We retrospectively analysed 18 chronic HCV-1b patients (9 with rapid virological response and 9 non-response to therapy) treated with PEG-IFN-α2a plus RBV for 48 weeks. The nearly full-length HCV genome sequence was amplified by reverse transcription (RT)-PCR followed by cloning and sequencing. Genetic diversity differences between two groups were analysed including the number of amino acid variations in the HCV polyprotein and the mean pair-wise protein distance. No single amino acid variations were closely associated with treatment outcome. However, the number of amino acid mutations in the NS5B region especially in the thumb domain and in the NS5A-V3 region was associated with the response to PEG-IFN-α/RBV therapy (P=0.002 and P=0.029, respectively). The number of substitutions in the NS5B region was significantly correlated with the numbers of substitutions in the V3 region (r=0.568, P=0.027). Amino acid substitutions in the NS5B region especially in the thumb domain and the NS5A-V3 region may play a role in the response to combined PEG-IFN-α2a and RBV therapy in HCV-1b patients.

913 THE MECHANISMS AND RISK FACTORS FOR FLUOROQUINOLONE-RESISTANCE IN ENTEROCOCCUS FAECALIS STRAINS CLINICALLY ISOLATED FROM URINARY TRACT INFECTION PATIENTS

The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities required for sequential methylation of the cap structure present at the 5' end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-l-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-l-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-l-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-l-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-l-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.

These are exciting times, with a plethora of new technologies that are expediting discovery of the genetic underpinnings of human disease. Comprehensive resequencing of the human genome is now feasible and affordable, allowing each person's entire genetic makeup to be revealed. The major focus of

BackgroundA substantial proportion of hepatitis C virus (HCV)-1b infected patients do not response to pegylated interferon-α plus ribavirin (PegIFNα/RBV) combination therapy that was partially associated with mutations in the non-structural 5A (NS5A) protein.ObjectivesAnalysis of NS5A polymorphisms in HCV genotype 1b pre-treatment serum samples from Estonian patients and their effect on the treatment response.Patients and MethodsTwenty-nine complete NS5A sequences obtained from patients with chronic HCV-1b infection who had received combined therapy with PegIFNα-2a/RBV were analyzed and compared with the prototype strain HCV-J. Twelve patients achieved a sustained virological response (SVR), 15 were non-SVR and 2 patients stopped treatment because of side effects.ResultsNo significant difference in total number of amino acid mutations was observed between isolates from SVR and non-SVR patients in any known regions of the NS5A protein. However, specific amino acid substitutions at positions 1989 and 2283 correlated significantly with SVR, mutations at positions 1979, 2107, 2171 and 2382 were associated with non-response to treatment and amino acid substitution at position 2319 was observed in relapsers. At phylogenetic analysis, NS5A nucleotide sequences have been subdivided into four groups characterized by the different treatment response. Twenty-four novel nucleotide polymorphisms and 11 novel amino acid polymorphisms were identified based on the phylogenetic tree topology.ConclusionsSpecific amino acid substitutions correlating with the treatment response were found. Polymorphisms revealed by phylogenetic analysis may define the signature patterns for treatment susceptible and treatment resistant strains prevalent in Estonia.

In our recent study of the full-length hepatitis A virus (HAV) genome from some patients with fulminant hepatitis and acute hepatitis, possible associations were suggested between the severity of hepatitis A and the amino acid substitutions in the nonstructural protein 2B. We therefore analyzed HAV 2B from many patients with various clinical disease severities. Serum samples from 30 Japanese patients with sporadic hepatitis A from five widely separated regions of Japan, comprising nine patients with fulminant hepatitis (FH), six with severe acute hepatitis (AHs), and 15 with acute hepatitis (AH), were examined for HAV RNA. The entire sequences of HAV 2B were analyzed. Compared with the sequence of the wild-type HAV strain GBM, nucleotide sequences of 2B had homology of 94.5 +/- 1.0% in FH, 95.2 +/- 1.2% in AHs, and 95.1 +/- 1.8% in AH. Deduced amino acid sequences had homology of 97.5 +/- 2.1% in FH, 97.9 +/- 2.4% in AHs, and 98.5 +/- 1.3% in AH. Differences were not statistically significant among the three groups. The average number of amino acid mutations between amino acids 100 and 200 was 5.0 +/- 5.2 per case in FH, 4.0 +/- 6.0 in AHs, and 1.9 +/- 2.9 in AH. The differences between FH and AH, AHs and AH, and between severe cases (FH and AHs) and nonsevere cases (AH) were not statistically significant (P = 0.13, P = 0.45, and P = 0.10, respectively). There were no obvious differences in the sequences among FH, AHs, and AH throughout the 2B region, but there seemed to be more mutations in the strains obtained from FH and AHs patients than in those obtained from AH patients in the central part of HAV 2B.

We monitored the circulating strains and genetic variation among seasonal influenza A and B viruses in Thailand between July 2017 and March 2020. The hemagglutinin gene was amplified and sequenced. We identified amino acid (AA) changes and computed antigenic relatedness using the Pepitope model. Phylogenetic analyses revealed multiple clades/subclades of influenza A(H1N1)pdm09 and A(H3N2) were circulating simultaneously and evolved away from their vaccine strain, but not the influenza B virus. The predominant circulating strains of A(H1N1)pdm09 belonged to 6B.1A1 (2017–2018) and 6B.1A5 (2019–2020) with additional AA substitutions. Clade 3C.2a1b and 3C.2a2 viruses co-circulated in A(H3N2) and clade 3C.3a virus was found in 2020. The B/Victoria-like lineage predominated since 2019 with an additional three AA deletions. Antigenic drift was dominantly facilitated at epitopes Sa and Sb of A(H1N1)pdm09, epitopes A, B, D and E of A(H3N2), and the 120 loop and 190 helix of influenza B virus. Moderate computed antigenic relatedness was observed in A(H1N1)pdm09. The computed antigenic relatedness of A(H3N2) indicated a significant decline in 2019 (9.17%) and 2020 (− 18.94%) whereas the circulating influenza B virus was antigenically similar (94.81%) with its vaccine strain. Our findings offer insights into the genetic divergence from vaccine strains, which could aid vaccine updating.

The 2017–2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017–2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017–2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017–2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33–47% and 24–30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017–2018 influenza epidemic season in Russia.

Inferring Protein Sequence-Function Relationships with Large-Scale Positive-Unlabeled Learning.