Seiichiro Fujisaki

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Characterization of a Large Cluster of Influenza A(H1N1)pdm09 Viruses Cross-Resistant to Oseltamivir and Peramivir during the 2013-2014 Influenza Season in Japan

ABSTRACT Between September 2013 and July 2014, 2,482 influenza 2009 pandemic A(H1N1) [A(H1N1)pdm09] viruses were screened in Japan for the H275Y substitution in their neuraminidase (NA) protein, which confers cross-resistance to oseltamivir and peramivir. We found that a large cluster of the H275Y mutant virus was present prior to the main influenza season in Sapporo/Hokkaido, with the detection rate for this mutant virus reaching 29% in this area. Phylogenetic analysis suggested the clonal expansion of a single mutant virus in Sapporo/Hokkaido. To understand the reason for this large cluster, we examined the in vitro and in vivo properties of the mutant virus. We found that it grew well in cell culture, with growth comparable to that of the wild-type virus. The cluster virus also replicated well in the upper respiratory tract of ferrets and was transmitted efficiently between ferrets by way of respiratory droplets. Almost all recently circulating A(H1N1)pdm09 viruses, including the cluster virus, possessed two substitutions in NA, V241I and N369K, which are known to increase replication and transmission fitness. A structural analysis of NA predicted that a third substitution (N386K) in the NA of the cluster virus destabilized the mutant NA structure in the presence of the V241I and N369K substitutions. Our results suggest that the cluster virus retained viral fitness to spread among humans and, accordingly, caused the large cluster in Sapporo/Hokkaido. However, the mutant NA structure was less stable than that of the wild-type virus. Therefore, once the wild-type virus began to circulate in the community, the mutant virus could not compete and faded out.

Monitoring and Characterization of Oseltamivir-Resistant Pandemic (H1N1) 2009 Virus, Japan, 2009–2010

No evidence of sustained spread was found, but 2 incidents of human-to-human transmission were suspected.

A single E105K mutation far from the active site of influenza B virus neuraminidase contributes to reduced susceptibility to multiple neuraminidase-inhibitor drugs

Drugs inhibiting the enzymatic activity of influenza virus neuraminidase (NA) are the cornerstone of therapy for influenza virus infection. The emergence of drug-resistant variants may limit the benefits of antiviral therapy. Here we report the recovery of an influenza B virus with reduced susceptibilities to NA inhibitors from a human patient with no history of antiviral drug treatment. The virus, designated B/Kochi/61/2011, was isolated by inoculating Madin-Darby canine kidney (MDCK) cells with respiratory specimens from the patient. NA inhibition assays demonstrated that the B/Kochi/61/2011 isolate showed a remarkable reduction in susceptibility to peramivir. The isolate also exhibited low to moderately reduced sensitivity to oseltamivir, laninamivir, and zanamivir. A sequence analysis of viruses propagated in MDCK cells revealed that the isolate contained a mutation (E105K) not previously associated with reduced susceptibility to NA inhibitors. However, pyrosequencing analysis showed that the NA E105K mutation was below a detectable level in the original clinical specimens, suggesting that the mutant virus may be preferably selected during propagation in MDCK cells. Analysis of the three-dimensional model of E105 and K105 NAs with peramivir suggested that the E105K mutation at the monomer-monomer interface of the NA tetramer may destabilize the tetrameric form of NA, leading to decreased susceptibility to NA inhibitors. These results have implications for understanding the mechanism of resistance against NA-inhibitor drugs.

Evaluation of Influenza Virus A/H3N2 and B Vaccines on the Basis of Cross-Reactivity of Postvaccination Human Serum Antibodies against Influenza Viruses A/H3N2 and B Isolated in MDCK Cells and Embryonated Hen Eggs

ABSTRACT The vaccine strains against influenza virus A/H3N2 for the 2010-2011 season and influenza virus B for the 2009-2010 and 2010-2011 seasons in Japan are a high-growth reassortant A/Victoria/210/2009 (X-187) strain and an egg-adapted B/Brisbane/60/2008 (Victoria lineage) strain, respectively. Hemagglutination inhibition (HI) tests with postinfection ferret antisera indicated that the antisera raised against the X-187 and egg-adapted B/Brisbane/60/2008 vaccine production strains poorly inhibited recent epidemic isolates of MDCK-grown A/H3N2 and B/Victoria lineage viruses, respectively. The low reactivity of the ferret antisera may be attributable to changes in the hemagglutinin (HA) protein of production strains during egg adaptation. To evaluate the efficacy of A/H3N2 and B vaccines, the cross-reactivities of postvaccination human serum antibodies against A/H3N2 and B/Victoria lineage epidemic isolates were assessed by a comparison of the geometric mean titers (GMTs) of HI and neutralization (NT) tests. Serum antibodies elicited by the X-187 vaccine had low cross-reactivity to both MDCK- and egg-grown A/H3N2 isolates by HI test and narrow cross-reactivity by NT test in all age groups. On the other hand, the GMTs to B viruses detected by HI test were below the marginal level, so the cross-reactivity was assessed by NT test. The serum neutralizing antibodies elicited by the B/Brisbane/60/2008 vaccine reacted well with egg-grown B viruses but exhibited remarkably low reactivity to MDCK-grown B viruses. The results of these human serological studies suggest that the influenza A/H3N2 vaccine for the 2010-2011 season and B vaccine for the 2009-2010 and 2010-2011 seasons may possess insufficient efficacy and low efficacy, respectively.

Characterization of neuraminidase inhibitor-resistant influenza A(H1N1)pdm09 viruses isolated in four seasons during pandemic and post-pandemic periods in Japan

Japan has the highest frequency of neuraminidase (NA) inhibitor use against influenza in the world. Therefore, Japan could be at high risk of the emergence and spread of NA inhibitor‐resistant viruses. The aim of this study was to monitor the emergence of NA inhibitor‐resistant viruses and the possibility of human‐to‐human transmission during four influenza seasons in Japan.

Antiviral susceptibility of influenza viruses isolated from patients pre- and post-administration of favipiravir.

Favipiravir, a viral RNA-dependent RNA polymerase inhibitor, has recently been approved in Japan for influenza pandemic preparedness. Here, we conducted a cell-based screening system to evaluate the susceptibility of influenza viruses to favipiravir. In this assay, the antiviral activity of favipiravir is determined by inhibition of virus-induced cytopathic effect, which can be measured by using a colorimetric cell proliferation assay. To demonstrate the robustness of the assay, we compared the favipiravir susceptibilities of neuraminidase (NA) inhibitor-resistant influenza A(H1N1)pdm09, A(H3N2), A(H7N9) and B viruses and their sensitive counterparts. No significant differences in the favipiravir susceptibilities were found between NA inhibitor-resistant and sensitive viruses. We, then, examined the antiviral susceptibility of 57 pairs of influenza viruses isolated from patients pre- and post-administration of favipiravir in phase 3 clinical trials. We found that there were no viruses with statistically significant reduced susceptibility to favipiravir or NA inhibitors, although two of 20 paired A(H1N1)pdm09, one of 17 paired A(H3N2) and one of 20 paired B viruses possessed amino acid substitutions in the RNA-dependent RNA polymerase subunits, PB1, PB2 and PA, after favipiravir administration. This is the first report on the antiviral susceptibility of influenza viruses isolated from patients after favipiravir treatment.

Influenza A(H1N1)pdm09 virus exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual H275Y/G147R substitution, Japan, March 2016

An influenza A(H1N1)pdm09 virus carrying a G147R substitution in combination with an H275Y substitution in the neuraminidase protein, which confers cross-resistance to oseltamivir and peramivir, was detected from an immunocompromised inpatient in Japan, March 2016. This dual H275Y/G147R mutant virus exhibited enhanced cross-resistance to both drugs compared with the single H275Y mutant virus and reduced susceptibility to zanamivir, although it showed normal inhibition by laninamivir.

Rapid detection of the S247N neuraminidase mutation in influenza A(H1N1)pdm09 virus by one-step duplex RT-PCR assay

Neuraminidase inhibitors, especially oseltamivir, have been used worldwide for the prophylaxis or treatment of pandemic influenza H1N1 2009 (A(H1N1)pdm09) viruses. The most commonly detected oseltamivir-resistant A(H1N1)pdm09 viruses possess the H275Y mutation in neuraminidase (NA). Recently, a novel A(H1N1)pdm09 variant containing the S247N mutation in NA was found mainly in the Asia-Pacific area. This mutation reduces sensitivity to oseltamivir and confers extremely high resistance to oseltamivir in association with the H275Y mutation compared with the resistance caused by the H275Y mutation alone. In this study, a rapid and simple one-step duplex RT-PCR assay for identifying A(H1N1)pdm09 viruses possessing the S247N mutation was developed. This assay is based on an endpoint genotyping analysis method and can use isolates from cell culture supernatants without RNA extraction, similar to the H275Y RT-PCR assay reported previously. The combination of the S247N and H275Y RT-PCR assays is a powerful surveillance method for determining whether A(H1N1)pdm09 viruses with the S247N mutation acquire the H275Y mutation.

Mutations at the monomer–monomer interface away from the active site of influenza B virus neuraminidase reduces susceptibility to neuraminidase inhibitor drugs

Amino acid changes in or near the active site of neuraminidase (NA) in influenza viruses reduce the susceptibility to NA inhibitor drugs. Here, we report the recovery of three influenza B viruses with reduced susceptibilities to NA inhibitors from human patients with no history of antiviral drug treatment. The three viruses were isolated by inoculating Madin–Darby canine kidney (MDCK) cells with respiratory specimens from the patients. NA inhibition assays demonstrated that two of the three isolates showed a highly reduced susceptibility to peramivir and moderately reduced susceptibility to oseltamivir, zanamivir, and laninamivir. The remaining one isolate exhibited moderately reduced sensitivity to peramivir, zanamivir, and laninamivir but was susceptible to oseltamivir. A sequence analysis of viruses propagated in MDCK cells revealed that all three isolates contained a single mutation (Q138R, P139S, or G140R) in NA not previously associated with reduced susceptibility to NA inhibitors. However, pyrosequencing analyses showed that the Q138R and G140R mutations were below a detectable level in the original clinical specimens; the P139S mutation was detected at a very low level, suggesting that the mutant viruses may be preferably selected during propagation in MDCK cells. The NA crystallographic structure showed that these mutations were located at the interface between the two monomers of the NA tetramer, away from the NA active site. In addition to amino acid substitutions around the active site of NA, these observations suggest that alterations in the monomer–monomer interface region of NA may contribute to reduced sensitivity to NA inhibitors.