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Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the in vitro susceptibility of multi-NAI-resistant AIVs is associated with in vivo susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3 R292K ) or Q136K (ma81K-N8 Q136K ) NA substitutions, which impart in vitro susceptibility only to LAN or OS, respectively. Both ma81K-N3 R292K and ma81K-N8 Q136K virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the in vitro susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of in vitro and in vivo NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs. IMPORTANCE The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that in vitro data predicted in vivo behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections., (Copyright © 2020 American Society for Microbiology.)

Objective: Direct comparison of the clinical outcomes between nucleos(t)ide analogue (NA) discontinuation versus NA continuation has not been performed in patients with chronic hepatitis B who achieved HBsAg-seroclearance. Whether NA discontinuation was as safe as NA continuation after NA-induced surface antigen of HBV (HBsAg) seroclearance was investigated in the present study., Designs: This multicentre study included 276 patients from 16 hospitals in Korea who achieved NA-induced HBsAg seroclearance: 131 (47.5%) discontinued NA treatment within 6 months after HBsAg seroclearance (NA discontinuation group) and 145 (52.5%) continued NA treatment (NA continuation group). Primary endpoint was HBsAg reversion and secondary endpoints included serum HBV DNA redetection and development of hepatocellular carcinoma (HCC)., Results: During follow-up (median=26.9 months, IQR=12.2-49.2 months), 10 patients (3.6%) experienced HBsAg reversion, 6 (2.2%) showed HBV DNA redetection and 8 (2.9%) developed HCC. Compared with NA continuation, NA discontinuation was not associated with HBsAg reversion in both univariable (HR=0.45, 95% CI=0.12 to 1.76, log-rank p=0.24) and multivariable analyses (adjusted HR=0.65, 95% CI=0.16 to 2.59, p=0.54). The cumulative probabilities of HBsAg reversion at 1, 3 and 5 years were 0.8%, 2.3% and 5.0% in the NA discontinuation group, and 1.5%, 6.3% and 8.4% in the NA continuation group, respectively. NA discontinuation was not associated with higher risk of either HBV redetection (HR=0.83, 95% CI=0.16 to 4.16, log-rank p=0.82) or HCC development (HR=0.53, 95% CI=0.12 to 2.23, log-rank p=0.38)., Conclusion: The discontinuation of NA was not associated with a higher risk of either HBsAg reversion, serum HBV DNA redetection or HCC development compared with NA continuation among patients who achieved HBsAg seroclearance with NA., Competing Interests: Competing interests: SUK reports receiving grants from Yuhan Pharmaceuticals, and lecture fees from Bristol-Myers Squibb, Gilead Science and Yuhan Pharmaceuticals; JWJ reports receiving research grants from Yuhan Pharmaceuticals and lecture fees from Abbie, Bristol-Myers Squibb, Gilead Science and MSD Korea, and serving as an advisory board member of Abbie, Gilead Science and MSD Korea; Y-SL reports receiving research grants from Bayer HealthCare Pharmaceuticals and Gilead Science, and serving as an advisory board member of Bayer HealthCare Pharmaceuticals and Gilead Science; HWL reports receiving lecture fee from Abbie, Chongkundang Pharmaceuticals, Dong-A ST Pharmaceuticals, Ildong Pharmaceuticals and Yuhan Pharmaceuticals; JHK, lecture fee from Abbie, Bristol-Myers Squibb, Gilead Science, and MSD Korea; SWL reports receiving research grants from Yuhan Pharmaceuticals, lecture fees from Abbie, Bristol-Myers Squibb, Bukwang Pharmaceuticals, Daewoong Pharmaceuticals, MSD Korea and Yuhan Pharmaceuticals, and serving as an advisory board member of Eisai and Gilead Science; JYJ reports receiving research grants from Bukwang and Yuhan Pharmaceuticals, lecture fees from Bristol-Myers Squibb, Bukwang Pharmaceuticals, Bayer HealthCare Pharmaceuticals, Celltrion, Daewoong Pharmaceuticals, Gilead Science and Yuhan Pharmaceuticals; YJK reports receiving research grants from Bristol-Myers Squibb, Roche, JW Creagene, Bukwang Pharmaceuticals, Handok Pharmaceuticals, Hanmi Pharmaceuticals, Yuhan Pharmaceuticals and Pharmaking, and lecture fees from Bayer HealthCare Pharmaceuticals, Gilead Science, MSD Korea, Yuhan Pharmaceuticals, Samil Pharmaceuticals, CJ Pharmaceuticals, Bukwang Pharmaceuticals and Handok Pharmaceuticals; J-HY reports receiving research grant from Bayer HealthCare Pharmaceuticals, Bukwang Pharmaceuticals and Daewoong Pharmaceuticals, and J-HL reports receiving lecture fees from GreenCross Cell, Daewoong Pharmaceuticals and Gilead Korea., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

The resistance of influenza viruses to neuraminidase (NA) inhibitors (NAIs; i.e. oseltamivir, zanamivir, peramivir and laninamivir) can be associated with several NA substitutions, with differing effects on viral fitness. To identify novel molecular markers conferring multi-NAI resistance, the NA gene of oseltamivir-resistant (H275Y, N1 numbering) 2009 pandemic influenza [A(H1N1)pdm09] virus was enriched with random mutations. This randomly mutated viral library was propagated in Madin-Darby canine kidney (MDCK) cells under zanamivir pressure and gave rise to additional changes within NA, including an I436N substitution located outside the NA enzyme active site. We generated four recombinant A(H1N1)pdm09 viruses containing either wild-type NA or NA with single (I436N or H275Y) or double (H275Y-I436N) substitutions. The double H275Y-I436N mutation significantly reduced inhibition by oseltamivir and peramivir and reduced inhibition by zanamivir and laninamivir. I436N alone reduced inhibition by all NAIs, suggesting that it is a multi-NAI resistance marker. I436N did not affect viral fitness in vitro or in a murine model; however, H275Y and I436N together had a negative impact on viral fitness. Further, I436N alone did not have an appreciable impact on viral replication in the upper respiratory tract or transmissibility in ferrets. However, the rg-H275Y-I436N double mutant transmitted less efficiently than either single mutant via the direct contact and respiratory droplet routes in ferrets. Overall, these results highlight the usefulness of a random mutagenesis approach for identifying potential molecular markers of resistance and the importance of I436N NA substitution in A(H1N1)pdm09 virus as a marker for multi-NAI resistance.

The development of boceprevir and telaprevir was a major step forward in the treatment of chronic hepatitis C. In addition, the treatment of these infections has been recently revolutionized by the approval of sofosbuvir and simeprevir. However, there are several challenges associated with the application of novel drugs, such as new and more frequent adverse events, new drug interactions, and excessively high treatment costs. An additional concern is viral resistance. These considerations highlight the fact that direct-acting antiviral agents are not a panacea and may not be the best option for all patients who are in need of therapy. This retrospective study revealed that the sustained virologic response was not significantly reduced following peginterferon and ribavirin retreatment compared with the new therapy. We suggest that patients who experience relapse shortly after completing treatment with peginterferon and ribavirin have a reasonable chance of achieving a sustained virologic response when retreated with these drugs alone.

Unlabelled: Neuraminidase inhibitors (NAIs) have been widely used to control influenza virus infection, but their increased use could promote the global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened a known mutation(s) that could confer multidrug resistance to the currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA-encoding genes (catalytic residues R152K and R292K, framework residues E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single and double mutant viruses recovered in the backbone of pH1N1, four variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition by all of the NAIs and two variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the nine recombinant H5N1 viruses, four variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition by all of the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance on pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection., Importance: There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition by all of the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo. Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza virus variants, as well as the development of alternative therapeutics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background/aims: Carnitine and vitamin complex (Godex®) is widely used in patients with chronic liver disease who show elevated liver enzyme in South Korea. The purpose of this study is to identify the efficacy and safety of carnitine from entecavir combination therapy in Alanine aminotransferase (ALT) elevated Chronic Hepatitis B (CHB) patients., Methods: 130 treatment-naïve patients with CHB were enrolled from 13 sites. The patients were randomly selected to the entecavir and the complex of entecavir and carnitine. The primary endpoint of the study is ALT normalization level after 12 months., Results: Among the 130 patients, 119 patients completed the study treatment. The ALT normalization at 3 months was 58.9% for the monotherapy and 95.2% for the combination therapy (P<0.0001). ALT normalization rate at 12 months was 85.7% for the monotherapy and 100% for the combination group (P=0.0019). The rate of less than HBV DNA 300 copies/mL at 12 months was not statistically significant (P=0.5318) 75.9% for the monotherapy, 70.7% for the combination and it was. Quantification of HBsAg level was not different from the monotherapy to combination at 12 months. Changes of ELISPOT value to evaluate the INF-γ secretion by HBsAg showed the increasing trend of combination therapy compare to mono-treatment., Conclusions: ALT normalization rate was higher in carnitine complex combination group than entecavir group in CHB. Combination group was faster than entecavir mono-treatment group on ALT normalization rate. HBV DNA normalization rate and the serum HBV-DNA level were not changed by carnitine complex treatment.

Herbal medicine is used to treat many conditions such as asthma, eczema, premenstrual syndrome, rheumatoid arthritis, migraine, headaches, menopausal symptoms, chronic fatigue, irritable bowel syndrome, cancer, and viral infections such as influenza. In this study, we investigated the antiviral effect of KIOM-C for the treatment of influenza A virus infection. Our results show that oral administration of KIOM-C conferred a survival benefit to mice infected with the 2009 pandemic H1N1 [A(H1N1)pdm09] virus, and resulted in a 10- to 100-fold attenuation of viral replication in ferrets in a dose-dependent manner. Additionally, oral administration of KIOM-C increased the production of antiviral cytokines, including IFN-γ and TNF-α, and decreased levels of pro-inflammatory cytokines (IL-6) and chemokines (KC, MCP-1) in the Bronchoalveolar lavage fluid (BALF) of A(H1N1)pdm-infected mice. These results indicate that KIOM-C can promote clearance of influenza virus in the respiratory tracts of mice and ferrets by modulating cytokine production in hosts. Taken together, our results suggest that KIOM-C is a potential therapeutic compound mixture for the treatment of influenza virus infection in humans., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background/aims: clevudine is a potent antiviral agent that has demonstrated efficacy in patients with chronic hepatitis B. This study compared the efficacy of clevudine (C), entecavir (E) and lamivudine (L) in treatment-naive patient with HBeAg-positive chronic hepatitis B., Methods: a total of 146 treatment-naive patients with HBeAg-positive chronic hepatitis B received clevudine, entecavir or lamivudine. C group (n=39) received 30 mg of clevudine, E group (n=39) received 0.5 mg of entecavir and L group (n=68) received 100 mg of lamivudine once a day for more than 48 weeks. The efficacy analysis estimated the mean changes of the HBV DNA levels as a virologic response, the normalization of the ALT levels (less than 35 IU/L) as a biochemical response and loss of HBeAg or seroconversion as a serologic response. The serum HBV DNA level was quantified by hybrid capture and real-time PCR assay., Results: before the administration of clevudine, entecavir and lamivudine, the mean HBV DNA and ALT levels and the gender and age were well balanced among the three groups (p>0.05). For the virologic response at 48 weeks, the mean changes of the HBV DNA levels from baseline of the C, E and L groups were -3.8+/-2.2, -4.5+/-1.9 and -2.5+/-2.1 log copies/mL. C and E group showed superior antiviral activity compared to that of L group (p<0.0001), but no significant differences in antiviral response were noted between C and E groups. For the biochemical response at 48 weeks, the normalization of the ALT levels (less than 35 IU/L) among the C, E and L groups was 82%, 74% and 71%, respectively (p=0.46). The rates of undetectable serum HBV DNA (less than 300 copies/mL) of the C, E and L groups were 39%, 69% and 27%, respectively (p<0.0001). For the serologic response at 48 weeks, the loss of HBeAg was 13%, 31% and 24% and the seroconversion was 10%, 23% and 17%, respectively. There was no difference of efficacy among the three groups regarding ALT normalization or serologic response (p>0.05). Viral breakthrough in C group was noted at 24 weeks (5%) and 48 weeks (21%), but no biochemical breakthrough was noted. The elevation of the serum CK level was noted in only 1 patient of group C at 48 weeks (2.56%) after therapy. For the patients without or with liver cirrhosis (LC), C and E group showed superior antiviral activity compared to that of the L group, but the antiviral activity was more effective in non- LC group than LC group (p<0.0001 vs p=0.036)., Conclusions: clevudine therapy compared with lamivudine for 48 weeks showed significantly potent antiviral efficacy in treatment-naive patients with HBeAg-positive chronic hepatitis B, and especially in the non-LC patients. However, the antiviral efficacy of clevudine was similar to that of entecavir even though taking into account relatively short follow up period and retrospective study.

We found a relatively high frequency of unique amantadine-resistant H3N2 and H9N2 avian influenza viruses (Val27Ile on M2 protein) isolated from live poultry markets in South Korea and confirmed that a Val27Ile single substitution in the M2 protein is enough to acquire the amantadine resistance phenotype by using reverse-genetically created human-avian reassortant viruses.

Background/aims: The purpose of this study is to elucidate the efficacy and safety of combined peginterferon and ribavirin therapy in Korean patients with chronic HCV infection., Methods: We retrospectively analyzed the clinical records of 84 patients. Thirty five patients with genotype 1 HCV infection were treated with peginterferon alpha-2a 180 microg/week and ribavirin 1,000-1,200 mg/day for 48 weeks, and 49 patients with genotype non-1 were treated with peginterferon alpha-2a 180 microg/week and ribavirin 800 mg/day for 24 weeks., Results: An early virologic response was seen in 87.0% of patients with genotype 1 HCV. An end of treatment response (ETR) was seen in 82.6% and 97.6% of patients with genotype 1 and genotype non-1, respectively. An overall sustained virologic response (SVR) was seen in 53 patients (82.8%) of the 64 patients: in 16 (69.6%) of 23 patients with genotype 1 and in 37 (90.2%) of 41 patients with genotype non-1. An end of treatment biochemical response was seen in 58 patients (90.6%) [genotype 1, 20 patients (87.0%); genotype non-1, 38 patients (92.7%)], and a sustained biochemical response was achieved in 49 patients (76.6%) [genotype 1, 14 patients (60.9%); genotype non-1, 35 patients (85.4%)]. Independent factors affecting an SVR were HCV genotype and the baseline HCV RNA level., Conclusions: This study shows that a combination therapy of peginterferon and ribavirin is highly effective for chronic HCV infection, producing a high SVR and ETR.

Hyung Joon Yim,1,&ast; Ji Hoon Kim,2,&ast; Yong Kyun Cho,3 Young Oh Kweon,4 Hyun Chin Cho,5 Jae Seok Hwang,6 Changhyeong Lee,7 Moon Soo Koh,8 Yang-Hyun Baek,9 Young-Min Park,10 Jeong-Hoon Lee,11 Seung Up Kim,12 Min-Kyu Kang,13 Neung Hwa Park,14 June Sung Lee,15 Young Eun Chon,16 Gab Jin Cheon,17 Hee Bok Chae,18 Joo Hyun Sohn,19 Young-Suk Lim20 1Department of Internal Medicine, Korea University Ansan Hospital, Korea University College of Medicine, Seoul, Korea; 2Department of Internal Medicine, Korea University Guro Hospital, Korea University College of Medicine, Seoul, Korea; 3Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea; 4Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Korea; 5Department of Internal Medicine, Gyeongsang National University Hospital, Gyeongsang National University School of Medicine, Jinju, Korea; 6Department of Internal Medicine, Keimyung University School of Medicine, Daegu, Korea; 7Department of Internal Medicine, Daegu Catholic University School of Medicine, Daegu, Korea; 8Department of Internal Medicine, Dongguk University Ilsan Hospital, College of Medicine, Dongguk University, Goyang, Korea; 9Department of Gastroenterology, DongA University College of Medicine, Busan, Korea; 10Department of Internal Medicine, Bundang Jesaeng General Hospital, Seongnam, Korea and Hepatology Center, Bundang Jesaeng General Hospital, Seongnam, Korea; 11Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea; 12Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea, and Yonsei Liver Center, Severance Hospital, Seoul, Korea; 13Department of Internal Medicine, Yeungnam University College of Medicine, Daegu, Korea; 14Department of Internal Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea; 15Department of Internal Medicine, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, Korea; 16Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea; 17Department of Medicine, GangNeung Asan Hospital, Ulsan University College of Medicine, Gangwon-do, Korea; 18Department of Internal Medicine, Chungbuk National University Hospital, Cheongju, Korea; 19Department of Internal Medicine, Hanyang University College of Medicine, Seoul, Korea, and Hanyang University Guri Hospital, Guri, Korea; 20Department of Gastroenterology, Liver Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea&ast;These authors contributed equally to this workCorrespondence: Young-Suk Lim, Department of Gastroenterology, Liver Center, Asan Medical Center, University of Ulsan College of Medicine, 43-Gil 88, Olympic-Ro, Songpa-Gu, Seoul, Korea, Tel +82-2-3010-3190, Fax +82-2-485-5782, Email limys@amc.seoul.krPurpose: Tenofovir disoproxil (TD), modified from tenofovir disoproxil fumarate (TDF), was developed as a salt-free formulation, removing fumarate to improve the ease of oral intake by reducing the tablet’s size. We evaluated the maintenance of antiviral effects and overall safety profile of TD 245 mg after switching from TDF 300 mg in patients with chronic hepatitis B (CHB).Patients and Methods: CHB patients with HBV-DNA < 69 IU/mL after ≥ 24 weeks of TDF therapy were enrolled. The primary efficacy endpoint was the HBV-DNA suppression rate (HBV-DNA < 69 IU/mL) at week 48; We evaluated the non-inferiority (10% margin) of TD to TDF in terms of efficacy. Safety was assessed based on adverse events (AEs), laboratory tests, bone mineral density, and renal function abnormalities.Results: Overall, 189 subjects were randomized in a 2:1 ratio, and 117 and 66 subjects in the TD and TDF groups, respectively, completed the study. In the per-protocol set, the HBV-DNA suppression rate at week 48 was 99.1% and 100% in the TD and TDF groups, respectively. The lower limit of the 97.5% one-sided confidence interval for the intergroup difference in HBV-DNA suppression rate was − 2.8%, which was greater than the prespecified margin of non-inferiority. The changes in creatinine clearance from baseline to week 48 was significantly less in the TD group and in the TDF group; − 0.8 ± 9.8 versus − 2.4 ± 12.8 mL/min, respectively (P=0.017).Conclusion: TD was non-inferior to TDF for maintaining viral suppression in CHB patients, showing the less decline of renal function.Keywords: viral DNA, bone density, antiviral agents, viral suppression