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Can (or have) antiviral drugs created drug-resistant viruses?

Can (or have) antiviral drugs created drug-resistant viruses?



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Evolution/emergence of antibiotic-resistance in bacteria is a known effect of extensive use of pharmaceutical antibiotics.

Pharmaceutical antivirals have come into extensive use in recent decades - e.g., Tamiflu for influenza; Harvoni for hepatitis C; Valtrex for herpes. Some are even prescribed as ongoing "suppressants."

Has anything analogous to antibiotic resistance (i.e., antiviral resistance) been observed in viruses treated with pharmaceutical antivirals? If not, are there theoretical reasons this would be unlikely (or impossible) to occur?


Resistance to antiviral therapy is a problem in the treatment of many viral illnesses.

Influenza is particularly significant given the epidemiological characteristics of the disease. This Nature Medicine article gives a good overview of oseltamivir resistant pandemic H1N1, which is a useful example, since our assumption that resistant virus would not spread very effectively was (probably predictably) not valid.

HIV is an excellent case study as well, since antiretroviral resistance is such an important issue in the treatment of that particular infection, and demonstrates the challenges of outwitting a pathogen with such a high error rate. This became apparent quite soon after introduction of (initially successful) monotherapy with AZT. In fact, preventing the emergence of resistant virus within the population of virus in a given patient is typically the primary consideration in choosing a drug regimen.

With regard to the other examples of antiviral treatment you mentioned, HCV resistance is well reviewed here. HSV resistance is reviewed here. Resistance has been observed in both examples, though it is more often considered in the normal course of treatment for HCV than HSV. I'd note that Harvoni, which you mentioned, is a combination therapy (ledipasvir-sofosbuvir). Combination therapy is one common strategy for preventing the emergence of resistance


Antiviral resistance in herpes simplex virus and varicella-zoster virus infections: diagnosis and management

Purpose of review: Aciclovir (ACV) is the first-line drug for the management of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections. Long-term administration of ACV for the treatment of severe infections in immunocompromised patients can lead to the development of drug resistance. Furthermore, the emergence of isolates resistant to ACV is increasingly recognized in immunocompetent individuals with herpetic keratitis. This review describes the mechanisms involved in drug resistance for HSV and VZV, the laboratory diagnosis and management of patients with infections refractory to ACV therapy.

Recent findings: Genotypic testing is more frequently performed for the diagnosis of infections caused by drug-resistant HSV or VZV isolates. Molecular biology-based systems for the generation of recombinant viruses have been developed to link unknown mutations with their drug phenotypes. Fast and sensitive methods based on next-generation sequencing will improve the detection of heterogeneous viral populations of drug-resistant viruses and their temporal changes during antiviral therapy, which could allow better patient management. Novel promising compounds acting on targets that differ from the viral DNA polymerase are under clinical development.

Summary: Antiviral drug resistance monitoring for HSV and VZV is required for a rational use of antiviral therapy in high-risk populations.


Antiviral resistance and impact on viral replication capacity: evolution of viruses under antiviral pressure occurs in three phases

Resistance development is a major obstacle to antiviral therapy, and all active antiviral agents have shown to select for resistance mutations. Aspects of antiviral resistance development are discussed for specific compounds or drug classes in the previous chapters, while this chapter provides an overview regarding the evolution of different viruses (HIV, HBV, HCV, and Influenza) under pressure of antiviral therapy. Virus replication is an error prone process resulting in a large number of variants (quasispecies) in patients. Resistance evolution under suboptimal therapy can be schematically distinguished into three phases. (1) preexisting variants less sensitive to the respective drug are selected from the quasispecies population, (2) outgrowing variants acquire additional mutations increasing their resistance, and (3) compensatory mutations accumulate to overcome the generally reduced replicative capacity of resistant variants. Successful therapy should be aimed at suppression of all existing viral variants, thus preventing selection of minority species and their subsequent evolution. This implies that the amount of mutations required for first escape to the viral regimen (genetic barrier) should be larger than the expected number of mutations present in viruses in the quasispecies. Accordingly, combination therapy can achieve complete inhibition of replication for most HIV, HBV, and Influenza infected patients without resistance development. However, resistant viruses can become selected under circumstances of suboptimal antiviral therapy and these resistant viruses can be transmitted. Proper use of drugs and worldwide monitoring for the presence and spread of drug resistant viruses are therefore of utmost importance.


Conclusion

ACV and related nucleoside analogues have been gold standard molecules for the treatment of HSV infections during the past decades. However, the emergence of ACV drug-resistant HSV is rising rapidly with the increasing numbers of transplant and cancer patients. Therefore, new antiviral drugs with different antiviral actions, including new antiviral targets, new antiviral mechanisms, and new antiviral molecules, are required. Janus-type nucleosides have two different faces (mimicking the natural purine and pyrimidine systems) in one molecule, and these drugs may pair with diverse natural bases via rotation around the glycosyl bond, which further induces viral lethal mutation. Therefore, unique Janus-type nucleoside analogues possess great potential in the exploitation of new lethal mutagenesis drugs as novel strategies for antiviral chemotherapy.


Strategies against drug resistance

Drug-resistant HSV mutants may result in more severe and chronic infections in immunocompromised patients, given the increasing numbers of transplant and cancer patients. Therefore, the emergence of drug-resistant HSV infections is no longer a rare event. Antiviral drugs for the treatment of HSV infections have been developed over the past 40 years. However, most drug-resistant HSV isolates have been discovered in laboratories and clinics, which may contribute to the use of a single target (such as viral DNA polymerase) in all current antiviral drugs. The identification of novel strategies for the development of new antiherpetic molecules with different mechanisms of action that are highly effective and exhibit low toxicity against drug-resistant HSV isolates is challenging. Here, we summarise some of the strategies currently in development:

New target

The DNA helicase/primase (H/P) complex is a target for herpes viral infection. 56, 57, 58, 59, 60, 61, 62, 63, 64 The viral H/P complex is common to all members of the herpes virus family, and it may be a good target for the development of novel anti-HSV agents. The HSV-1 H/P complex includes three components (UL5, UL52, and UL8) that exhibit 5′𠄳′ helicase, primase, and single-stranded DNA-dependent NTPase activities, respectively. The new inhibitors of the H/P complex have diverse chemical structures, such as thiazole, thiazoleurea, and thiazolyphenyl derivatives. 63 BAY 57-1293 exhibits almost 200 times greater potency against HSV than ACV in vitro 65, 66, 67 ( Figure 2 ). ASP2151 has been shown to be a safe and effective treatment for genital HSV in Phase III clinical trials 59, 62, 68 ( Figure 2 ). Some promising compounds have been identified, and several of these compounds have undergone clinical trials. However, several problems still exist. For example, the Phase I clinical trial of ASP2151 was terminated because of adverse events. 69 Therefore, the development of this new type of drugs will require substantial work in the future.

The chemical structures of two potent HPI active compounds against HSV. HPI, helicase-primase inhibitor HSV, herpes simplex virus.

New types of molecules

Natural products are an important source of new molecules for use as anti-HSV agents, such as flavonoids, sugar-containing compounds, and peptides. 6, 70, 71, 72 Researchers have recently found that the notoginsenoside ST-4 inhibits the entry of HSV into cells in vitro, with concentrations for 50% of maximal effect (EC50s) of 16.47 μmol·L 𢄡 and 19.44 μmol·L 𢄡 for HSV-1 and HSV-2, respectively. 73, 74 Cheng and colleagues 75, 76 have found that putranjivain A and pterocarnin A (from Euphorbia jolkini and pterocaryastenoptera, respectively) inhibit the entry and replication of viruses at concentrations of 2𠄸 μmol·L 𢄡 . In the 1990s, Perry et al. 77 first reported that mycalamide A display antiviral activities. Mycalamide A has recently been shown to inhibit HSV-1 at 5 ng per disc. 78 Traditional Chinese medicine theory (in which one compound may target several proteins, or several compounds may target one protein) has allowed identification of a large number of natural products that inhibit HSV effectively these discoveries may hopefully solve the present problem of drug resistance. However, the development of natural antiviral drugs faces several challenges, such as the isolation and identification of the active components from complex products, large-scale production, and selective inhibition.

New antiviral mechanism

The lethal mutagenesis antiviral mechanism has been proposed as a novel chemotherapeutic strategy for drug resistance. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 Viruses survive on the basis of quasispeciestheory, 92, 93 which states that viruses must maintain high levels of potentially beneficial mutations to adapt to new environments quickly via immune responses and antiviral drug therapy. However, the high frequency of mutations in the viral genome also implies a large danger of genetic phenomena. There is an intrinsic limit to the maximum number of mutations in a viral genome before the virus loses its infection activities. The viral genetic information may be lost if the virus quasispecies exceeds the limitation, or it may result in a lethal accumulation of errors (termed lethal mutagenesis). Therefore, lethal mutagenesis may be effective not only in reducing viral infection activity but also in weakening the capacity of the virus for drug resistance. Only one nucleoside analogue, ribavirin, exhibits broad spectrum of antiviral activity against DNA- and RNA-based viruses. Ribavirin is also a classic example that is mutagenic in viral cell cultures. Crotty and colleagues 79, 80, 90, 94, 95 have demonstrated that ribavirin may be a template for uridine or cytidine with equal efficiency via rotation around the C 3 -carbonyl bond to give s-cis and s-trans conformers, which may have pushed the viral genome mutations beyond the error threshold ( Figure 3 ).

The lethal mutagenesis mechanism of ribavirin. The ribavirin cis conformer can pair with uridine by mimicking adenosine, and the trans conformer can pair with cytidine by mimicking guanosine.

However, the efficiency of ribavirin's incorporation into a viral genome is relatively low. The exploration of new mutagenic molecules to efficiently lead to the mutation of a viral genome is an excellent strategy to develop new antiviral drugs on the basis of lethal mutagenesis. Numerous researchers have focused on advancing the application of nucleoside molecules to induce viral lethal mutations 88, 91 ( Table 2 ). For example, 5-aza-5,6-dihydro-2′-deoxycytidine (KP-1212) pairs with different natural purines (guanosine and adenosine) by the diverse tautomerization of the nucleobase (amino and imino). 96, 97, 98 KP-1212 inhibits HIV with an EC50 of 10 nmol·L 𢄡 , which increases the mutation frequency of proviral HIV-1 DNA by 50%�% and does not result in resistance or genotoxicity to the host. 99 The prodrug of KP-1212, KP-1461, has been used as a monotherapy for the treatment of HIV-1 infection with significant resistance in Phase IIa clinical trials, which have provided critical insight for the translation to clinical use and new avenues for drug development. 91, 100

Table 2

Chemical nameMutation
5-aza-5,6-dihydro-2′-deoxycytidineC/T–U transitions
5-hydroxycytidineC/T–U transitions
5-aza-2′-deoxycytidineC/G transversions
2-amino-N6-hydroxyadenosineA/G transitions
8-oxiguanosineG/U transversions

Two different faces/base-pairing systems, a novel Janus-type pyrimido[4,5-d]pyrimidine guanosine𠄼ytosine (J-GC) ribonucleoside and 2′-deoxyribosenucleoside with a tridentate hydrogen bonding pattern, have been designed and synthesised for lethal mutagenesis. 101, 102 Cristol first proposed Janus molecules (from the two-faced Roman god Janus) to describe a new symmetrical carbocyclic system. The J-GC mimics natural nucleosides and has the structure of canonical pyrimidine and pure systems in a single molecule ( Figure 4 ). The Watson𠄼rick base-pairing pattern of J-GC can maintain the tridentate H-bond array. The base moiety of J-GC has one face with a Watson𠄼rick donor𠄽onor�ptor H-bond pattern of guanine and the other face with an acceptor�ptor𠄽onor array of cytosine. The J-GC has two different conformations (syn or anti), which allow pairing with diverse nucleosides in the viral genome via rotation around the glycosyl bond and further induces viral lethal mutations, in a similar manner to ribavirin ( Figure 4 ).

The potential mutagenic molecule. Janus nucleoside analogues (for example, J-GC) can pair with guanosine and cytidine by rotating around the glycosyl bond. J-GC, Janus-type pyrimido[4,5-d]pyrimidine guanosine𠄼ytosine.

Janus-type pyrimido[4,5-d]pyrimidine adenosine–thymidine (J-AT) nucleosides have been synthesised to expand this tridentate J-GC nucleoside system to a bidentate J-AT nucleoside system and obtain a combination of all four chemical letters of the genetic nucleoside alphabet. 103 The base moiety of J-AT has one face with a Watson𠄼rick H-bond acceptor𠄽onor pattern of thymidine and the other face with a donor�ptor pattern of adenine. J-AT may be able to pair with diverse nucleosides in the viral genome by rotation around the glycosyl bond. Different mono-substituted nucleosides have been synthesised by replacing one N–H on the thymine ring or the adenine ring with corresponding sugar residues attaching to N1, N3, or N8 of a Janus-type adenosine–thymidine system through divergent synthetic routes, such as Vorbruggen or transglycosylation reactions. 104, 105, 106, 107, 108 The preliminary antiviral activity testing has demonstrated that the J-GC ribonucleoside is active against the hepatitis B virus, which supports the application of Janus-type nucleosides in the field of drug-resistant HSV and the great potential for antiviral drug development. These researchers have also found that the Janus-type nucleosides form different morphogenesis nanostructures (flower-like superstructures, nanobundles, and nanoparticles) by self-assembling in solutions and have demonstrated that the novel self-assembled nucleoside nanoparticle can efficiently act as a drug delivery system in the treatment of oral cancer. 107, 108 These molecules for the development of this theory for antiviral use are just beginning. However, this subject will likely yield the best advances in strategies against drug resistance.


6. Compensatory mutations

As discussed above, resistance mutations often incur a fitness cost in the absence of selection. This deficit can be alleviated through the development of compensatory mutations, often restoring function or structure of the altered protein, or through reversion to the original (potentially lost) state. Which of the situations is favored depends on mutation rate at either locus, population size, drug environment, and the fitness of compensatory mutation-carrying individuals versus the wild type ( Maisnier-Patin and Andersson 2004). Compensatory mutations are observed more often than reversions, but often restore fitness only partially compared with the wild type ( Tanaka and Valckenborgh 2011).

In a fluctuating environment (e.g. incomplete adherence, see Section 8), the fate of compensatory mutations is uncertain. These mutations are generally either neutral or deleterious in the absence of the target mutation, and are thus likely to be lost if the primary resistance mutation is removed. However, if the compensatory mutations are antagonistically epistatic with the primary ones, then they may be maintained: by conferring a relatively higher fitness than if both the primary and compensatory mutations were absent, they may be conserved in the population ( Khudyakov 2010 zur Wiesch et al. 2011). These compensatory mutations could then act as permissive (secondary) mutations, allowing the frequency of the primary resistance mutation to fluctuate ( Bloom et al. 2010).


Can (or have) antiviral drugs created drug-resistant viruses? - Biology

COLUMBUS , Ohio Researchers at Ohio State will spend the next two years testing their theories about just how an AIDS-like virus in cats is able to resist the powerful medicines that are thrown against it.

It's one of the latest efforts at understanding one of the leading problem areas in medicine today -- antimicrobial drug resistance. When bacteria or viruses become resistant to drugs, they become more difficult, or even impossible, to treat.

The project, funded by the National Institute on Drug Abuse, could reveal how some viral infections become able to withstand antiviral medications and even thrive in the presence of some drugs.

If successful, the research might pave the way to smarter, more effective treatments for a host of pathogens that have learned to resist most therapeutic efforts.

The project grew from important discoveries made five years ago as part of a controversial research program investigating the impact of methamphetamine on feline immunodeficiency virus (FIV) one of only three animal viruses that can be used to mimick HIV (human immunodeficiency virus) infections in humans.

Surprisingly, that project showed that the virus was able reproduce itself 15 times faster when methamphetamine was present.

The work also showed that FIV mutated rapidly to adapt to grow in astrocytes, the dominant cell type within the brain, and that this phenomenon was accelerated by exposure to methamphetamine.

That observation led to an epiphany of sorts, explained Lawrence Mathes, professor of veterinary biosciences and associate dean for research and graduate studies in the College of Veterinary Medicine and principal investigator on the project.

If the virus becomes drug-resistant as it routinely mutates into this new form, would that drug resistance occur earlier if methamphetamine were present" he asked.

After an initial phase five years ago that used cats as the animal model for the study, research shifted to more refined work with cell cultures of astrocytes grown in the laboratory, focusing on the changes taking place in individual cells. Mathes reasoned that the same mutated form of FIV would probably be present in the brains of infected cats.

He and his colleagues turned to tissue stored from another decade-old unrelated project that looked at how the virus suppressed the animals' immune systems.

We went back to those tissues and, in fact, found that the same virus mutations we saw in the cultured cell experiments were present in that brain tissue but only after long-term infection, he said.

The new research grant will use tissue culture methods to look specifically at how the presence of methamphetamine may increase the virus' ability to resist anitiviral drugs, in this case, a powerful AIDS drug called azidothymidine, or AZT.

We know a lot about AZT, how it works and what mutations it causes in the virus, he said. The researchers will treat FIV-infected cell cultures with low concentrations of AZT, forcing it to develop a resistance to the drug, repeating the procedure in the presence of methamphetamine.

We know how long it normally takes the mutation to appear in the virus. We predict that it will appear earlier in cells exposed to both AZT and methamphetamine, he said.

Mathes said that the first year of the project is focused on continued in vitro studies using both FIV and cat cell lines as well as parallel experiments with HIV in human cell lines.

If the results are promising, the researchers will test the drugs' interactions with the virus in a small study using two dozen cats in the second year.


Study identifies 21 existing drugs that could treat COVID-19

A Nature study authored by a global team of scientists and led by Sumit Chanda, Ph.D., professor at Sanford Burnham Prebys Medical Discovery Institute, has identified 21 existing drugs that stop the replication of SARS-CoV-2, the virus that causes COVID-19.

The scientists analyzed one of the world's largest collections of known drugs for their ability to block the replication of SARS-CoV-2, and reported 100 molecules with confirmed antiviral activity in laboratory tests. Of these, 21 drugs were determined to be effective at concentrations that could be safely achieved in patients. Notably, four of these compounds were found to work synergistically with remdesivir, a current standard-of-care treatment for COVID-19.

"Remdesivir has proven successful at shortening the recovery time for patients in the hospital, but the drug doesn't work for everyone who receives it. That's not good enough," says Chanda, director of the Immunity and Pathogenesis Program at Sanford Burnham Prebys and senior author of the study. "As infection rates continue to rise in America and around the world, the urgency remains to find affordable, effective, and readily available drugs that can complement the use of remdesivir, as well as drugs that could be given prophylactically or at the first sign of infection on an outpatient basis."

Extensive testing conducted

In the study, the research team performed extensive testing and validation studies, including evaluating the drugs on human lung biopsies that were infected with the virus, evaluating the drugs for synergies with remdesivir, and establishing dose-response relationships between the drugs and antiviral activity.

Of the 21 drugs that were effective at blocking viral replication, the scientists found:

  • 13 have previously entered clinical trials for other indications and are effective at concentrations, or doses, that could potentially be safely achieved in COVID-19 patients.
  • Two are already FDA approved: astemizole (allergies), clofazamine (leprosy), and remdesivir has received Emergency Use Authorization from the agency (COVID-19).
  • Four worked synergistically with remdesivir, including the chloroquine derivative hanfangchin A (tetrandrine), an antimalarial drug that has reached Phase 3 clinical trials.

"This study significantly expands the possible therapeutic options for COVID-19 patients, especially since many of the molecules already have clinical safety data in humans," says Chanda. "This report provides the scientific community with a larger arsenal of potential weapons that may help bring the ongoing global pandemic to heel."

The researchers are currently testing all 21 compounds in small animal models and "mini lungs," or lung organoids, that mimic human tissue. If these studies are favorable, the team will approach the U.S. Food and Drug Administration (FDA) to discuss a clinical trial(s) evaluating the drugs as treatments for COVID-19.

"Based on our current analysis, clofazimine, hanfangchin A, apilimod and ONO 5334 represent the best near-term options for an effective COVID-19 treatment," says Chanda. "While some of these drugs are currently in clinical trials for COVID-19, we believe it's important to pursue additional drug candidates so we have multiple therapeutic options if SARS-CoV-2 becomes drug resistant."

Screening one of the world's largest drug libraries

The drugs were first identified by high-throughput screening of more than 12,000 drugs from the ReFRAME drug repurposing collection -- the most comprehensive drug repurposing collection of compounds that have been approved by the FDA for other diseases or that have been tested extensively for human safety.

Arnab Chatterjee, Ph.D., vice president of medicinal chemistry at Calibr and co-author on the paper, says ReFRAME was established to tackle areas of urgent unmet medical need, especially neglected tropical diseases. "We realized early in the COVID-19 pandemic that ReFRAME would be an invaluable resource for screening for drugs to repurpose against the novel coronavirus," says Chatterjee.

The drug screen was completed as rapidly as possible due to Chanda's partnership with the scientist who discovered the first SARS virus, Kwok-Yung Yuen, M.D., chair of Infectious Diseases at the University of Hong Kong and Shuofeng Yuan, Ph.D., assistant research professor in the Department of Microbiology at the University of Hong Kong, who had access to the SARS-CoV-2 virus in February 2020.

About the ReFrame library

ReFRAME was created by Calibr, the drug discovery division of Scripps Research, under the leadership of President Peter Shultz, Ph.D., with support from the Bill & Melinda Gates Foundation. It has been distributed broadly to nonprofit collaborators and used to identify repurposing opportunities for a range of disease, including tuberculosis, a parasite called Cryptosporidium and fibrosis.

A global team

The first authors of the study are Laura Riva, Ph.D., a postdoctoral research fellow in the Chanda lab at Sanford Burnham Prebys and Shuofeng Yuan at the University of Hong Kong, who contributed equally to the study. Additional study authors include Xin Yin, Laura Martin-Sancho, Naoko Matsunaga, Lars Pache, Paul De Jesus, Kristina Herbert, Peter Teriete, Yuan Pu, Courtney Nguyen and Andrey Rubanov of Sanford Burnham Prebys Jasper Fuk-Woo Chan, Jianli Cao, Vincent Poon, Ko-Yung Sit and Kwok-Yung Yuen of the University of Hong Kong Sebastian Burgstaller-Muehlbacher, Andrew Su, Mitchell V. Hull, Tu-Trinh Nguyen, Peter G. Schultz and Arnab K. Chatterjee of Scripps Research Max Chang and Christopher Benner of UC San Diego School of Medicine Luis Martinez-Sobrido, Wen-Chun Liu, Lisa Miorin, Kris M. White, Jeffrey R. Johnson, Randy Albrecht, Angela Choi, Raveen Rathnasinghe, Michael Schotsaert, Marion Dejosez, Thomas P. Zwaka and Adolfo Garcia-Sastre of the Icahn School of Medicine at Mount Sinai Ren Sun of UCLA Kuoyuan Cheng of the National Cancer Institute and the University of Maryland Eytan Ruppin of the National Cancer Institute Mackenzie E. Chapman, Emma K. Lendy and Andrew D. Mesecar of Purdue University and Richard J. Glynne of Inception Therapeutics.

Research reported in this press release was supported by the National Institutes of Health (NIH) (U19AI118610, U19AI135972, HHSN272201700060C, GM132024, HHSN272201400008C, HR0011-19-2-0020, U19AI142733), the Department of Defense (DoD) (W81XWH-20-1-0270), the Bill & Melinda Gates Foundation, Dinah Ruch, Susan and James Blair, Richard Yu and Carol Yu, the Shaw Foundation of Hong Kong, Michael Seak-Kan Tong, May Tam Mak Mei Yin, the Health and Medical Research Fund (COVID190121), the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region the National Program on Key Research Project of China (2020YFA0707500, 2020YFA0707504), Research Grants Council (T11/707/15), the Huffington Foundation, the JPB Foundation, the Open Philanthropy Project (2020-215611 [5384]) and anonymous donors.


Can (or have) antiviral drugs created drug-resistant viruses? - Biology

In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused by viruses harboring this mutation.

Influenza B viruses cause annual epidemics and contribute to ≈30% of influenza-associated deaths among children in the United States (1). Two lineages, B/Victoria/2/87 and B/Yamagata/16/88, have been co-circulating globally in recent years (2,3). Neuraminidase (NA) inhibitors (NAIs) are the only drugs available for treating influenza B virus infections, but NA mutations that emerge during treatment or due to natural variance can diminish the usefulness of NAIs.

The Study

For this study, the National Center for Laboratory and Epidemiology in Vientiane, Laos, a member of the World Health Organization Global Influenza Surveillance and Response System, provided influenza A and B viruses to the World Health Organization Collaborating Center at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA the viruses had been collected during October 1, 2015–February 29, 2016. We propagated the viruses and then used the CDC standardized NA inhibition assay to assess their susceptibility to NAIs (4). Compared with the median 50% inhibitory concentration (IC50) values for B-Victoria lineage viruses, IC50 values for 2 of the 24 B-Victoria lineage viruses, B/Laos/0406/2016 and B/Laos/0525/2016, were elevated for zanamivir (129- to 158-fold), oseltamivir (4-fold), peramivir (72- to 74-fold), and laninamivir (41- to 42-fold) (Table 1). These results were interpreted as highly reduced inhibition by zanamivir, normal inhibition by oseltamivir, and reduced inhibition by peramivir and laninamivir (Table 1) (5).

Figure 1. Neuraminidase gene segment (nts 399–497) of influenza B/Laos/0080/2016 virus carrying NA-H134 (A) and B/Laos/0654/2016, NA-N134 (B). RNA extracted from respiratory specimens was used for reverse transcription PCR (RT-PCR) amplification. Two primers.

This interpretation is useful but obscures the higher median oseltamivir IC50 value (9.67 nmol/L vs. 0.42–1.47 nmol/L for other NAIs Table 1) and the lower potency of oseltamivir in inhibiting NA activity of influenza B viruses (4,7). Moreover, reports from clinical studies indicate a lesser susceptibility of influenza B viruses to oseltamivir than to zanamivir (7–9). Although the laboratory criteria defining clinically relevant NAI resistance are not established, the inhibitory profiles of these 2 viruses suggest resistance to > 1 antiviral drugs. NA sequence analysis revealed that both viruses had an amino acid substitution, histidine (H)→asparagine (N), at the highly conserved residue 134 (NA-H134N) (6) the presence of H134N in the respiratory specimens was confirmed by pyrosequencing (Figure 1) (10). NA-H134Y was previously reported in influenza B virus displaying reduced inhibition by peramivir (11). The inhibition profile of influenza B viruses bearing NA-H134N resembles that of influenza A(H1N1) viruses carrying NA-Q136R (residue 134 in influenza B NA corresponds to 136 in N1 numbering) (12). Residue 134 (136) has been implicated in the conformational change of the 150-loop, which may adversely affect the interaction between the NA active site and NAIs, especially those containing the guanidyl group (Technical Appendix Figure).

To expand testing, the Laos National Center for Laboratory and Epidemiology provided 40 additional specimens that were positive for B-Victoria lineage virus by real-time reverse transcription PCR (13), bringing the total number tested to 64. The specimens were collected during October 2015–April 2016 in Champasack (n = 41), Vientiane (n = 12), Luangprabang (n = 7), and Saravanh (n = 5) Provinces from 28 male and 37 female patients (median age 7 [range 0–67] years). Pyrosequencing revealed NA-H134N in 1 specimen the respective isolate, B/Laos/0654/2016, displayed the expected NA inhibition profile (Table 1). In total, we found the NA-H134N substitution in 3 (4.6%) of the 65 tested B-Victoria viruses. Analysis of NA sequences deposited to the GISAID database (http://www.gisaid.org) revealed that among 8,601 sequences of influenza B viruses collected worldwide during October 2014–September 2016, only 3 other sequences contained a substitution at H134 (2 harbored H134Y and 1 H134L) the 3 sequences were for B-Victoria lineage viruses.

Epidemiologic data revealed that the NA-H134N viruses were collected from a young woman, a young man, and a 3-year-old girl residing in 2 distant provinces (Table 2). The 3 infections occurred 6–10 days apart in February 2016, and 1 of the patients received medical care for severe acute respiratory illness. No epidemiologic links were identified among the 3 patients infected with the drug-resistant viruses, and patients had no documented exposure to NAIs.

The 3 drug-resistant viruses were genetically similar to other B-Victoria lineage viruses circulating in Laos during 2015‒2016. Besides having the NA-H134N amino acid substitution, these viruses also shared the M1-H159Q amino acid substitution not identified in other virus sequences (Table 2). Also, these viruses have 3 synonymous nucleotide mutations: PB1-c93t, PB1-g1930a, and HA-g1520a. In addition, B/Laos/0406/2016, B/Laos/0525/2016, and B/Laos/0654/2016 harbored substitutions NA-D390D/E, HA-V225A, and NS1-V220I, respectively (Table 2). An analysis of influenza B NS1 sequences available in the GISAID database (as of September 12, 2016) indicated that NS1-V220I is rare, present in only 7 (0.1%) of 10,405 sequences. Taken together, the geographic distance between the sites where the drug-resistant viruses were collected and the differences in their genomes point toward the possibility of influenza NA-H134N viruses circulating in Laos communities.

Figure 2. Characterization of influenza B viruses detected in Laos, February 2016. A) Thermostability of neuraminidase (NA) determined after viruses were incubated for 15 min at 4°C or at 30°C–57°C. NA enzyme activity.

Results of the NA inhibition assay showed that NA-H134N impairs binding of NAIs to the active site of the enzyme. To determine whether this change also affects other properties (e.g., thermostability) of the enzyme, we incubated 3 H134N viruses at elevated temperatures for 15 min and then assessed their NA activity (Figure 2, panel A). The H134N substitution reduced the thermostability of the enzyme. This was evident from the undetectable activity levels starting at 47.5°C, which was 7.5°C lower than that for the control virus, B/Laos/0880/2016, with H134 (p<0.001) (Figure 2, panel A).

To assess the replicative fitness of NA-H134N viruses, we used primary human differentiated normal human bronchial epithelial (NHBE) cells, a cell culture system that morphologically and functionally recapitulates the human airway. The NA-H134N viruses displayed ≈1–2 log10 lower titers at 24–72 h after inoculation (Figure 2, panel B). Although, the virus yield reduction (area under the curve) was evident for 2 of the NA-H134N viruses (AUC272 [p<0.05]) (Figure 2, panel B), the difference was not statistically significant for B/Laos/0654/2016 (Figure 2, panel B). The growth kinetics data in differentiated NHBE cells indicate an attenuated phenotype for NA-H134N viruses in vitro. Unlike the other 2 drug-resistant viruses, B/Laos/0654/2016 had substitution NS1-V220I, which resides at the recently discovered second RNA binding site of the NS1 protein of influenza B viruses (15). This finding suggests a possible compensatory effect of NS1-V220I on the in vitro replicative capacity of B/Laos/0654/2016.

We assessed the replicative fitness of drug-resistant B/Laos/0654/2016 in three 4- to 6-month-old male ferrets (Mustela putorius furo) (Triple F Farms, Sayre, PA, USA) that were serologically negative by HI assay for currently circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses. At 48 h after inoculation with virus (10 4 50% tissue culture infectious dose/mL), ferrets displayed fever ( > 1.5°C above baseline) that lasted 21.8 ± 5.1 h on average. Virus shedding lasted 6 days nasal wash virus titers, which were determined daily, were 4.2 ± 0.4 6.0 ± 0.2 4.8 ± 0.4, 4.7 ± 0.4, 4.7 ± 0.6, and 2.8 ± 0.4 log10 50% tissue culture infectious doses/mL, respectively. These data suggest that the drug-resistant virus can replicate to high titers in the upper respiratory tract of ferrets and induce persistent fever.

Conclusions

In February 2016, we detected 3 influenza B viruses in Laos bearing a rare NA-H134N substitution. Current antiviral medications may not effectively control infections caused by such viruses. Virus harboring NA-H134N and NS1-V220I replicated efficiently in NHBE cells and in the ferret upper respiratory tract. Studies to ascertain the effect of NA-H134N and NS1-V220I on influenza B virus virulence and transmissibility in a mammalian host are needed.

Dr. Baranovich worked in the Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, during the conduct of this study. Her research interests include the molecular mechanisms of influenza virus resistance to antiviral medications and the effect of resistance mutations on viral fitness and evolution.

Acknowledgments

We thank the laboratories that and clinicians who submit specimens and isolates to the World Health Organization Collaborating Center for Influenza in Atlanta, Georgia, USA. We greatly value the technical assistance provided by Michelle Adamczyk, Lori Lollis, Juan De la Cruz, Anton Chesnokov, and members of Reference and Genomic Teams in the Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention. We thank Hoffmann-La Roche Ltd, Switzerland, for providing oseltamivir carboxylate, the active form of the ethyl ester prodrug oseltamivir phosphate GlaxoSmithKline, Australia, for providing zanamivir BioCryst Pharmaceuticals, USA, for providing peramivir and Biota, Australia, for providing laninamivir.