Guidance for Industry

Antiviral Product 

Development — Conducting 

and Submitting Virology 

Studies to the Agency 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER) 

June 2006 

Clinical Antimicrobial 
Guidance for Industry

Antiviral Product 

Development — Conducting 

and Submitting Virology 

Studies to the Agency 

Additional copies are available from: 

Office of Training and Communications 

Division of Drug Information, HFD-240 

Center for Drug Evaluation and Research 

Food and Drug Administration 

5600 Fishers Lane 

Rockville, MD 20857 

(Tel) 301-827-4573 

http://www.fda.gov/cder/guidance/index.htm 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER) 

June 2006 

Clinical Antimicrobial 
TABLE OF CONTENTS 

I. INTRODUCTION............................................................................................................. 1

II. BACKGROUND ............................................................................................................... 2

III. NONCLINICAL VIROLOGY STUDIES ...................................................................... 3

A. Mechanism of Action Studies........................................................................................................3

B. Antiviral Activity ...........................................................................................................................4

1. Antiviral Activity in Vitro.................................................................................................................4

2. Antiviral Activity in Vitro in the Presence of Serum Proteins .........................................................6

3. Inhibitory Quotient ..........................................................................................................................7

4. Antiviral Activity in Vivo..................................................................................................................7

C. Cytotoxicity and Therapeutic Indexes .........................................................................................7

D. In Vitro Combination Activity Analysis ......................................................................................8

E. Resistance .......................................................................................................................................8

1. Selection of Resistant Virus in Vitro ................................................................................................8

2. Genotypic Analysis.........................................................................................................................10

3. Phenotypic Analysis.......................................................................................................................10

4. Cross-Resistance............................................................................................................................11

IV. PROPOSAL FOR MONITORING RESISTANCE DEVELOPMENT.................... 11

V. VIROLOGY STUDY REPORTS.................................................................................. 13

VI. SUMMARY ..................................................................................................................... 14
Contains Nonbinding Recommendations 

Guidance for Industry1 

Antiviral Product Development — Conducting and Submitting 

Virology Studies to the Agency 

This guidance represents the Food and Drug Administration’s (FDA’s) current thinking on this topic. It 

does not create or confer any rights for or on any person and does not operate to bind FDA or the public. 

You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 

and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for 

implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate 

number listed on the title page of this guidance. 

I. INTRODUCTION 

The purpose of this guidance is to assist sponsors in the development of antiviral drugs and 

biological products (i.e., therapeutic proteins and monoclonal antibodies) from the initial preIND through the new drug application (NDA) and postmarketing stages. This guidance should 

serve as a starting point for understanding what nonclinical and clinical virology data are 

important to support the submission of an investigational new drug application (IND), NDA, or 

biologics license application (BLA) for approval of an antiviral product. This guidance focuses 

on nonclinical and clinical virology study reports and makes recommendations for collecting and 

submitting resistance data to the Food and Drug Administration (FDA). Nonclinical and clinical 

virology study reports, based on collected data, are essential for the FDA’s review of antiviral 

drug investigational and marketing applications. Specific topics discussed in this guidance 

include: 

• Defining the mechanism of action 

• Establishing specific antiviral activity of the investigational product 

• Assessing the potential for antagonism of other antiviral products that might be used in 

combination with the investigational product 

• Providing data on the development of viral resistance to the investigational product 

• Providing data that identify cross-resistance to approved antiviral products having the 

same target 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and should be 

viewed only as recommendations, unless specific regulatory or statutory requirements are cited. 

 

1

 This guidance has been prepared by the Division of Antiviral Products in the Center for Drug Evaluation and 

Research (CDER) at the Food and Drug Administration. 

1
Contains Nonbinding Recommendations 

The use of the word should in FDA guidances means that something is suggested or 

recommended, but not required. 

II. BACKGROUND 

The recommendations in this guidance are based on the antiviral product review experience of 

the Division of Antiviral Products and input from pharmaceutical sponsors and the scientific 

community. Because of the experience, history, and lessons learned with HIV-1 studies, this 

guidance employs studies commonly used to evaluate HIV-1 products as a paradigm for studies 

of products to treat other viruses. Although assays and model systems vary with different 

viruses, many of the principles in this guidance can be applied to antiviral products in 

development for the treatment of other viral infections (e.g., hepatitis B virus, hepatitis C virus, 

herpes simplex virus, varicella zoster virus, influenza virus, rhinovirus, cytomegalovirus, and 

human papillomavirus). Since the field of virology is dynamic and continually evolving, this 

guidance will be revised as new information accumulates and as circumstances warrant. 

Sample formats have been developed to assist sponsors in providing resistance data to the 

Agency (see the stand-alone documents accompanying this guidance). The sample formats are 

being provided as stand-alone documents to help sponsors provide the appropriate information to 

the Agency. These sample formats will be updated as needed, and additional formats for other 

viruses may be provided. 

Guidances on the overall organization of INDs and NDAs can be found at 

www.fda.gov/cder/regulatory/applications/default.htm. Sponsors are encouraged to contact the 

division early in the development of an investigational antiviral product to facilitate the review 

and approval process. To assist prospective sponsors, the FDA accepts submissions in an 

abbreviated IND format (i.e., pre-INDs) for review and comment. Pre-INDs are especially 

useful in instances when sponsors are unfamiliar with the process for evaluating investigational 

products in humans. Information about submitting pre-INDs to the division (“Getting Started 

with the Pre-IND Process”) can be found at www.fda.gov/cder/ode4/preind/getting.htm. The 

FDA accepts electronic submissions to expedite the review process. FDA Web sites can be 

consulted for information on electronic application submission. 

Sponsors are advised to consult the division for additional guidance on the development of 

investigational products against orthopoxviruses, influenza virus, severe acute respiratory 

syndrome (SARS) virus, or other emerging infections. For antiviral products that treat smallpox 

and other orthopoxviruses, related information can be found in the draft guidance for industry 

Vaccinia Virus — Developing Drugs to Mitigate Complications from Smallpox Vaccination.

2

 

Sponsors should give careful attention to observing all provisions of the Select Agent rule3

 and 

other applicable governmental and institutional biosafety and biosecurity provisions. 

 

2

 When final, this guidance will represent the FDA’s current thinking on this topic. For the most recent version of a 

guidance, check the CDER guidance Web page at http://www.fda.gov/cder/guidance/index.htm. 

3

 See 42 CFR part 73 and http://www.cdc.gov/od/sap/sitemap.htm. 

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Contains Nonbinding Recommendations 

III. NONCLINICAL VIROLOGY STUDIES 

Nonclinical virology studies aid in the evaluation of the safety and efficacy of an investigational 

product before it is tested in humans. Recommended studies can identify the mechanism of 

action, help establish specific antiviral activity of the product in a model system, and provide 

data on the development of viral resistance to the investigational product. Additionally, coadministration of an investigational product with other products approved for the same indication 

is often necessary to treat viral infections in clinical settings. In these cases, it is desirable to 

have in vitro combination activity studies designed to identify possible negative interactions on 

antiviral activity (i.e., antagonism) of the investigational product with other antiviral products. 

As more antiviral products are developed to treat particular viral diseases, cross-resistance (i.e., 

viral resistance to one product causing resistance to more than one product within that drug 

class) can become a major issue in clinical settings. Therefore, from a scientific perspective, the 

following information is critical in the development of antiviral products: 

• Determining the antiviral activity of an investigational product against relevant viruses 

resistant to other approved products with the same target molecule or complex 

• Determining the antiviral activity of approved products against viruses resistant to an 

investigational product with the same target molecule or complex 

We recommend conducting nonclinical studies (i.e., mechanism of action, antiviral activity in 

vitro, cytotoxicity and therapeutic indexes, and effects of serum protein binding on antiviral 

activity) before the initiation of phase 1 clinical studies. When developing products for viruses 

where in vitro infection systems exist, sponsors should complete in vitro drug combination 

activity studies of the investigational product with other approved products against the same 

virus (i.e., all approved and available investigational products that target the same protein and at 

least one representative product from each of the other existent drug classes) before the initiation 

of clinical trials that will examine the efficacy of the investigational product in combination with 

other antiviral products. Furthermore, we recommend examining the in vitro selection of 

resistant viruses to the investigational product, the phenotypic and genotypic characterization of 

resistant viruses, and cross-resistance before initiation of clinical studies in patients infected with 

a particular virus. Complete study reports on nonclinical and clinical virology studies can be 

submitted to the FDA upon their completion and need not be held until submission of the NDA. 

Specific details of each of the nonclinical studies recommended by the division are discussed in 

the following sections. 

A. Mechanism of Action Studies 

Mechanism of action studies should be conducted before the initiation of phase 1 clinical studies. 

There are many steps in a virus life cycle that can be targeted by potential antiviral product 

candidates. Products can act directly to inhibit a virus by targeting a specific viral-encoded 

function (e.g., an enzyme inhibitor) or act indirectly (e.g., interferon induction of host cell 

response). We recommend that mechanism of action studies: 

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Contains Nonbinding Recommendations 

• Demonstrate the investigational product’s ability to specifically inhibit viral replication 

or a virus-specific function 

• Establish the site of the product’s action (e.g., viral replicase, protease) 

Biochemical, structural, cellular, or genetic data can be presented to support the proposed 

mechanism of action. Data that demonstrate the mechanism of action include, but are not limited 

to, receptor binding, inhibition of enzymatic activity, X-ray crystallographic structure 

determination of bound inhibitor complex, and characterization of resistance mutations in the 

gene encoding the target. 

A well-characterized mechanism of action is useful in predicting toxicities and in designing 

studies to assess the development of resistance. A clear understanding of the mechanism of 

action for an investigational antiviral product can provide insight into the regions of the viral 

genome where resistance mutations could develop. These regions are not limited to the site of 

action (viral-encoded target) of the investigational product but can include the enzyme substrate 

or another viral-encoded protein existing in a complex with the target protein. Characterization 

of the resistance mutations can provide in vivo validation of the mechanism of action studies. 

The specificity of the investigational product should be demonstrated for the viral target over 

cellular or host proteins, especially in those cases in which a viral enzyme has a cellular 

counterpart. For example, if the investigational product targets a viral polymerase, we 

recommend showing the activity of the product against the viral polymerase in comparison with 

its activity against host DNA polymerases such as DNA polymerase α, β, and γ. 

Immunomodulatory products raise additional issues. These products can have the potential for 

unintended effects on viral replication and other adverse effects resulting from actions on the 

immune system. For investigational products that act through a general immune stimulatory 

mechanism, we recommend that sponsors show a reduction in antiviral activity and identify the 

specific immune system components that are involved. Sponsors should consult the division for 

specific advice regarding the development of immunomodulatory products for the treatment of 

viral diseases as well as products with other nonviral host targets. 

B. Antiviral Activity 

1. Antiviral Activity in Vitro

For many human viruses, there are cell culture systems or animal hosts in which the infectious 

agent can undergo a complete virus life cycle. In these cases, we recommend that the sponsor 

document that the investigational product and/or its metabolites show specific, quantifiable 

antiviral activity in vitro before initiating tests in humans (i.e., before initiation of phase 1 

studies). It is important that these data support clinical testing in humans by providing clear 

evidence of antiviral effects at drug concentrations that can be achieved in vivo with acceptable 

risk-benefit. Additionally, in vitro antiviral activity and cytotoxicity assessments (see Section 

III.C., Cytotoxicity and Therapeutic Indexes) using relevant cell types and virus isolates can be 

4
Contains Nonbinding Recommendations 

used to guide the selection of appropriate dose ranges in early clinical trials. Sponsors are 

encouraged to obtain antiviral activity data using primary human target cells, if possible. 

Because of viral genetic variation, the antiviral activity of the investigational product should be 

examined for multiple clinical isolates and viral isolates representative of the virus population in 

clinical trials. Antiviral activity evaluations that are recommended to support the development 

of the investigational product include: 

• Assessing specific antiviral activity of the investigational product against a broad range 

of clinical and laboratory viral isolates including different clades, subtypes, or genotypes 

• Evaluating the antiviral activity of the investigational product against mutant viruses that 

are resistant to products with the same target molecule or complex as the investigational 

product as well as a representative sample of viruses resistant to other approved products 

for the same indication 

We recommend determining specific antiviral activity using a quantitative assay to measure 

virus replication in the presence of increasing concentrations of the product compared to 

replication in the absence of the product. The effective concentration is the concentration of 

product at which virus replication is inhibited by 50 percent (e.g., EC50 for cell-based assays; 

IC50 for biochemical or subcellular assays). Assays that evaluate antiviral activity and 

cytotoxicity include, but are not limited to, virus inactivation assays, plaque reduction assays, 

cytopathic effect inhibition assays, peripheral blood mononuclear cell (PBMC) assays, and 

binding and fusion assays. Other factors that can be assessed in these assays include the effect of 

an increasing multiplicity of infection and the effect of pretreatment of virus or cells before 

infection versus treatment post-infection. We suggest that host cell lines be low in passage 

number for reproducible results. 

It is important that the effective concentration be consistent with data supporting the mechanism 

of action. An investigational product that inhibits virus replication at concentrations lower than 

biochemical data for the proposed mechanism indicates that another target or mechanism of 

inhibition may be affected. Resistance analyses can provide in vivo validation of the proposed 

mechanism of action when biochemical data are not consistent with antiviral activity data. For 

nucleoside or nucleotide analogs, we recommend that sponsors determine the intracellular halflife (t1/2) of the triphosphate form of the active drug moiety in stationary and dividing cells from 

the target tissue. 

For some human viruses (e.g., hepatitis B and hepatitis C viruses), no satisfactory cell culture or 

animal model exists, and in these cases, inhibition of an essential viral function or activity 

against related viruses can be used to indicate potential activity. When no satisfactory cell 

culture or animal model exists for the target human virus, it is particularly important to know 

whether or not the active moiety of an antiviral product enters cells, if it has a proposed 

intracellular site of action, and if the intracellular concentration correlates with biochemical 

studies. Cell-based assays and host cell lines for studying viruses such as hepatitis B virus 

(HBV) and hepatitis C virus (HCV) replication have advanced, but at the present time are 

limited. Currently, assays that examine HBV replication include, but are not limited to: 

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Contains Nonbinding Recommendations 

• Measuring HBV DNA polymerase activity in biochemical assays 

• Cell culture assays with baculovirus-mediated cell transfer or transfection of HBV 

genomes into human hepatoma cell lines followed by quantitative Southern blot analysis 

with HBV DNA probes 

• Cell culture assays with stably-transfected cell lines containing the HBV genome 

• Quantitative PCR of extracellular HBV DNA 

For analyses of HCV replication, replicon systems have been developed that permit studies of 

viral replication and can be used to assess antiviral activity of some anti-HCV products.4,5 In 

addition, recent studies indicate that cell-based systems for the replication of HCV in vitro are in 

development and might be available to assess antiviral activity.6 Currently, assays that examine 

HCV replication using the HCV replicon system include, but are not limited to: 

• Measuring the level of HCV RNA by RT-PCR in HCV replicon cells 

• Measuring HCV RNA polymerase, HCV serine protease, or reporter enzyme (e.g., 

luciferase) activity in biochemical assays 

2. Antiviral Activity in Vitro in the Presence of Serum Proteins 

Serum proteins can bind to and sequester many products and interfere with a product’s antiviral 

activity. We recommend that sponsors ascertain if the investigational product is significantly 

bound by serum proteins. Common methods for determining protein binding include 

equilibrium dialysis, ultrafiltration methods, and fluorescence-based high throughput human 

serum albumin and α-acidic glycoprotein protein binding. If the investigational product is 

highly protein bound, sponsors are encouraged to examine the in vitro antiviral activity of the 

investigational product in the presence of a series of dilutions of human serum up to 40 percent 

(e.g., 5 percent, 10 percent, 20 percent, 40 percent). An EC50 value for 100 percent human 

serum can be extrapolated from these data and the serum-adjusted EC50 values reported. In 

addition, sponsors are encouraged to determine EC50 values in the presence of physiological 

concentrations of α-acidic glycoprotein and human serum albumin. 

 

4

 Lohmann V, F Korner, J-O Koch, U Herian, L Theilmann, and R Bartenschlager, 1999, Replication of 

Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line, Science 285:110-113. 

5

 Blight K, A Kolykhalov, and C Rice, 2000, Efficient Initiation of HCV RNA Replication in Cell Culture, Science 

290:1972-1974. 

6

 Wakita T, T Pietsschmann, T Kato, T Date, M Miyamoto, A Zhao et al., 2005, Production of Infectious Hepatitis 

C Virus in Tissue Culture from a Cloned Viral Genome, Nat Med. 11:791-6. 

6
Contains Nonbinding Recommendations 

3. Inhibitory Quotient 

Information on plasma and intracellular product concentrations is important in assessing the 

dose-response of antiviral therapy and evaluating the potential for resistance development; 

therefore, it is useful to determine an inhibitory quotient (IQ). An IQ is the Cmin/serum-adjusted 

EC50 value. (For more information on determining EC50 values, see Section III.B.1., Antiviral 

Activity in Vitro.) We view IQ values as a useful tool integrating in vivo product concentrations 

and antiviral activity for an individual product. It is a measure that characterizes the relationship 

between product exposure and the susceptibility of a virus to a product. A high IQ indicates that 

an effective product concentration can be achieved in a patient to inhibit the virus and minimize 

the development of resistance. Since one dose may not be adequate for all patient populations, 

IQ values can be used to aid in the selection of doses to further evaluate in phase 3 and phase 4 

clinical studies. 

4. Antiviral Activity in Vivo 

In cases where no in vitro cell culture or replicon system has been shown to be predictive of 

antiviral activity in humans, measurement of viral titers after treatment in animal model systems 

can be used to assess antiviral activity of the investigational product. Analyses from animal 

models include the morbidity and mortality of animals following documented infection, 

histological examination of tissues, quantification of viral titers over time, isolation and 

characterization of resistant isolates in animals that experience viral rebound, quantification of 

viral antigens and antibodies, pharmacokinetics of the investigational product, and a description 

of symptoms (e.g., neurological, weight loss). 

C. Cytotoxicity and Therapeutic Indexes 

It is important to establish that an investigational product has antiviral activity at concentrations 

that can be achieved in vivo without inducing toxic effects to cells. Furthermore, in a cell 

culture model, apparent antiviral activity of an investigational product can be the result of host 

cell death after exposure to the product. Cytotoxicity tests use a series of increasing 

concentrations of the antiviral product to determine what concentration results in the death of 50 

percent of the host cells (see also Section III.B.1., Antiviral Activity in Vitro). This value is 

referred to as the median cellular cytotoxicity concentration and is identified by CC50 or CCIC50. 

The relative effectiveness of the investigational product in inhibiting viral replication compared 

to inducing cell death is defined as the therapeutic or selectivity index (i.e., CC50 value/EC50

value). It is desirable to have a high therapeutic index giving maximum antiviral activity with 

minimal cell toxicity. We recommend determining CC50 values in both stationary and dividing 

cells from multiple relevant human cell types and tissues to ascertain the potential for cell-cycle, 

species, or tissue-specific toxicities. Studies determining cytotoxicity and therapeutic indexes 

should be conducted before the initiation of phase 1 clinical studies. 

Because of the myelosuppressive effects of some antiviral products, we recommend assessing 

the potential effects of certain investigational products (e.g., nucleoside analogs) on the growth 

of human bone marrow progenitor cells in colony formation assays. Additionally, some 

investigational antiviral products are potential inhibitors of cellular DNA polymerases, which are 

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Contains Nonbinding Recommendations 

responsible for normal nuclear and mitochondrial DNA synthesis and repair. Sponsors should 

determine IC50 values for investigational antiviral products against cellular polymerases and 

show specificity for the viral target over cellular polymerases. The inhibition of human pol γ, 

the enzyme responsible for mitochondrial DNA synthesis, has been linked to defects in 

mitochondrial function that can lead to adverse events in humans. Therefore, it is important to 

examine the effects of certain investigational products (e.g., nucleoside analogs) on human pol γ

activity and on mitochondrial toxicity (e.g., lactic acid production, mitochondrial DNA content, 

mitochondrial morphology, glucose utilization). 

D. In Vitro Combination Activity Analysis 

Within an infected individual, viruses can exist as a heterogeneous population of variant viruses, 

some of which may show reduced susceptibility to one or more antiviral products. Therefore, for 

some viruses, administration of multiple antiviral products (e.g., three-drug combination 

antiretroviral therapy against HIV-1) can be more effective than a single product in establishing 

and maintaining inhibition of virus replication. However, the interactions of products are 

complex and can result in antagonistic, additive, or synergistic effects with respect to antiviral 

activity. For this reason, sponsors should evaluate the in vitro antiviral activity of investigational 

products in two-drug combinations with other products approved for the same indication. 

Specifically, combinations that should be tested include the investigational product with all 

approved products that target the same protein and at least two appropriate products from each 

class of products approved for the same indication. We recommend completing the in vitro drug 

combination activity studies of the investigational product with approved products before 

initiation of clinical trials that will evaluate the efficacy of the investigational product in 

combination with other antiviral products. Often patients are infected with two or more viral 

diseases (e.g., HIV and HBV or HCV); therefore, we also recommend that the in vitro antiviral 

activity of antiviral products used in co-infected patients for different indications be assessed in 

in vitro combination activity studies. 

E. Resistance 

1. Selection of Resistant Virus in Vitro 

This guidance focuses on resistance to antiviral products caused by mutations in viral genomes 

that result in reduced phenotypic susceptibility to a given antiviral product. Resistance, as it is 

used here, is not an absolute term, but relative. We recommend that the in vitro selection of 

resistant viruses to the investigational product, the phenotypic and genotypic characterization of 

resistant viruses, and cross-resistance analyses be examined before initiation of clinical studies in 

patients infected with the particular virus. We understand that the resistance data generated in 

vitro are not necessarily predictive of clinical resistance. However, in vitro resistance selection 

studies are recommended to assess the potential barrier of a target virus to develop reduced 

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Contains Nonbinding Recommendations 

susceptibility (i.e., resistance) to the investigational product and to aid in designing clinical 

studies.7

 

Selection in cell culture of virus resistant to the investigational product can provide insight into 

whether the genetic threshold for resistance development is high or low. A product with a low 

genetic threshold may select for resistance with only one or two mutations. In contrast, a 

product with a high genetic threshold may require multiple mutations to select for resistance. 

Several factors specific to the investigational product and the target virus affect the development 

of resistance (e.g., product concentration). The rate of appearance of mutant viruses depends on 

the rate of replication of the virus, the number of virus genomes produced, the fidelity of the 

replicative machinery, and host factors. Consideration of these factors can help in designing 

tests to detect resistant virus in vitro. For example, when multiple mutations are required to 

develop resistance to high concentrations of the investigational product, many cell culture 

systems do not produce sufficient virus titers to select resistant virus. In these instances, serial 

passage of the virus in cell culture under conditions of increasing concentrations of the 

investigational product can lead to the isolation of resistant virus. Sponsors are encouraged to 

assess the development of resistance in vitro over the concentration range spanning the 

anticipated in vivo concentration. Selection of variants resistant to the investigational product 

should be repeated more than once (e.g., with different strains of wild-type, with resistant strains, 

under high and low selective pressure) to determine if the same or different patterns of resistance 

mutations develop, and to assess the relationship of product concentration to the genetic barrier 

to resistance. 

If the targeted virus replicates in a cell culture system, two basic methods can be employed to 

isolate viruses that have reduced susceptibility to the investigational product: 

• A high initial virus inoculum is propagated for several passages at a fixed product 

concentration, using multiple cultures to test different concentrations. 

• A low initial virus inoculum is passaged in the presence of increasing product 

concentrations starting near the EC50 value for the parental virus. 

Virus production is monitored by determining the genotype and phenotype of isolates throughout 

the selection process to detect the outgrowth of resistant viruses. 

HCV resistance to investigational products can be examined using HCV replicon systems. 

Methods that select HCV resistance using HCV replicon cells include the following: 

• HCV replicon cells are cultured at low density in the presence of neomycin and a fixed 

concentration of the investigational product using multiple cultures to test different 

 

7

 For an in-depth review of resistance issues relating to HIV-1, see the draft guidance for industry Role of HIV Drug 

Resistance Testing in Antiretroviral Drug Development. When final, the guidance will represent the FDA’s current 

thinking on this topic. For the most recent version of a guidance, check the CDER guidance Web page at 

http://www.fda.gov/cder/guidance/index.htm. 

9
Contains Nonbinding Recommendations 

concentrations. The cells containing resistant replicons will form colonies that are then 

expanded for genotypic and phenotypic characterization. 

• HCV replicon cells are passaged in the presence of a fixed product concentration, but in 

the absence of neomycin, using multiple cultures to test different concentrations. The 

HCV replicon cells from each passage are harvested and stored for phenotypic and 

genotypic characterization. 

2. Genotypic Analysis 

Genotypic analysis of resistant viruses selected in vitro determines the mutations that might 

contribute to reduced susceptibility to the investigational product. Identifying resistance 

mutations by DNA sequence analysis of the relevant portions of the virus genome can be useful 

in predicting clinical outcomes and supporting the proposed mechanism of action of the 

investigational product. Sponsors should determine the entire coding sequence of the gene for 

the target protein and compare the pattern of mutations leading to resistance of the 

investigational product with the pattern of mutations of other products in the same class. For 

larger viruses (e.g., herpesviruses, poxviruses), the relevant portions of the viral gene targeted by 

the investigational product should be sequenced and analyzed for mutations that could contribute 

to product resistance (e.g., marker rescue). We recommend that resistance pathways be 

characterized in several genetic backgrounds (i.e., strains, subtypes, genotypes) and that isolates 

be obtained throughout the selection process to identify the order in which multiple mutations 

appear. 

For genotypic assays, sponsors are encouraged to identify sequencing primers, state how many 

bases can be read from the primer accurately, and define the sensitivity of the genotypic assay 

used for detecting minority viral subpopulations. It is important that sponsors define what 

percentage of the population a mutation has to represent to be detected in their genotypic assay. 

3. Phenotypic Analysis 

Phenotypic analysis determines if mutant viruses have reduced susceptibility to the 

investigational product. When mutations that may be associated with resistance are identified by 

genotypic analysis, the ability of each of these mutations to confer phenotypic resistance should 

be evaluated in a recombinant virus system if possible (e.g., by using site-directed mutagenesis, 

PCR amplification of relevant portions of virus genome to introduce these mutations into a 

standard laboratory genetic background, or other suitable system). Recombinant viruses can 

then be tested in vitro for susceptibility to the product to determine an EC50 value. The fold 

resistant change should be calculated as the EC50 value of the isolate/EC50 value of the reference 

or parental strain. Phenotypic results can be determined with any standard virus assay (e.g., 

protein assay, viral RNA assay, polymerase assay, MTT cytotoxic assay, reporter gene 

expression). The shift in susceptibility (or fold resistant change) for a viral isolate should be 

measured by determining the EC50 values for the isolate and comparing it to the EC50 value of a 

reference (well-characterized wild-type laboratory strain) or parental virus done under the same 

conditions and at the same time. The use of the EC50 value for determining shifts in 

susceptibility is preferred because it can be determined with greater precision than an EC90 or 

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Contains Nonbinding Recommendations 

EC95 value. The utility of a phenotypic assay depends on its sensitivity (i.e., its ability to 

measure shifts in susceptibility (fold resistance change) in comparison to reference, parental 

strains, or baseline clinical isolates). Calculating the fold resistant change (EC50 value of 

isolate/EC50 value of reference strain) allows for comparisons among phenotypic assays. 

4. Cross-Resistance 

Antiviral products targeting the same protein (typically products of the same drug class) may 

develop mutations that lead to reduced susceptibility to one antiviral product and can result in 

decreased or loss of susceptibility to other antiviral products in the same drug class. This 

observation is referred to as cross-resistance. Cross-resistance is not necessarily reciprocal, so it 

is important to evaluate both possibilities. For example, if virus X is resistant to drug A and drug 

B, and virus Y is also resistant to drug A, virus Y may still be sensitive to drug B. We 

recommend that the effectiveness of the investigational product against viruses resistant to other 

approved products in the same drug class and the effectiveness of approved products against 

viruses resistant to the investigational product be evaluated by phenotypic analyses. 

Additionally, we recommend that cross-resistance be analyzed between drug classes in instances 

where more than one drug class targets a single protein or protein complex (e.g., nucleoside 

reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors 

(NNRTIs), which both target the HIV-encoded reverse transcriptase). We suggest that multiple 

recombinant and clinical isolates representative of the breadth of diverse mutations and 

combinations of mutations known to confer reduced susceptibility to products in the same drug 

class be tested for phenotypic susceptibility to the investigational product. If phenotyping is 

performed in cell lines with recombinant viruses, the EC50 value should be validated with 

clinical isolates. 

IV. PROPOSAL FOR MONITORING RESISTANCE DEVELOPMENT 

It is important to disseminate information about an antiviral product’s resistance profile to health 

care professionals and patients so that they can make optimal treatment decisions. Furthermore, 

crucial decisions in protocol design and product development plans often hinge on resistance and 

cross-resistance data. Therefore, it is strongly recommended that comprehensive resistance 

testing be undertaken during all phases of product development consistent with the way the 

product will be used in clinical practice. 

For some viruses, measurements of changes in viral concentrations are accepted endpoints to 

determine clinical effectiveness of antiviral products. In these cases, assays that measure and 

monitor viral load can be used. Well-characterized genotypic and phenotypic assays provide the 

basis for examination of the emergence of resistant virus to investigational products and attempt 

to show a relationship between viral resistance and clinical virologic failure. In addition, 

phenotypic and genotypic results are used to define treatment options and predict the utility of 

treating an individual with an investigational product. Genotypic analysis of viral isolates from 

patients failing to respond to therapy or undergoing viral rebound can help identify mutations 

that contribute to reduced susceptibility to the investigational product. In addition, we 

recommend genotypic and phenotypic analyses of baseline isolates be used to determine 

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Contains Nonbinding Recommendations 

response to treatment outcomes based on baseline mutations and polymorphisms and baseline 

phenotypic drug susceptibilities. In cases where measurements of viral concentrations have not 

been established as principal endpoints, development of assays and monitoring of resistance 

emergence will be important for exploratory analyses of relationships between virologic 

measurements and clinical outcomes and can assist in refining designs of future studies. 

We recommend that sponsors develop and submit a plan for monitoring the development of 

resistant viruses in clinical studies before the initiation of clinical studies in virus-infected 

individuals. A resistance monitoring plan should include, but not be limited to: 

• A description of the assays that will be used to monitor viral loads 

• Viral load assay protocols and performance characteristics (if required) 

• The genotypic and phenotypic assays that will be used 

• Genotypic and phenotypic assay protocols and performance characteristics (if required) 

• The methods for sample collection and storage 

• The methods for sample handling and shipping (frozen or ambient) 

• A description of additional resistance analyses 

• Time points when samples for viral loads, genotypic and phenotypic assays, and other 

resistance analyses will be collected (i.e., baseline, week 24, week 48, following regimen 

failure or discontinuation) 

The resistance monitoring plan should be included with the overall clinical development plan in 

the IND. 

We also recommend developing and submitting with the IND plans for genotypic and 

phenotypic baseline studies and resistance substudies early in product development. We suggest 

that genotypic and phenotypic analyses of baseline and post-treatment isolates be completed in a 

timely manner to characterize the resistance profile of the investigational product and its crossresistance potential with other antiviral products. For viruses such as HBV and HCV, baseline 

and post-treatment genotypic analyses are key to monitoring the development of genotypic 

resistance. Comparing genotypic analyses of samples from patients exhibiting virologic 

breakthrough (or rebound) with baseline samples can identify mutations associated with 

resistance. The extent and type of resistance monitoring and analysis should be discussed and 

agreed to with the division in advance. 

Generally, in studies of treatment-experienced patients, sponsors are strongly encouraged to 

collect phenotypic and genotypic data for baseline isolates from all patients and endpoint isolates 

from all virologic failures and discontinuations (not suppressed). Virologic failure and 

discontinuation samples should be collected when the patient is still on the study product. In 

studies of treatment-naïve patients, phenotypic and genotypic data for baseline and endpoint 

isolates from all virologic failures and discontinuations (not suppressed) should be obtained. 

Therefore, in treatment-naïve studies, a baseline sample should be collected and stored from all 

patients for potential phenotypic and genotypic analysis of virologic failures. Additional 

genotypic and phenotypic assessments and subset analyses may be appropriate depending on the 

clinical study protocol or population, thus re-emphasizing the need to collect and store baseline 

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samples and treatment samples throughout studies. In select cases, sponsors may propose to 

collect samples at baseline and at the time of failure on a subset of treatment-naïve patients when 

in vitro data indicate that acquisition of a single mutation results in a high degree of phenotypic 

resistance. However, any such proposal should be discussed with the division in advance. 

Virologic failures and discontinuation definitions are protocol defined. Sponsors with 

investigational products can solicit advice from the division early in product development on 

definitions of clinical response-failure and plans to monitor resistance in clinical studies. We 

recommend that sponsors consult with the division for detailed descriptions and examples of 

how to submit clinical viral resistance data. In conjunction with this guidance, we are providing 

stand-alone documents to aid sponsors in submitting resistance data for HIV, HBV, HCV, and 

influenza studies. These sample formats may be updated periodically and additional formats for 

other viruses added as needed. 

Sponsors can choose to quantify viral loads and conduct phenotypic and genotypic analyses 

themselves or send samples to companies that have been certified by Clinical Laboratory 

Improvement Amendments (CLIA). Proper handling procedures should be followed for 

laboratory samples. If an assay is not performed according to the manufacturer’s specifications, 

the assay results might not be acceptable. Sponsors are encouraged to use approved assays (if 

possible) that are characterized and validated. If the assay is investigational, we recommend that 

sponsors provide the performance characteristics of the assay (e.g., accuracy, precision, limits of 

detection and quantification, specificity, linearity, range, robustness, stability), as well as sources 

of viruses (e.g., blood, plasma), their storage and stability, and cell culture procedures. For 

definitions on assay validation, refer to the guidance for industry Bioanalytical Method 

Validation, the guidance for industry Antiretroviral Drugs Using Plasma HIV RNA 

Measurements — Clinical Considerations for Accelerated and Traditional Approval, and the 

ICH guideline for industry Q2A Text on Validation of Analytical Procedures. Assays that will 

be used in clinical practice should have more extensive validation than exploratory assays that 

are used for the characterization of antiviral activity and resistance of investigational products. 

Commercially available assays that are routinely used should be identified, but it may not be 

necessary to provide the performance characteristics. The amount and nature of validation 

necessary for an assay and mechanisms of submitting assay performance characteristics (e.g., 

Data Master File) should be discussed with the division. We recommend that sponsors 

consistently use the same assay for any particular analysis or measurement in phase 3 studies and 

that the same assay be used for a particular patient throughout the study. Sponsors should 

provide data supporting the implementation and use of any new assays that become available 

during product development. 

V. VIROLOGY STUDY REPORTS 

Complete virology study reports can be extensive and should include the primary data and 

derived data, the procedures used to obtain the data, and information necessary to evaluate the 

data. Virology study reports should contain information on nonclinical studies, clinical antiviral 

activity of the investigational product, resistance development to the investigational product in 

treated patients, cross-resistance with other products in the same drug class, and baseline 

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genotypic and phenotypic virologic response analyses (if applicable). The formats of virology 

study reports should be similar to that of a scientific publication and typically should include the 

following sections: summary, introduction, materials and methods, results, and discussion. The 

methods section should describe all the protocols employed and include a description of the 

statistical analyses used. Virology study reports on nonclinical and clinical studies can be 

submitted upon their completion and need not be held until submission of the NDA. If the NDA 

is submitted in the common technical document (CTD) format, sponsors should submit virology 

study reports and datasets in Module 5, Section 5.3.5.4, Other Studies, under the specific 

heading, Antiviral Reports. 

VI. SUMMARY 

This guidance identifies virology studies relevant to the development and application review of 

antiviral products for the treatment of viral infections. The goal of this guidance is to stimulate 

the generation of more complete analyses for antiviral products. Such analyses help provide data 

that support the introduction of an investigational product into humans and provide the data 

necessary for determining dose-response relationships, designing clinical trials, and selecting 

appropriate patient populations. Thus, the data collected during these studies can affect the 

therapeutic success of a given product. 

Because nonclinical in vitro virology studies can provide useful information for the design of in 

vivo studies and can help predict the development of resistant viruses in vivo, we recommend 

conducting nonclinical studies before the initiation of phase 1 clinical studies. In vitro selected 

resistant viruses should be analyzed carefully before initiation of studies in patients infected with 

a particular virus. This guidance includes recommendations for how and when to perform 

virology studies. Such information could be included in product labeling to facilitate appropriate 

prescribing of antiviral products and maximize the chance for therapeutic success. To assist 

sponsors in providing data from clinical resistance studies, we have developed stand-alone 

documents that accompany this guidance and provide a format for submitting resistance data to 

the Agency. 

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