Existing research has established two categories of foundational evidence: first, viral replication inhibition and host cell tolerance windows observed in in vitro experiments with wild-type SARS-CoV-2; second, changes in viral load, lymphocyte subsets, and immune-related indicators observed in SIV-infected monkey experiments. Subsequent research will establish a complete evidence chain covering sample standards, in vitro efficacy, mechanisms of action, viral resistance, animal validation, and human studies.
01 Sample Standardization and Antiviral Activity Window
Establish standards for raw material sources, preparation processes, component fingerprints, batch consistency, and key activity indicators. Different batches of samples will be tested separately for cytotoxic concentration, viral replication inhibition concentration, and selectivity index to confirm whether there is a stable and reproducible window between the effective concentration and the host cell safety concentration.
Research will simultaneously set negative controls, positive antiviral drug controls, and parallel repeats of different batches to avoid inferring stable antiviral activity based on single experimental results.
02 Multi-Cell Systems and Multi-Virus Model Validation
Building on existing Vero cell experiments, introduce human-derived airway epithelial cells, air-liquid interface culture models, and lung organoids to compare changes in viral entry, replication, and release across different cell systems. Human lung organoids and airway models can more closely resemble the human respiratory tissue environment and can be used to verify whether cell line experimental results have cross-model consistency.
The viral range will be expanded from SARS-CoV-2 to representative respiratory RNA viruses or other viral models, and research subjects will be selected based on clear mechanisms of action to determine whether existing results are specific to a single virus or represent broad-spectrum inhibition signals worthy of further exploration.
03 Viral Life Cycle Action Link Analysis
Through pre-treatment, synchronous infection, post-infection sampling, and elution experiments, determine whether the sample primarily affects viral adsorption, cellular entry, genome replication, protein synthesis, viral assembly, or release processes.
Further combine viral RNA, infectious viral titer, viral protein expression, intracellular localization, and host antiviral gene changes to establish a "time of action—action link—virological outcome" mechanism chain, clarifying the differences between direct action on the virus and regulation of host cell responses.
04 Viral Resistance Barrier and Mutation Risk Research
Conduct continuous low-dose passage experiments to observe whether viral sensitivity decreases after long-term exposure, and perform whole-genome sequencing of pre- and post-exposure viruses to identify potential adaptive mutations and resistance sites.
Simultaneously test cross-activity against different viral variants and whether synergistic, antagonistic, or cross-resistance effects occur when used in combination with existing antiviral drugs. The FDA antiviral product development guidelines include non-clinical and clinical virological research, resistance data collection, and genotype-phenotype associations as important components of antiviral development.
05 Non-Human Primate Model Research
Building on existing SIV-infected monkey results, use randomized grouping, blinded detection, and preset endpoints to continuously observe plasma viral load, area under the viral load curve, CD4/CD8 ratio, T cell activation and exhaustion, NK cells, inflammatory factors, lymph node pathology, and tissue viral reservoirs.
Primate research should also compare intervention start time, duration, changes after discontinuation, and dose-effect relationships.
06 Pharmacokinetic Basis and Tissue Exposure Research
Conduct pharmacokinetic studies focusing on blood components, metabolic products, tissue distribution, and exposure time, with a particular analysis of active substances in lung tissue, lymphatic tissue, intestines, and other virus-related target organs.
Correlate in vivo exposure concentrations with in vitro viral inhibition concentrations to determine whether effective exposure levels of virological significance are achieved in animals, providing a basis for dose design and subsequent translational research.
07 Prospective Human Studies
After obtaining relatively complete in vitro mechanism, animal efficacy, and safety results, conduct prospective, registered human studies. Studies should predefine populations, intervention timing, control methods, and primary endpoints. Virological endpoints may include viral load reduction, time to negative conversion, and recurrence; clinical endpoints may include symptom duration, oxygenation status, hospitalization or severe disease risk, and safety.