Plaque Assay Method Research Articles

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Isolation of Escherichia coli bacteriophages from Domestic Wastewater in Tangerang Selatan

In recent years, there has been a continuous increase in the prevalence of antibiotic resistance among bacteria responsible for infectious diseases. Several studies have shown that the occurrence of the COVID-19 pandemic is a major factor contributing to this rapid increase. To overcome this challenge, one promising approach is the use of bacteriophages as a treatment option. Therefore, this study aims to isolate E. coli lytic bacteriophages as an alternative antibiotic for treating secondary bacterial infections of COVID-19. Isolation of bacteriophages was carried out using the plaque assay method with double layer agar technique. The isolate obtained was purified and tested for effectiveness in lysing E. coli ATCC 25922 bacterial cells. The results showed that E. coli had resistance to antibiotics, such as Amoxicillin, Clindamycin, Ciprofloxacin, Azithromycin, and Trimethoprim/sulfamethoxazole. BEB1 isolate obtained from domestic waste had activity in inhibiting the growth of the test bacteria with an effectiveness of 72% at the 25th hour. Based on these findings, it had the potential to be developed as an alternative antibiotic agent to overcome infectious diseases caused by E. coli ATCC 25922.

Loss of TRPM8 Exacerbate Herpes Simplex Keratitis Infection in Mice by Promoting the Infiltration of CD11b+ Ly6G+ Cells and Increasing the Viral Load in the Cornea.

To reveal the role of transient receptor potential cation subfamily M member 8 (TRPM8) channels in herpes simplex keratitis (HSK). HSK models were established using TRPM8 knockout (TRPM8-/-) mice and their wild-type (WT) littermates. The infected corneas were graded and harvested to evaluate the mRNA levels of inflammatory factors through quantitative real-time polymerase chain reaction (RT-PCR), as well as the infiltration of inflammatory cells through immunofluorescence staining and flow cytometry. Viral titers were determined by plaque assay and absolute quantitative method. RNA-sequencing was conducted to elucidate the transcriptome of corneal epithelium in response to TRPM8 knockout after infection. The anti-inflammatory effect of TRPM8 agonist menthol was documented via subconjunctival administration. Compared to their wild-type counterparts, TRPM8-deficient mice exhibited exacerbated infection symptoms and thicker corneas in HSK models. Infection in TRPM8-deficient mice resulted in significant lymphocyte infiltration, primarily consisting of Ly6G+ CD11b+ cells. Additionally, TRPM8-deficient mice displayed increased levels of corneal viral titers after infection, along with decreased expression of interferon-stimulated genes (ISGs). Subconjunctival administration of menthol effectively alleviated infection-induced symptoms and Ly6G+ CD11b+ cell infiltration in herpes simplex virus type 1 (HSV-1)-treated mice. TRPM8 promoted host resistance to HSV-1 infection by suppressing the accumulation of Ly6G+ CD11b+ cells and virus replication. These findings suggest that targeting TRPM8 could be valuable for therapeutic interventions against HSV-1 infections.

Antiviral Effect of Candies Containing Persimmon-Derived Tannin against SARS-CoV-2 Delta Strain.

Inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the mouth has the potential to reduce the spread of coronavirus disease 2019 (COVID-19), due to the virus being readily transmitted by dispersed saliva. Persimmon-derived tannin has strong antioxidant and antimicrobial activity owing to its strong adhesion to proteins, and it also exhibited antiviral effects against non-variant and Alpha-variant SARS-CoV-2 in our previous study. In this study, we first demonstrated the antiviral effects of persimmon-derived tannin against the Delta variant of SARS-CoV-2 in vitro via the plaque assay method. We then examined the effects of candy containing persimmon-derived tannin. Remarkably, the saliva samples provided by healthy volunteers while they were eating tannin-containing candy showed that the virus titers of the SARS-CoV-2 Delta variant were suppressed. In addition, we found that the SARS-CoV-2 viral load in saliva from patients with COVID-19 collected immediately after they had eaten the tannin-containing candy was below the level of detection via PCR for SARS-CoV-2. These data suggest that adding persimmon-derived tannin to candy and holding such candy in the mouth is an effective method for inactivating SARS-CoV-2 in saliva, and the application of this approach shows potential for inhibiting the transmission of COVID-19.

Rapid Bacteriophage Quantification by Digital Biosensing on a SlipChip Microfluidic Device.

The rise of antimicrobial resistance (AMR) is a major global public health concern, and it is urgent to develop new antimicrobial drugs and alternative therapies. There has been growing interest in the use of phage therapy as an alternative to treat AMR, and it has shown promising results in early studies and clinical trials. Phage quantification is a crucial step in the development and application of phage therapy. The traditional double-layer plaque assay requires cumbersome manual operations and typically takes up to 18 h to yield a rough phage estimation. Spectrophotometry, flow cytometry, and PCR-based methods cannot distinguish between infectious and noninfectious phages. Here, we developed a digital biosensing method for rapid bacteriophage quantification on a digital phage SlipChip (dp-SlipChip) microfluidic device containing 2304 microdroplets in 3 nL. By compartmentalizing the phages and bacteria in nanoliter droplets and analyzing the growth profile of bacteria at 3 h, the number of infectious phages can be precisely quantified. The results from the dp-SlipChip were consistent with the traditional double-layer plaque assay method and exhibited higher consistency and repeatability. The dp-SlipChip does not require a complex fluidic handling instrument to generate and manipulate droplets. This SlipChip-based digital biosensing method not only provides a promising tool for rapid phage quantification, which is important for the use of phages in clinical practice to treat antimicrobial-resistant bacteria, but can also be used as an ultrasensitive, high-specificity method to detect bacteria. Furthermore, this approach can be applied to other digital biology studies that require analysis at the single-object level.

MP05-12 IN VITRO EFFECT OF E.COLI PHAGES ON E.COLI BIOFILM MODEL BUILT ON URETERAL STENT

You have accessJournal of UrologyCME1 Apr 2023MP05-12 IN VITRO EFFECT OF E.COLI PHAGES ON E.COLI BIOFILM MODEL BUILT ON URETERAL STENT Leonid Boyarsky, Ortal Yerushalmi, Ronen Hazan, Amitay Lorber, Vladimir Yutkin, Ofer Gofrit, and Mordechai Duvdevani Leonid BoyarskyLeonid Boyarsky More articles by this author , Ortal YerushalmiOrtal Yerushalmi More articles by this author , Ronen HazanRonen Hazan More articles by this author , Amitay LorberAmitay Lorber More articles by this author , Vladimir YutkinVladimir Yutkin More articles by this author , Ofer GofritOfer Gofrit More articles by this author , and Mordechai DuvdevaniMordechai Duvdevani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To assess, in vitro, the ability of E.coli phages to eradicate biofilms built on ureteral stents with an intention to develop an additional treatment option for chronic urinary infection in patients with indwelling ureteral stent. METHODS: Phages were grown on isolated E.coli cultures. Phage host range of infection was determined with plaque assay method. The urine stability of phages was assessed. Biofilm model was built separately on 96 well plates and on ureteral stent. Following biofilm infection, the phage lytic with and without ampicillin was assessed by colony forming units (CFU) test and scanning electron microscopy (SEM). RESULTS: Ampicillin resistant, E.coli strain O5 was found as sensitive to a group of phages designated ECOY 1-8 and was therefore selected as a study strain. ECOY5 was found to be stable in urine and was selected as a study phage. In the study group, the CFU test showed 10- fold and 100- fold decrease in E.coli CFU growth for well plate and stent models respectively. The lytic effect of phage treatment was visualized on SEM images. CONCLUSIONS: Use of the ECYO5 phage alone or in combination with ampicillin appears to be very effective in breaking down the biofilm of E.coli. Further research is needed to develop phage-based products for stent associated infections including other uropathogens. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e48 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Leonid Boyarsky More articles by this author Ortal Yerushalmi More articles by this author Ronen Hazan More articles by this author Amitay Lorber More articles by this author Vladimir Yutkin More articles by this author Ofer Gofrit More articles by this author Mordechai Duvdevani More articles by this author Expand All Advertisement PDF downloadLoading ...

Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Evaluation of the virucidal efficacy of Klaran UVC LEDs against surface-dried norovirus.

Human norovirus (HuNoV) is a highly contagious pathogenic virus that is transmitted through contaminated food, water, high-touch surfaces and aerosols. Globally, there are an estimated 685 million infections annually due to norovirus, including 200 million affecting children under the age of 5. HuNoV causes approximately 50, 000 child deaths per year and costs an estimated USD $60 billion annually in healthcare. This study sought to determine the inactivation profile of ultraviolet subtype C (UVC) against norovirus using a UVC light-emitting diode (LED) array, KL265-50V-SM-WD. The array emitted radiation at 269 nm peak wavelength and a measured fluence of 1.25 mW cm−2 at a 7 cm source–surface distance. Since the HuNoV is not cultivable, the study utilized feline calicivirus (FCV) ATCC VR-782, a recommended surrogate as challenge organism. The test followed modified ASTM E2197. Assessment of virus inactivation was performed using a plaque assay method. With irradiance at a UVC dose of 22.5 mJ cm−2, the study obtained 99.9 % virus reduction (3 log reduction). The results demonstrate that the UVC LED array can provide effective inactivation of HuNoV.

Cellular miR-101-1 Reduces Efficiently the Replication of HSV-1 in HeLa Cells

Introduction: Herpes simplex viruses (HSVs) are widely distributed in the human population. HSV type 1 (HSV-1) is responsible for a spectrum of diseases, ranging from gingivostomatitis to keratoconjunctivitis, and encephalitis. The HSVs establish latent infections in nerve cells, and recurrences are common. Their frequent reactivation in elderly and immunosuppressed patients causes serious health complications. Objectives: Due to the growing resistance to its main drug, acyclovir, alternative treatments with different mechanisms of action are required. MicroRNAs regulate host and viral gene expression posttranscriptionally. Previous studies reported that mir-101-2 expression has widely participated in the regulation of HSV-1 replication. In this study, we investigate the effect of hsa-miR-101-1 in the replication of HSV-1. Methods: We found that transfection of miR-101-1 into HeLa cells could reduce effectively HSV-1 replication using plaque assay and real-time PCR methods. Results: We showed that overexpression of miR-10-1 produced less viral progeny and manifested a weaker cytopathic effect, without affecting cell viability. Discussion/Conclusion: This result can give us new insights into the control of HSV-1 infections.

Prevention of Candida biofilm formation over polystyrene by plasma polymerization technique

This work investigates the antifungal effect of plasma polymer films produced by low-pressure RF-generated plasma system using acrylic acid, 2–hydroxyethyl methacrylate, and diethyl phosphite (DEP). Unmodified and plasma-modified polystyrene (PS) microplate wells were tested by 30 biofilm-positive Candida spp. isolated from blood samples and two control strains using a quantitative plaque assay method. Regardless of the precursors and plasma parameters, biofilm formation was inhibited for all plasma-modified microplate wells. The most significant anti-biofilm effect was observed on PS modified by DEP at 90 W plasma power with the inhibition of all Candida species’ biofilm formation.

Evaluation of filter media covered with spun fibres and containing thyme essential oil with antimicrobial properties

ABSTRACT Inhalation of bioaerosols has been linked to many health problems. Filter media impregnated with antimicrobial material can provide effective removal and inactivation of bioaerosols. In this study, fibres were spun on a substrate by centrifugal spinning, obtaining filter media denoted 5THY and THY. Thyme essential oil was used as an antimicrobial agent. For 5THY, the thyme essential oil was added to the polyacrylonitrile (PAN) solution, while for THY, it was sprayed onto the medium after the fibres had been produced. The THY medium presented a higher collection efficiency, compared to the substrate or 5THY, with efficiencies of 99% for microparticles and 58% for nanoparticles. Using the plaque assay method, THY provided the highest reductions of the bacteria Escherichia coli and Staphylococcus aureus, with efficiency of 99.999%. The findings demonstrated that filter media covered with spun fibres and containing thyme essential oil provided excellent antimicrobial action and filtration performance.

Norovirus and human adenovirus occurrence and diversity in recreational water in a karst aquifer in the Yucatan Peninsula, Mexico

To determine the seasonal occurrence and diversity of norovirus (NoV) and human adenovirus (HAdV) in groundwater from sinkholes, and brackish water used for recreational activities in the karst aquifer of the Yucatan Peninsula, Mexico. Hollow fibre ultrafiltration was used to concentrate viruses and standard plaque assay methods were used to enumerate somatic and F+ specific coliphages as viral indicators. Real-time quantitative polymerase chain reaction assays were used to estimate the number of genome copies for NoV strains GI, and GII, and HAdVs. The predominant NoV genotypes and HAdV serotypes were identified by comparative sequence analysis. Somatic and male F+ specific coliphages were detected at concentrations up to 94 and 60 plaque-forming units per 100ml respectively. The NoV genogroup I (GI) was associated with 50% of the sampled sites during the rainy season only, at concentrations ranging from 120 to 1600 genome copies per litre (GCl-1 ). The NoV genogroup II (GII) was detected in 30 and 40% of the sampled sites during the rainy and dry seasons, respectively, at concentrations ranging from 10 to 290GCl-1 . During the rainy and dry seasons, HAdVs were detected in 20% of the sites, at concentrations ranging from 24 to 690 GCl-1 . Identification of viral types revealed the presence of NoV GI.2, GII.Pe, GII.P16 and GII.P17, and HAdV F serotypes 40 and 41. These findings demonstrate that NoVs and HAdVs are prevalent as virus contaminants in the karst aquifer, representing potential health risks particularly during the rainy season, in one of the most important areas used for tourism in Mexico. This is one of the few studies conducted in karst aquifers that provide a foundational baseline of the distribution, concentrations and diversity of NoVs and HadVs in these particular environments.

A Functional Ubiquitin-Proteasome System is Required for Efficient Replication of New World Mayaro and Una Alphaviruses.

Mayaro (MAYV) and Una (UNAV) are emerging arboviruses belonging to the Alphavirus genus of the Togaviridae family. These viruses can produce febrile disease with symptoms such as fever, headache, myalgia, skin rash and incapacitating poly-arthralgia. Serological studies indicate that both viruses are circulating in different countries in Latin America. Viruses need the host cell machinery and resources to replicate effectively. One strategy to find new antivirals consists of identifying key cellular pathways or factors that are essential for virus replication. In this study, we analyzed the role of the ubiquitin-proteasome system (UPS) in MAYV and UNAV replication. Vero-E6 or HeLa cells were treated with the proteasome inhibitors MG132 or Lactacystin, and viral progeny production was quantified using a plaque assay method. In addition, the synthesis of viral proteins was analyzed by Western blot and confocal microscopy. Our results indicate that treatment with proteasome inhibitors decreases MAYV and UNAV protein synthesis, and also causes a significant dose-dependent decrease in MAYV and UNAV replication. Proteasome activity seems to be important at the early stages of MAYV replication. These findings suggest that the ubiquitin-proteasome system is a possible pharmacological target to inhibit these neglected alphaviruses.

Growth Characteristics of Chikungunya Virus Isolate from Indonesia in Various Human Cell Lines in vitro

Chikungunya (CHIK) fever, a febrile illness caused by Chikungunya virus (CHIKV) infection, is one of mosquito-borne viral diseases affecting people living in the tropical and subtropical regions in the world. The pathogenesis of the disease is yet to be completely unraveled, and research on CHIK has been conducted by employing various methods, including using cell lines to investigate the biological characteristics of CHIKV in vitro. To assess the suitability of human cell line model for CHIK study, various human cell lines including A549, Huh7, and HepG2 were infected with CHIKV and assayed for their susceptibility to infection. The MTT and plaque assay methods were performed to measure cell viability and virus growth kinetics, respectively. Fluorescence-activated Cell Sorting (FACS) and immunofluorescence assay were performed to measure the proportion of infected cells in the system and their morphological visualization. Both A549 and Huh7 human cell lines showed stable high cell viability upon infection while CHIKV growth kinetics were significantly lower in these cells compared to Vero-CCL81, a monkey cell line that is routinely used in other arboviruses research. Interestingly, we observed significantly different results in HepG2 human cell line, in which cell viability and CHIKV growth kinetics were significantly higher. FACS and immunofluorescence assay confirm the higher infection rate of CHIKV in HepG2 than A549 human cell line. We concluded herethat human hepatocytes HepG2 cell line was susceptible to Asian Genotype of CHIKV and proposed as an alternative cell for the in vitro CHIKV studies to the commonly used A549 and Vero cells.

Study of characteristics of mycobacteriophage - A novel tool to treat Mycobacterium spp.

Mycobacteriophages are viruses that infect Mycobacterium spp. Till date, 10427 mycobacteriophages have been isolated and 1670 mycobacteriophage genomes have been sequenced https://phagesdb.org/hosts/genera/1/ (cited on 30th December,2018). In the previous study, 10 different mycobacteriophages from 14 soil samples were isolated, by qualitative plaque formation method using Mycobacterium smegmatis as host. Among these, three phages were found to infect four different species of Mycobacterium, i.e., Mycobacterium fortuitum subsp. fortuitum MTCC993, Mycobacterium kansasii MTCC3058, Mycobacterium avium subsp. avium MTCC1723, and Mycobacterium tuberculosis MTCC300, besides the host M. smegmatis. The phage lysates were concentrated by polyethylene glycol (PEG) precipitation. One of the three phages showing host diversity was selected for further study. The various phage growth parameters such as incubation temperature, time of adsorption, host cell density and effect of cations were standardised. The studies were done by qualitative and quantitative plaque assay method. The phage selected for further study showed an optimum adsorption time of 15 min. The optimum temperature for propagation was found to be 37°C. The phage was found to be stable at 42°C. In the presence of calcium, the phage showed a higher rate of infectivity. Understanding the biology of mycobacteriophages and their host diversity is the key to understanding mycobacterial systems. This could be the first step toward exploiting the potential of phages as therapeutic agents.

Influence of water antimicrobials and storage conditions on inactivating MS2 bacteriophage on strawberries

Foodborne illnesses caused by norovirus contaminated fresh produce remain a food safety concern worldwide. In the present study, the impacts of commercial and home processing conditions of strawberries were evaluated for inactivation of the MS2 bacteriophage. MS2 was used as a surrogate of norovirus and was spot inoculated onto strawberries to achieve 6.6 log PFU/g. The inoculated strawberries were washed with tap water, electrolyzed water, or 50 ppm chlorine for 90 s prior to and after storage. After initial washing, the strawberries were separately stored at −20 °C and −80 °C for 30 days. Change in MS2 populations on strawberries was evaluated by plaque assay method on day 1, 15, and 30 for −20 °C and −80 °C groups. The results showed that washing strawberries prior to storage resulted in a significant decrease (approximately 1 log PFU/g) of MS2 population regardless of the treatment (p < 0.05). Frozen storage had minor effects on inactivating MS2, which resulted in approximately a 0.5 log PFU/g reduction at the end of storage. Washing frozen berries in electrolyzed water or 50 ppm chlorine on day 30 resulted in an additional 1 log PFU/g decrease in MS2 compared to water alone. These results suggest that washing strawberries with a chemical antimicrobial prior to and post frozen storage may enhance microbial safety.

Isolation and Enrichment of Bacteriophages by Membrane Filtration Immobilization Technique

The method described here enables rapid bacteriophage isolation and enrichment of host-specific bacteriophages from an environmental sample. This is achieved by using a simple 0.45-µm Millipore membrane where a specific host is immobilized on the membrane and a sample suspected of containing bacteriophages is exposed to the immobilized cells with the help of a membrane filtration unit. This filtration step facilitates host-specific interaction of bacteriophages with the host and maximization of this interaction using a classic membrane filtration method. Under the effect of vacuum from a vacuum pump, a filter assembly provides a chance for every bacteriophage in the sample to interact with the specific host on the membrane filter. Our technique allows retaining specific bacteriophages on the membrane along with its host cells via adsorption; these adsorbed bacteriophages (along with their hosts) on a filter disc are then enriched in regular nutritive broth, tryptone soya broth (TSB), by incubation. With help of a plaque assay method, host-specific phages of various bacterial species can be isolated, segregated, and enriched. © 2018 by John Wiley & Sons, Inc.

Microcosm environment models for studying the stability of adenovirus and murine norovirus in water and sediment.

Microcosms are useful tools for understanding the survival and fate of enteric viruses in aquatic environments. This study set out to determine the stability of infectious enteric viruses in an aquatic environment using a laboratory scale microcosm. Sediment and overlaying water were collected from a lagoon and inoculated with known concentrations of recombinant adenovirus (AdV-GFP) and murine norovirus (MNV-1). Infectious particles of these viruses were measured using fluorescence microscopy (AdV-GFP) or the plaque assay method (MNV-1), over 85 days in two different conditions: under natural sunlight and in fully darkened environments. The time required to reach one log reduction in viral titres (T90) of viable viruses in a natural condition microcosm for AdV-GFP and MNV-1 was shorter than in a dark condition microcosm. There was also a negative correlation between the temperature and infectivity of these viruses in both water and sediment samples. Considering that microcosms aim to mimic natural environment conditions and that AdV-GFP and MNV-1 are excellent surrogates for measuring the infectivity of the respective viruses strains, the results presented here have the potential to be applied in future health hazard studies and also would be useful for future climate scenarios.

Fage Litik Spesifik Escherichia coli pada Limbah Cair Pasar Tradisional di Kota Banda Aceh

Changes in environmental conditions and behavior in patients taking antibiotics causes some pathogenic bacteria become resistant. Increased bacterial resistance to antibiotics encourages applications as biocontrol phage to reduce pathogenic bacteria. This research aims to detect lytic phage on liquid waste traditional market in lysizing Escherichia coli bacteria. Plaque assay method was used in detecting lytic phage in wastewater. The Phage assay results from samples of wastewater traditional markets in Banda Aceh showed that specific lytic phage of E. coli was not detected; it is shown by the absence of clear zone formation on double-layer media (plaque assay negative).

Mechanism of Action of the Anti-Influenza Virus Active Kampo (Traditional Japanese Herbal) Medicine, Hochuekkito

When the Kampo medicine, Hochuekkito (Hochu), was administered to normal mice for 2 weeks, influenza virus titer was reduced. The mechanism of action of Hochu was examined using the plaque assay method. It was suggested that Hochu may either obstruct the first stage of the infection process (adsorption and entry) or may directly target viral particles. Using the plaque assay method, these 2 modes of action could not be differentiated. Virus RNA in the infected cell was verified by quantitative real-time polymerase chain reaction. An equal inhibition effect was obtained when Hochu was preprocessed for normal cells and when they were made to act simultaneously with virus adsorption. The viral load at the cell surface following UV irradiation was higher in the Hochu-administered group as compared with that of the control. Moreover, the affinity of Hochu for the influenza virus was hundred times higher than its affinity for the host cell. The effect of entry obstruction by Hochu was observed via image analysis, where the amount of virus nucleocapsid protein (NP) invading the cell was visualized with FITC-labeled NP antibody. Hochu does not seem to have an effect on nucleic acid synthesis, viral release from infected cells, and on the subsequent second round of infection. In conclusion, Hochu binds to viral particles and forms complexes that can obstruct the entry of influenza virus into cells.