Comparison of extraction-based and elution-based polymerase chain reaction testing, and automated and rapid antigen testing for the diagnosis of severe acute respiratory syndrome coronavirus 2.

We aimed to compare the differences in testing performance of extraction-based polymerase chain reaction (PCR) assays, elution-based direct PCR assay, and rapid antigen detection tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used nasopharyngeal swab samples of patients with coronavirus disease 2019 (COVID-19). We used the MagNA Pure 24 System (Roche Diagnostics K.K.) or magLEAD 12gC (Precision System Science Co., Ltd.) for RNA extraction, mixed the concentrates with either the LightMix Modular SARS-CoV PCR mixture (Roche Diagnostics K.K.) or Takara SARS-CoV-2 direct PCR detection kit (Takara Bio Inc.), and amplified it using COBAS® z480 (Roche Diagnostics K.K.). For elution-based PCR, we directly applied clinical samples to the Takara SARS-CoV-2 direct PCR detection kit before the same amplification step. Additionally, we performed Espline SARS-CoV-2 (Fuji Rebio Co., Ltd.) for rapid diagnostic test (RDT), and used Lumipulse SARS-CoV-2 antigen (Fuji Rebio Co., Ltd.) and Elecsys SARS-CoV-2 antigen (Roche Diagnostics K.K.) for automated antigen tests (ATs). Extraction-based and elution-based PCR tests detected the virus up to 214-216 and 210 times dilution, respectively. ATs remained positive up to 24-26 times dilution, while RDT became negative after 22 dilutions. For 153 positive samples, positivity rates of the extraction-based PCR assay were 85.6% to 98.0%, while that of the elution-based PCR assay was 73.2%. Based on the RNA concentration process, extraction-based PCR assays were superior to elution-based direct PCR assays for detecting SARS-CoV-2.

10-Year survey on serum antibody positivity rates and titers for measles and rubella in healthcare workers; an observational study at a Japanese university hospital.

BACKGROUND: We evaluated the effect of the two-dose vaccination strategy, which has been a widely adopted as childhood routine schedule worldwide to acquire herd immunity, on healthcare workers (HCWs) in Japan. METHODS: Between 2010 and 2019, antibody titers for measles and rubella were measured annually among newly employed HCWs at Osaka University Hospital, Japan, using Enzygnost® assays (Siemens Healthcare Diagnostics Co. Ltd., Marburg, Germany). The data were categorized by age to compare the antibody positivity rates and antibody titers among no-vaccine, single-dose, and two-dose groups. RESULTS: Over the 10-year period, the annual antibody positivity rates for measles and rubella were 84.0%-95.3% and 90.0%-94.5%, respectively, without any particular trend. The antibody titers for measles (median [interquartile range]: 8.4 [3.9, 20] vs. 6.1 [3.5, 12]) and rubella (11 [5.5, 20] vs. 6 [3.7, 11]) were statistically lower (p < 0.001) in the two-dose generation than in the single-dose generation. DISCUSSION: A shift from single-dose to two-dose vaccination did not yield an increase in antibody positivity rates for both measles and rubella among HCWs. Notably, antibody titers were significantly lower in the two-dose generation. CONCLUSION: Despite several limitations, our data suggests a paradoxical vulnerability in young HCWs who received the two-dose vaccination in a view of sero-positivity rates.

Risk for the occupational infection by cytomegalovirus among health-care workers.

BACKGROUND: Cytomegalovirus (CMV) are ubiquitously distributed worldwide, causing a wide range of clinical manifestations from congenital infection to a life-threatening disease in immunocompromised individuals. CMV can be transmitted via human-to-human contact through body fluids; however, the risk of CMV infection among healthcare workers (HCWs) has not been fully evaluated. AIM: This study aimed to assess the risk of CMV infection among HCWs through daily medical practices. METHODS: Serum samples from HCWs at Osaka University Hospital (Japan) were analysed. Initially, we compared CMV IgG seropositivity among HCWs (medical doctors, nurses, and others) in 2017, which was examined after 1 year to evaluate seroconversion rates among those with seronegative results. Then, we examined CMV seroconversion rates in HCWs who were exposed to blood and body fluids. FINDINGS: We analysed 1153 samples of HCWs (386 medical doctors, 468 nurses, and 299 others), of which CMV seropositivity rates were not significantly different (68.9%, 70.3%, and 70.9%, respectively). Of these, 63.9% (221/346) of CMV seronegative HCWs were followed after 1 year, with CMV seroconversion rates of 3.2% (7/221). Among 72 HCWs who tested negative for CMV IgG when exposed to blood and body fluids, the CMV seroconversion rate was 2.8% (2/72). The CMV seroconversion rates between the two situations were not significantly different. CONCLUSION: Our study indicated that CMV infection through daily patient care seems quite rare. Further well-designed studies with a large sample size are warranted to verify our finding.

Vaccination strategy for epidemic viral diseases in healthcare workers: Cut-off for optimal immunization.

Healthcare workers (HCWs) are at an increased risk of being exposed to epidemic viral diseases (EVDs), such as measles, rubella, mumps, and varicella-zoster. Currently, in case of the absence of written records on previous immunizations, the Japanese Society for Infection Prevention and Control guidelines require HCWs to have antibody titers higher than laboratory thresholds, possibly leading to over-immunization. We report our vaccination strategy and the consequent incidences of EVDs at the Osaka University Hospital between 2000 and 2016. In 2001, we initiated an annual serology check of antibody titers against EVDs and immunization for newly employed HCWs. As an additional vaccination program, all HCWs with low antibody titers were vaccinated in 2005 and 2010. Antibody titers were determined by an enzyme immunoassay (EIA), with a positive range of >2.0 cut-off index. After implementing the vaccination strategy to keep the laboratory threshold, there were only sporadic cases of EVDs among HCWs. More than 99% of individuals who had positive titers in 2005 remained the positive antibody titers in 2010, indicating that a minimum interval of 5 years is enough to measure immunity. Unprotected workers can, even silently, transmit the contagious viruses to patients and coworkers, possibly resulting in a nosocomial outbreak. However, over-vaccination may yield adverse effects and financial burdens. Our observational data indicate that the laboratory cut-off index of >2.0 by EIA may provide a sufficient herd immunity to prevent EVDs among HCWs.

Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb-/-Runx2+/- Murine Incisors.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.

BACKGROUND: Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". METHODS: Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. RESULTS: The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. CONCLUSIONS: According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients.

Durability of immunity by hepatitis B vaccine in Japanese health care workers depends on primary response titers and durations.

BACKGROUND: Health care workers (HCWs) are frequently exposed to hepatitis B virus (HBV) infection. The efficacy and safety of immunization with the hepatitis B (HB) vaccine are well recognized, but the durability of immunity and need for booster doses in those with secondary vaccine response failure remains controversial. METHODS: This was a retrospective cohort study performed at Osaka University Hospital, Japan. We examined antibodies against HB surface antigen (anti-HBs) titers annually after immunization for previously non-immunized HCWs. Primary responders were categorized by their sero-positive durations as short responders (those whose anti-HBs titers declined to negative range within 3 years), and long responders (those who retained positive anti-HBs levels for 3 years and more). We re-immunized short responders with either single or 3-dose boosters, the long responders with a single booster when their titers dropped below protective levels, and examined their sero-protection rates over time thereafter. RESULTS: From 2001 to 2012, data of 264 HCWs with a median age of 25.3 were collected. The rate of anti-HBs positivity after primary vaccination were 93.0% after three doses (n = 229), 54.5% after two doses (n = 11), and 4.2% after a single dose (n = 24). Of 213 primary responders, the anti-HBs levels of 95 participants (44.6%) fell below the protective levels, including 46 short responders and 49 long responders. HCWs with higher initial anti-HBs titers after primary vaccination had significantly longer durations of sero-positivity. For short responders, 3-dose booster vaccination induced a longer duration of anti-HBs positivity compared to a single-dose booster, whereas for long responders, a single-dose booster alone could induce prolonged anti-HBs positivity. CONCLUSION: Our preliminary data suggested that it may be useful to differentiate HB vaccine responders based on their primary response durations to maintain protective levels of anti-HBs efficiently. A randomized, prospective, large-scale study is warranted to support our findings.

Novel and Simple Approach to Estimating the Actual Incidence of Blood and Body Fluid Exposure.

BACKGROUND: There is no current way to determine the actual blood and body fluid exposure (BBFE) incidence in hospitals. We propose a simple, reliable, and widely available method for the accurate estimation of BBFE. METHODS: Data for BBFE for healthcare workers between 2006 and 2015 at Osaka University Hospital were retrospectively extracted from the electronic records. Annual positivity of hepatitis C virus (HCV) antibody in the source individuals and overall patient population were calculated over time. We created an estimation formula focusing on the difference in HCV positivity between the source individuals and overall patient population for the actual number of BBFEs. A linear regression model was used to evaluate the temporal change in the reported and estimated BBFEs. RESULTS: During the study period, 937 BBFEs were reported. HCV positivity between the post-BBFE cohort and overall patient population greatly differed; the incidence ratio ranged from 2.1 to 5.7. The linear regression model revealed that the reported BBFEs did not significantly change during the study period (the slope, 1.315 [95% confidence interval (C.I.): -0.849 to 3.480, p = 0.199]). The annual incidence ratio of the estimated and reported BBFEs significantly reduced over time (the slope, -0.287 [95% C.I.: -0.488 to -0.086, p = 0.011]), indicating that, although the reported number of BBFEs seemed unchanged, the estimated incidence decreased. CONCLUSIONS: We propose a novel and simple approach to estimating the actual incidence of BBFEs in hospitals using the difference in HCV positivity between the post-BBFE cohort and overall patient population.

Antagonistic Functions of USAG-1 and RUNX2 during Tooth Development.

Supernumerary teeth and tooth agenesis are common morphological anomalies in humans. We previously obtained evidence that supernumerary maxillary incisors form as a result of the successive development of the rudimentary maxillary incisor tooth germ in Usag-1 null mice. The development of tooth germs is arrested in Runx2 null mice, and such mice also exhibit lingual epithelial buds associated with the upper molars and incisors. The aim of this study is to investigate the potential crosstalk between Usag-1 and Runx2 during tooth development. In the present study, three interesting phenomena were observed in double null Usag-1-/-/Runx2-/- mice: the prevalence of supernumerary teeth was lower than in Usag-1 null mice; tooth development progressed further compared than in Runx2 null mice; and the frequency of molar lingual buds was lower than in Runx2 null mice. Therefore, we suggest that RUNX2 and USAG-1 act in an antagonistic manner. The lingual bud was completely filled with odontogenic epithelial Sox2-positive cells in the Usag-1+/+/Runx2-/- mice, whereas almost no odontogenic epithelial Sox2-positive cells contributed to supernumerary tooth formation in the rudimentary maxillary incisors of the Usag-1-/-/Runx2+/+ mice. Our findings suggest that RUNX2 directly or indirectly prevents the differentiation and/or proliferation of odontogenic epithelial Sox2-positive cells. We hypothesize that RUNX2 inhibits the bone morphogenetic protein (BMP) and/or Wnt signaling pathways regulated by USAG-1, whereas RUNX2 expression is induced by BMP signaling independently of USAG-1.

Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis.

BACKGROUND: Wnt5a and Mrfzb1 genes are involved in the regulation of tooth size, and their expression levels are similar to that of Bmp7 during morphogenesis, including during the cap and early bell stages of tooth formation. We previously reported that Usag-1-deficient mice form supernumerary maxillary incisors. Thus, we hypothesized that BMP7 and USAG-1 signaling molecules may play important roles in tooth morphogenesis. In this study, we established double genetically modified mice to examine the in vivo inter-relationships between Bmp7 and Usag-1. RESULTS: We measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography (micro-CT) in adult Bmp7- and Usag-1-LacZ knock-in mice and their F2 generation upon interbreeding. The mandibular incisors of adult Bmp7+/- mice were significantly larger than those of wild-type (WT) mice. The mandibular incisors of adult Usag-1-/- mice were the largest of all genotypes examined. In the F2 generation, the effects of these genes were additive; Bmp7+/- was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of Usag-1-/-Bmp7+/- mice was significantly larger than that of Usag-1-/-Bmp7 +/+ mice. At embryonic day 15 (E15), BrdU assays demonstrated that the labeling index of Bmp7+/- embryos was significantly higher than that of WT embryos in the cervical loop. Additionally, the labeling index of Usag-1-/- embryos was significantly the highest of all genotypes examined in dental papilla. CONCLUSIONS: Bmp7 heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. Our findings may be useful in applications of regenerative medicine and dentistry.

Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1) regulate supernumerary organ formations.

Bone morphogenetic proteins (BMPs) are highly conserved signaling molecules that are part of the transforming growth factor (TGF)-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1) suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.

MLC1 mutations in Japanese patients with megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive neurological disorder manifesting early onset macrocephaly and delayed-onset neurological deterioration. Characteristic radiological findings revealed by brain magnetic resonance imaging are the most important factors for obtaining a clinical diagnosis. In this study, we analyzed the causative gene, MLC1, in seven unrelated Japanese patients. The most common mutation in our study was p.S93L; this mutation was observed in 11 alleles (78.6%). The second most common mutation, p.A275D, was observed in two alleles (14.3%). A novel single-nucleotide deletion, c.578delG (p.V194Sfs*2), was identified in one allele. As the clinical severities of patients with MLC were variable even among those sharing identical genotypes, this condition may be modified by environmental factors, modifier genes or epigenetic factors.

Aldehyded Dextran and ε -Poly(L-lysine) Hydrogel as Nonviral Gene Carrier.

Background. The expression term of the gene transfected in cells needs to belong enough inorder to make a gene therapy clinically effective. The controlled release of the transfected gene can be utilized. The new biodegradable hydrogel material created by 20 w/w% aldehyded dextran and 10 w/w% ε -poly(L-lysine) (ald-dex/PLL) was developed. We examined whether it could be as a nonviral carrier of the gene transfer. Methods. A plasmid (Lac-Z) was mixed with ald-dex/PLL. An in vitro study was performed to assess the expression of Lac-Z with X-gal stain after gene transfer into the cultured 293 cells and bone marrow cells. As a control group, PLL was used as a cationic polymer. Results. We confirmed that the transfection efficiency of the ald-dex/PLL had a higher transfection efficiency than PLL in 293 cells (plasmid of 2 µ g: ald-dex/PLL 1.1%, PLL 0.23%, plasmid of 16 µ g: ald-dex/PLL 1.23%, PLL 0.48%). In bone marrow cells, we confirmed the expression of Lac-Z by changing the quantity of aldehyded dextran. In the groups using ald-dextran of the quantity of 1/4 and 1/12 of PLL, their transfection efficiency was 0.43% and 0.41%, respectively. Conclusions. This study suggested a potential of using ald-dex/PLL as a non-carrier for gene transfer.

Id2 controls chondrogenesis acting downstream of BMP signaling during maxillary morphogenesis.

Maxillofacial dysmorphogenesis is found in 5% of the population. To begin to understand the mechanisms required for maxillofacial morphogenesis, we employed the inhibitors of the differentiation 2 (Id2) knock-out mouse model, in which Id proteins, members of the regulator of basic helix-loop-helix (bHLH) transcription factors, modulate cell proliferation, apoptosis, and differentiation. We now report that spatially-restricted growth defects are localized at the skull base of Id2 KO mice. Curiously, at birth, neither the mutant Id2 KO nor wild-type (WT) mice differed, based upon cephalometric and histological analyses of cranial base synchondroses. In postnatal week 2, a narrower hypertrophic zone and an inhibited proliferative zone in presphenoid synchondrosis (PSS) and spheno-occipital synchondrosis (SOS) with maxillary hypoplasia were identified in the Id2 mutant mice. Complementary studies revealed that exogenous bone morphogenetic proteins (BMPs) enhanced cartilage growth, matrix deposition, and chondrocyte proliferation in the WT but not in the mutant model. Id2-deficient chondrocytes expressed more Smad7 transcripts. Based on our results, we assert that Id2 plays an essential role, acting downstream of BMP signaling, to regulate cartilage formation at the postnatal stage by enhancing BMP signals through inhibiting Smad7 expression. As a consequence, abnormal endochondral ossification was observed in cranial base synchondroses during the postnatal growth period, resulting in the clinical phenotype of maxillofacial dysmorphogenesis.

Calcium bridge triggers capsid disassembly in the cell entry process of simian virus 40.

The calcium bridge between the pentamers of polyoma viruses maintains capsid metastability. It has been shown that viral infection is profoundly inhibited by the substitution of lysine for glutamate in one calcium-binding residue of the SV40 capsid protein, VP1. However, it is unclear how the calcium bridge affects SV40 infectivity. In this in vitro study, we analyzed the influence of host cell components on SV40 capsid stability. We used an SV40 mutant capsid (E330K) in which lysine had been substituted for glutamate 330 in protein VP1. The mutant capsid retained the ability to interact with the SV40 cellular receptor GM1, and the internalized mutant capsid accumulated in caveolin-1-mediated endocytic vesicles and was then translocated to the endoplasmic reticulum (ER) region. However, when placed in ER-rich microsome, the mutant capsid retained its spherical structure in contrast to the wild type, which disassembled. Structural analysis of the mutant capsid with cryo-electron microscopy and image reconstruction revealed altered pentamer coordination, possibly as a result of electrostatic interaction, although its overall structure resembled that of the wild type. These results indicate that the calcium ion serves as a trigger at the pentamer interface, which switches on capsid disassembly, and that the failure of the E330K mutant capsid to disassemble is attributable to an inadequate triggering system. Our data also indicate that calcium depletion-induced SV40 capsid disassembly may occur in the ER region and that this is essential for successful SV40 infection.

Presentation of functional foreign peptides on the surface of SV40 virus-like particles.

Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus-cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.

Enhanced BMP signaling results in supernumerary tooth formation in USAG-1 deficient mouse.

Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We previously reported that USAG-1 deficient mice have supernumerary teeth. The supernumerary maxillary incisor appears to form as a result of the successive development of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. We confirmed that BMPs were expressed in both the epithelium and mesenchyme of the rudimentary incisor at E14 and E15. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Wnt signaling as demonstrated by the nuclear localization of beta-catenin was also up-regulated. Inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signaling results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1 deficient mouse model.

Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3.

The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells.