ABSTRACT
Background: The PROTECT study is a longitudinal cohort study initiated in July 2021 with weekly testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 4 states: Arizona, Florida, exas, and Utah. This study aims to examine vaccine-elicited antibody response against postvaccination SARS-CoV-2 infections. Methods: Children aged 5-11 years had serum collected 14-59 days after their second dose of monovalent Pfizer-BioNTech coronavirus disease 2019 messenger RNA vaccine. Vaccine-elicited antibodies were measured using the area under the curve (AUC) and end-point titer using enzyme-linked immunosorbent assay (receptor-binding domain [RBD] and S2) and surrogate neutralization assays against ancestral (WA1) and Omicron (BA.2). Results: 79 vaccinated participants (33 [41.7%] female; median age, 8.8 years [standard deviation, 1.9 years]), 48 (60.8%) were from Tucson, Arizona; 64 (81.0%) were non-Hispanic white; 63 (80.8%) attended school in person; 68 (86.1%) did not have any chronic conditions; and 47 (59.5%) were infected after vaccination. Uninfected children had higher AUCs against WA1 (P = .009) and Omicron (P = .02). The geometric mean and surrogate neutralization titer above the limit of detection was 346.0 for WA1 and 39.7 for Omicron, an 8.7-fold decrease (P < .001). After adjustment of covariates in the WA1-specific model, we observed a 47% reduction in the odds of postvaccination infection for every standard deviation increase in RBD AUC (aOR, 0.53 [95% confidence interval, .29-.97) and a 69% reduction in the odds of infection for every 3-fold increase in RBD end titer (0.31 [.06-1.57]). Conclusions: Children with higher antibody levels experienced a lower incidence of postvaccination SARS-CoV-2 infection.
ABSTRACT
Infections with Chikungunya virus, a mosquito-borne alphavirus, cause an acute febrile syndrome often followed by chronic arthritis that persists for months to years post-infection. Neutralizing antibodies are the primary immune correlate of protection elicited by infection, and the major goal of vaccinations in development. Using convalescent blood samples collected from both endemic and non-endemic human subjects at multiple timepoints following suspected or confirmed chikungunya infection, we identified antibodies with broad neutralizing properties against other alphaviruses within the Semliki Forest complex. Cross-neutralization generally did not extend to the Venezuelan Equine Encephalitis virus (VEEV) complex, although some subjects had low levels of VEEV-neutralizing antibodies. This suggests that broadly neutralizing antibodies elicited following natural infection are largely complex restricted. In addition to serology, we also performed memory B-cell analysis, finding chikungunya-specific memory B-cells in all subjects in this study as remotely as 24 years post-infection. We functionally assessed the ability of memory B-cell derived antibodies to bind to chikungunya virus, and related Mayaro virus, as well as the highly conserved B domain of the E2 glycoprotein thought to contribute to cross-reactivity between related Old-World alphaviruses. To specifically assess the role of the E2 B domain in cross-neutralization, we depleted Mayaro and Chikungunya virus E2 B domain specific antibodies from convalescent sera, finding E2B depletion significantly decreases Mayaro virus specific cross-neutralizing antibody titers with no significant effect on chikungunya virus neutralization, indicating that the E2 B domain is a key target of cross-neutralizing and potentially cross-protective neutralizing antibodies.
Subject(s)
Chikungunya Fever , Chikungunya virus , Animals , Humans , Broadly Neutralizing Antibodies , Antibodies, Viral , Antibodies, Neutralizing , GlycoproteinsABSTRACT
As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be globally important. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants have created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explore the impact of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies produced by a cohort of 96 health care workers. We find robust neutralizing antibody responses among those with hybrid immunity against all variants, including Omicron BA.2, and we further found significantly improved neutralizing titers with longer vaccine-infection intervals up to 400 days. These results indicate that anti-SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting with greater impact seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.
ABSTRACT
As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be important globally. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants has created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explored the effect of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies elicited in a cohort of 96 health care workers. We found robust neutralizing antibody responses among those with hybrid immunity; these hybrid immune responses neutralized all variants, including BA.2. Neutralizing titers were significantly improved for those with longer vaccine-infection intervals of up to 400 days compared with those with shorter intervals. These results indicate that anti-SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting, with greater potency seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/prevention & control , Pandemics , Vaccination , Antibodies, Neutralizing , Adaptive ImmunityABSTRACT
Individuals with chronic lymphocytic leukemia (CLL) have significant immune disfunction, often further disrupted by treatment. While currently available COVID-19 vaccinations are highly effective in immunocompetent individuals, they are often poorly immunogenic in CLL patients. It is important to understand the role a heterologous boost would have in patients who did not respond to the initial two-dose mRNA vaccine series. SARS-CoV-2 specific immune responses, including antibodies and memory B-cells, CD4 and CD8 T-cells were assessed prior to vaccination, as well as postinitial vaccination series and post-third dose in two subjects. One subject seroconverted, had RBD-specific memory B-cells and spike-specific CD4 T-cells while the other did not. Both subjects had a spike-specific CD8 T-cell response after the original mRNA vaccination series that was further boosted after the third dose or remained stable. The results of this study, however small, are especially promising to CLL individuals who did not seroconvert following the initial mRNA vaccination series.
ABSTRACT
Current coronavirus disease 2019 (COVID-19) vaccines effectively reduce overall morbidity and mortality and are vitally important to controlling the pandemic. Individuals who previously recovered from COVID-19 have enhanced immune responses after vaccination (hybrid immunity) compared with their naïve-vaccinated peers; however, the effects of post-vaccination breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals, including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera after breakthrough infection and vaccination after natural infection broadly neutralize SARS-CoV-2 (severe acute respiratory coronavirus 2) variants to a similar degree. Although age negatively correlates with antibody response after vaccination alone, no correlation with age was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination.
Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Vaccination , Adult , Aged , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/epidemiology , COVID-19/immunology , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Humans , Immunogenicity, Vaccine , Middle Aged , Phagocytosis , SARS-CoV-2/growth & development , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , THP-1 Cells , Time Factors , Vero Cells , Viral LoadABSTRACT
The unprecedented severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has called for substantial investigations into the capacity of the human immune system to protect against reinfection and keep pace with the evolution of SARS-CoV-2. We evaluated the magnitude and durability of the SARS-CoV-2-specific antibody responses against parental WA-1 SARS-CoV-2 receptor-binding domain (RBD) and a representative variant of concern (VoC) RBD using antibodies from 2 antibody compartments: long-lived plasma cell-derived plasma antibodies and antibodies encoded by SARS-CoV-2-specific memory B cells (MBCs). Thirty-five participants naturally infected with SARS-CoV-2 were evaluated; although only 25 of 35 participants had VoC RBD-reactive plasma antibodies, 34 of 35 (97%) participants had VoC RBD-reactive MBC-derived antibodies. Our finding that 97% of previously infected individuals have MBCs specific for variant RBDs provides reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to elicit immunity with the capacity to limit disease severity and transmission of VoCs as they arise and circulate.
Subject(s)
COVID-19 , Memory B Cells , SARS-CoV-2/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Humans , Severity of Illness Index , Spike Glycoprotein, CoronavirusABSTRACT
IMPORTANCE: Individuals with Chronic Lymphocytic Leukemia have significant immune disfunction, often further disrupted by treatment. While currently available COVID-19 vaccinations are highly effective in immunocompetent individuals, they are often poorly immunogenic in CLL patients. It is important to understand the role heterologous boost would have in patients who did not respond to the recommended two-dose mRNA vaccine series with a SARS-CoV-2 specific immune response. OBJECTIVE: To characterize the immune response of two CLL patients who failed to seroconvert after initial mRNA vaccine series following a third, heterologous, COVID-19 vaccination with Ad26.COV2.S. DESIGN: Two subjects with CLL were enrolled in an IRB-approved observational longitudinal cohort study of the immune response to COVID-19 vaccination. After enrollment, they received a third vaccination with Ad26.COV2.S. Blood was drawn prior to original vaccination series, four weeks after mRNA vaccination, and again four weeks after third vaccination. SETTING: Eligible subjects were approached by oncologist overseeing CLL treatment and informed about study, at time of enrollment subjects consented to join the cohort study. PARTICIPANTS: Sixteen subjects enrolled in the larger CLL cohort study, of whom two subjects received a third COVID-19 vaccination and were included in this analysis. Subject 1 is CLL treatment naive, while Subject 2 is currently on active treatment. MAIN OUTCOMES AND MEASURES: SARS-CoV-2 specific immune response, including plasma antibodies, memory B-cells, CD4 and CD8 T-cells were assessed prior to vaccination (baseline) as well as post vaccination series and post third dose. RESULTS: Of the two subjects who received Ad26.COV2.S doses, Subject 1 seroconverted, had RBD-specific memory B-cells as well as spike-specific CD4 T-cells while Subject 2 did not. Both subjects had a spike-specific CD8 T-cell response after original mRNA vaccination series that was further boosted after third dose (Subject 1), or remained stable (Subject 2). CONCLUSIONS AND RELEVANCE: The results of this study, however small, is especially promising to CLL individuals who did not seroconvert following initial mRNA vaccination series. Especially those that are treatment naive, not currently in active treatment, or who may consider vaccination before beginning active treatment.
ABSTRACT
SARS-CoV-2 and its variants continue to infect hundreds of thousands every day despite the rollout of effective vaccines. Therefore, it is essential to understand the levels of protection that these vaccines provide in the face of emerging variants. Here, we report two demographically balanced cohorts of BNT162b2 vaccine recipients and COVID-19 patients, from which we evaluate neutralizing antibody titers against SARS-CoV-2 as well as the B.1.1.7 (alpha) and B.1.351 (beta) variants. We show that both B.1.1.7 and B.1.351 are less well neutralized by serum from vaccinated individuals, and that B.1.351, but not B.1.1.7, is less well neutralized by convalescent serum. We also find that the levels of variant-specific anti-spike antibodies are proportional to neutralizing activities. Together, our results demonstrate the escape of the emerging SARS-CoV-2 variants from neutralization by serum antibodies, which may lead to reduced protection from re-infection or increased risk of vaccine breakthrough.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , BNT162 Vaccine , COVID-19/blood , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Child , Child, Preschool , Cohort Studies , Female , Humans , Infant , Male , Middle Aged , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Young AdultABSTRACT
In this investigation we examined the magnitude, breadth, and durability of SARS-CoV-2 specific antibodies in two distinct B-cell compartments: long-lived plasma cell-derived antibodies in the plasma, and peripheral memory B-cells along with their associated antibody profiles elicited after in vitro stimulation. We found that magnitude varied amongst individuals, but was the highest in hospitalized subjects. Variants of concern (VoC) -RBD-reactive antibodies were found in the plasma of 72% of samples in this investigation, and VoC-RBD-reactive memory B-cells were found in all but 1 subject at a single time-point. This finding, that VoC-RBD-reactive MBCs are present in the peripheral blood of all subjects including those that experienced asymptomatic or mild disease, provides a reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to limit disease severity and transmission of variants of concern as they continue to arise and circulate.
ABSTRACT
The COVID-19 pandemic is a global health emergency, and the development of a successful vaccine will ultimately be required to prevent the continued spread and seasonal recurrence of this disease within the human population. However, very little is known about either the quality of the adaptive immune response or the viral Ag targets that will be necessary to prevent the spread of the infection. In this study, we generated recombinant Vaccinia virus expressing the full-length spike protein from SARS-CoV-2 (VacV-S) to evaluate the cellular and humoral immune response mounted against this viral Ag in mice. Both CD8+ and CD4+ T cells specific to the SARS-CoV-2 spike protein underwent robust expansion, contraction, and persisted for at least 40 d following a single immunization with VacV-S. Vaccination also caused the rapid emergence of spike-specific IgG-neutralizing Abs. Interestingly, both the cellular and humoral immune responses strongly targeted the S1 domain of spike following VacV-S immunization. Notably, immunization with VacV-expressing spike conjugated to the MHC class II invariant chain, a strategy previously reported by us and others to enhance the immunogenicity of antigenic peptides, did not promote stronger spike-specific T cell or Ab responses in vivo. Overall, these findings demonstrate that an immunization approach using VacV or attenuated versions of VacV expressing the native, full-length SARS-CoV-2 spike protein could be used for further vaccine development to prevent the spread of COVID-19.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunity, Cellular , Immunity, Humoral , Immunoglobulin G/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Vaccinia virus , Animals , Cell Line , Immunization , Mice , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccinia virus/genetics , Vaccinia virus/immunologyABSTRACT
We compared the serum neutralizing antibody titers before and after two doses of the BNT162b2 COVID-19 vaccine in ten individuals who recovered from SARS-CoV-2 infection prior to vaccination to 20 individuals with no history of infection, against clinical isolates of B.1.1.7, B.1.351, P.1, and the original SARS-CoV-2 virus. Vaccination boosted pre-existing levels of anti-SARS-CoV-2 spike antibodies 10-fold in previously infected individuals, but not to levels significantly higher than those of uninfected vaccinees. However, neutralizing antibody titers increased in previously infected vaccinees relative to uninfected vaccinees against every variant tested: 5.2-fold against B.1.1.7, 6.5-fold against B.1.351, 4.3-fold against P.1, and 3.4-fold against original SARS-CoV-2. Our study indicates that a first-generation COVID-19 vaccine provides broad protection from SARS-CoV-2 variants in individuals with previous infection.
ABSTRACT
There is growing interest in understanding antibody (Ab) function beyond neutralization. The non-structural protein 1 (NS1) of Zika virus (ZIKV) is an attractive candidate for an effective vaccine as Abs against NS1, unlike the envelope or premembrane, do not carry the risk of mediating antibody-dependent enhancement. Our aim was to evaluate whether ZIKV NS1 Abs elicited following natural infection in humans can mediate antibody-dependent cellular cytotoxicity (ADCC). We evaluated the isotype specificity of ZIKV-specific Abs in immune sera and supernatants from stimulated immune PBMC and found that Abs against ZIKV NS1 and virus-like particles were predominantly of the IgG1 isotype. Using a recently developed FluoroSpot assay, we found robust frequencies of NS1-specific Ab-secreting cells in PBMC of individuals who were naturally infected with ZIKV. We developed assays to measure both natural killer cell activation by flow cytometry and target cell lysis of ZIKV NS1-expressing cells using an image cytometry assay in the presence of ZIKV NS1 Abs. Our data indicate efficient opsonization of ZIKV NS1-expressing CEM-NKR cell lines using ZIKV-immune but not ZIKV-naïve sera, a prerequisite of ADCC. Furthermore, sera from immune donors were able to induce both NK cell degranulation and lysis of ZIKV NS1 CEM-NKR cells in vitro. Our data suggest that ADCC is a possible mechanism for ZIKV NS1 Abs to eliminate virally infected target cells.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Viral Nonstructural Proteins/immunology , Zika Virus Infection/immunology , Zika Virus/immunology , Cells, Cultured , Cross Reactions/immunology , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Viral Vaccines/immunology , Zika Virus Infection/virologyABSTRACT
We tested human sera from large, demographically balanced cohorts of BNT162b2 vaccine recipients (n=51) and COVID-19 patients (n=44) for neutralizing antibodies against SARS-CoV-2 variants B.1.1.7 and B.1.351. Although the effect is more pronounced in the vaccine cohort, both B.1.1.7 and B.1.351 show significantly reduced levels of neutralization by vaccinated and convalescent sera. Age is negatively correlated with neutralization in vaccinee, and levels of variant-specific RBD antibodies are proportional to neutralizing activities.
ABSTRACT
BACKGROUND: Serological confirmation of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for understanding the dynamics of the pandemic and determining seroprevalence rates within afflicted communities. Common challenges with SARS-CoV-2 serological assays include poor analytical specificity and sensitivity and lack of a serological standard for quantitative assessment of antibody titers. METHODS: To overcome these obstacles, we developed a quantitative enzyme-linked immunosorbent assay based on an optimized 2-dimensional screening assay that utilizes SARS-CoV-2 receptor binding domain (RBD) of spike protein and SARS-CoV-2 spike S1 subunit. RESULTS: A total of 4 SARS-CoV-2-reactive monoclonal antibodies were evaluated for use as serum standards for calibrating assays performed on different days or by different laboratories. This approach provided quantitative analysis of hospitalized reverse transcription polymerase chain reaction-confirmed COVID-19 cases that in some cases reached >100 µg/mL. The assay demonstrated 72% sensitivity based on time points ranging from 2 to 52 days post-symptom onset, with 100% sensitivity at time points measured ≥13 days post-symptom onset and 100% specificity. CONCLUSIONS: Using these optimized reagents and serological standards, we believe this approach will be useful for sensitive and specific determination of seroconversion rates and quantitatively measuring the durability of antiviral antibody responses following SARS-CoV-2 infection or vaccination.
ABSTRACT
Under-recognition of dengue infection may lead to increased morbidity and mortality, whereas early detection is shown to help improve patient outcomes. Recent incidence and outbreak reports of dengue virus in the United States and other temperate regions where dengue was not typically seen have raised concerns regarding appropriate diagnosis and management by healthcare providers unfamiliar with the disease. This study aimed to describe self-reported clinical symptoms of dengue fever in a non-endemic cohort and to establish a clinically useful predictive algorithm based on presenting features that can assist in the early evaluation of potential dengue infection. Volunteers who experienced febrile illness while traveling in dengue-endemic countries were recruited for this study. History of illness and blood samples were collected at enrollment. Participants were classified as dengue naive or dengue exposed based on neutralizing antibody titers. Statistical analysis was performed to compare characteristics between the two groups. A regression model including joint/muscle/bone pain, rash, dyspnea, and rhinorrhea predicts dengue infection with 78% sensitivity, 63% specificity, 80% positive predictive value, and 61% negative predictive value. A decision tree model including joint/muscle/bone pain, dyspnea, and rash yields 77% sensitivity and 67% specificity. Diagnosis of dengue fever is challenging because of the nonspecific nature of clinical presentation. A sensitive predicting model can be helpful to triage suspected dengue infection in the non-endemic setting, but specificity requires additional testing including laboratory evaluation.
Subject(s)
Dengue/diagnosis , Dengue/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cohort Studies , Cross-Sectional Studies , Dengue/virology , Female , Humans , Logistic Models , Male , Middle Aged , Models, Biological , Multivariate Analysis , Oregon , Travel , Young AdultABSTRACT
Zika virus (ZIKV) emerged as a global public health threat throughout the Americas since 2014. Phylogenetically, the virus is composed of three main lineages, an African, Asian, and American lineage. The recent emergence and spread of ZIKV has raised questions regarding the breadth and potency of human primary ZIKV immune sera against antigenically diverse ZIKV. Although ZIKV is thought to compose a single antigenic serotype, in-depth evaluation of the antigenic relatedness of ZIKV across genetic variants has been limited to a relatively small series of early convalescent human immune sera (4-12 weeks) against a limited number (3) of genetic variants. Using virus neutralization assays, we characterize the potency and breadth of twelve primary ZIKV immune sera from adults infected 5 to 38 months previously against a panel of 11 ZIKV isolates from the African, Asian and American lineages. We assess the variability of neutralization potency of immune sera from these subjects and the variability of susceptibility to neutralization for each virus isolate. Overall, we found all sera neutralized all viruses at FRNT50 ranging from 1:271 to 1:4271, a 15.8-fold range, with only small differences between subject geometric mean titers (GMT) against all viruses and small differences between each ZIKV isolate and sensitivity to neutralization by all sera: when pooled, African strains were 1.3-fold more sensitive to neutralization by subject immune sera compared to pooled American strains. Finally, we subjected our data to analysis using antigenic cartography, finding that ZIKV are highly antigenically similar, with only a ~4-fold range across all antigenic distances between viruses, consistent with a single serotype.
Subject(s)
Immune Sera/immunology , Serogroup , Zika Virus Infection/virology , Zika Virus/classification , Zika Virus/immunology , Adult , Africa , Aged , Americas , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Asia , Female , Genotype , Humans , Male , Middle Aged , Neutralization Tests , Young Adult , Zika Virus/genetics , Zika Virus/isolation & purificationABSTRACT
Biological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.