Oropouche virus (OROV) spread across the Americas in 2024, yet Panama’s Darién migration corridor saw no outbreak until nearly a year after Brazil’s January 2024 peak, raising two hypotheses: cryptic circulation masked by diagnostic gaps, or recent introduction under permissive climatic conditions. Here we resolve this paradox using integrated clinical, genomic, and climate-informed surveillance. Among 1,040 individuals tested, 43% were OROV-positive and showed a clinical signature distinct from co-circulating arboviruses, including headache more frequent than in dengue (RR 2.38, 95% CI 1.74-3.24). The household secondary attack rate was 56%, and waste burning independently predicted infection. Phylogeographic reconstruction identified a single recent introduction in October 2024 with no evidence of adaptive evolution, excluding prolonged cryptic persistence. Climate-informed models indicate broad outbreak susceptibility across Panama, with Bocas del Toro and Los Santos as the next highest-risk provinces. These findings identify a Central American foothold for OROV with potential for further northward spread.

Viruses that cause the clinical syndrome referred to as viral hemorrhagic fever (VHF) are responsible for numerous infectious disease outbreaks. High-priority emerging viruses include orthoebolaviruses, orthomarburgviruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). Many of these viruses cause a similar clinical presentation in infected humans and have an overlapping geographic distribution with a risk of coemergence. As such, an antigen rapid diagnostic test to distinguish between these viruses would be beneficial in low-resource settings. In this study, we developed single-plex and multiplex antigen detection lateral flow immunoassays (LFIs) to rapidly detect and distinguish between emerging viruses that can cause VHF. We evaluated two antibody-labeling methods, colloidal gold nanoparticles and cellulose nanobeads (CNBs), to determine which approach would increase assay performance and multiplexing capabilities. Assay performance was evaluated by determining their sensitivity, specificity, matrix evaluation, and stability testing. All assays were highly specific, with no crossreactivity observed for the single-plex assays. Several of the assays performed better with the CNBs, including the DENV, YFV, RVFV, and orthomarburgvirus LFIs. No matrix effect was observed with most of the assays except that serum did impact the RVFV and DENV assays. In general, the multiplex assays were less sensitive compared with their respective single-plex assay. The most successful assays were the single-plex CNB LFIs assembled into an eight-plex cartridge, which allows for rapid and simultaneous testing of antigen to seven viruses.

ABSTRACT Antigen-based rapid diagnostic tests (Ag-RDTs) provide timely results, are simple to use, and are less expensive than molecular assays. Recent studies suggest that antigen-based testing aligns with virus culture-based results (a proxy of contagiousness at the peak viral phase of illness); however, the performance of Ag-RDTs for newer SARS-CoV-2 variants is unclear. In this study, we (i) assessed the performance of Ag-RDTs and diagnostic antibodies to detect a range of SARS-CoV-2 variants and (ii) determined whether Ag-RDT results correlated with culture positivity. We noted only minor differences in the limit of detection by variant for all assays, and we demonstrated consistent antibody affinity to the N protein among the different variants. We observed moderate to high sensitivity (46.8%–83.9%) for Ag-RDTs when compared to PCR positivity (100%), and all variants were assessed on each assay. Ag-RDT sensitivity and PCR Ct showed an inverse correlation with the detection of viable virus. Collectively, our results demonstrate that commercially available Ag-RDTs offer variable sensitivity compared to PCR, show similar diagnostic validity across variants, and may predict the risk of transmissibility. These findings may be used to support more tailored SARS-CoV-2 isolation strategies, particularly if other studies clarify the direct association between Ag-RDT positivity and transmission risk. The apparent trade-off between sensitivity in the detection of any PCR-positive infection and concordance with infectious virus positivity may also inform new RDT diagnostic development strategies for SARS-CoV-2 and other epidemic respiratory pathogens. IMPORTANCE Despite the availability of vaccines, COVID-19 continues to be a major health concern, and antigen-based rapid diagnostic tests (Ag-RDTs) are commonly used as point-of-care or at-home diagnostic tests. In this study, we evaluated the performance of two commercially available Ag-RDTs and a research Ag-RDT to detect multiple SARS-CoV-2 variants using upper respiratory tract swab samples from clinical COVID-19 cases. Furthermore, we determined whether Ag-RDT results correlated with culture positivity, a potential proxy of viral transmissibility. Our results have important implications to inform future testing and response strategies during periods of high COVID-19 transmission with new variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) global pandemic. Rapid and sensitive detection of the virus soon after infection is important for the treatment and prevention of transmission of COVID-19, and detection of antibodies is important for epidemiology, assessment of vaccine immunogenicity, and identification of the natural reservoir and intermediate host(s). Patient nasal or oropharyngeal swabs or saliva used in conjunction with polymerase chain reaction (PCR) detect SARS-CoV-2 RNA, whereas lateral flow immunoassays (LFI) detect SARS-CoV-2 proteins. Enzyme-linked immunosorbent assays (ELISA) detect anti-SARS-CoV-2 antibodies in blood. Although effective, these assays have poor sensitivity (e.g., LFI) or are labor intensive and time consuming (PCR and ELISA). Here we describe the development of rapid, automated ELISA-based immunoassays to detect SARS-CoV-2 antigens and antibodies against the virus. The Simple Plex™ platform uses rapid microfluidic reaction kinetics for sensitive analyte detection with small sample volumes. We developed three sensitive <90-min Simple Plex immunoassays that measure either the SARS-CoV-2 antigens or the immune response to SARS-CoV-2, including neutralizing antibodies, in serum from COVID-19 patients.

The ongoing emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants underscores the need for rapid, adaptable, high-throughput testing. However, assays for neutralizing antibodies, which are a good measure of viral protection, usually require cell culture and either infectious SARS-CoV-2 or pseudotyped viral particles. To circumvent the challenges of cell-based assays, SARS-CoV-2 surrogate virus neutralization tests (sVNTs) measure inhibition of the binding of the spike (S) protein receptor binding domain (RBD) to its receptor, human angiotensin-converting enzyme 2 (hACE2) by neutralizing antibodies. Here we tested a prototype automated microfluidic cartridge-based sVNT platform using SARS-CoV-2 wild-type (WT) and B.1.617.2 (Delta) variant RBDs. This sVNT showed a high correlation with cell-based neutralization assays for biospecimens collected post-COVID-19 vaccination and post-SARS-CoV-2 infection as well as for pre-pandemic SARS-CoV-2 negative sera. Thus, this assay, which takes less than 80 min, is a relatively simple, safe, and accurate alternative to traditional VNTs.

Employing assays approved by the U.S. Food and Drug Administration (FDA) to assist in detection of brain injury in mild traumatic brain injury (TBI) patients, this study demonstrated that the astroglial protein, glial fibrillary acidic protein (GFAP) and the neuronal protein, ubiquitin C-terminal hydrolase (UCH-L1) were positively associated with age in COVID-19 patients. Controlling for age, UCH-L1 and GFAP were significantly elevated in COVID-19 patients compared to non-COVID-19 controls, and UCH-L1, but not GFAP, was elevated in patients with neurological alterations. Data from this study are also compared to historical data on levels of UCH-L1 and GFAP in brain injured and healthy normal patients. These data support further studies of an FDA approved assay format that could facilitate timely development, validation, and FDA approval of blood tests to detect neuronal and glial cell injuries following infection by SARS-CoV-2. Moreover, appropriately validated blood tests could detect brain injury originating from any systemic pathogen. ![Figure][1]</img> ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The research leading to the results of this study was not supported by extramural funding. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Barnes-Jewish Hospital ED-in St. Louis (IRB#202007018) IRB of University of Florida (IRB#202003085, IRB#202001993) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The assay results, patient clinical characteristics and selected unidentified demographic data are provided as supplemental materials. [1]: pending:yes

Background Blood biomarkers of neurological injury could provide a rapid diagnosis of central nervous system (CNS) injury caused by infections. An FDA-approved assay for mild traumatic brain injury (TBI) measures glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which signal astrocyte and neuronal injury, respectively. Here, we assessed the applicability of this biomarker assay for determining infection-induced brain injury. Methods We measured serum levels of GFAP and UCH-L1 retrospectively in serum samples from three study populations: 1) human cases infected with Venezuelan equine encephalitis virus (VEEV) and Madariaga virus (MADV) (n = 73), 2) human sepsis patients who were severely ill or diagnosed with encephalitis (n = 66), and 3) sepsis cases that were subsequently evaluated for cognitive impairment (n = 64). Results In the virus infection group, we found elevated GFAP for VEEV (p = 0.014) and MADV (p = 0.011) infections, which correlated with seizures (p = 0.006). In the bacterial sepsis group, GFAP was elevated in cases diagnosed with encephalitis (p = 0.0007) and correlated with headaches (p = 0.0002). In the bacterial sepsis cases with a later cognitive assessment, elevated GFAP (p = 0.0057) at study enrollment was associated with cognitive impairment six months later with a positive prognostic capacity of 79% (CI: 66–95%; p = 0.0068). Conclusions GFAP and UCH-L1 levels measured using an FDA-approved assay for TBI may indicate brain injury resulting from viral or bacterial infections and could predict the development of neurological sequelae.

Madariaga virus (MADV) and Venezuelan equine encephalitis virus (VEEV) are emerging arboviruses affecting rural and remote areas of Latin America. However, clinical and epidemiologic reports are limited, and outbreaks are occurring at an increasing frequency. We addressed the data gap by analyzing all available clinical and epidemiologic data of MADV and VEEV infections recorded since 1961 in Panama. A total of 168 human alphavirus encephalitis cases were detected in Panama during 1961‒2023. We described the clinical signs and symptoms and epidemiologic characteristics of those cases, and also explored signs and symptoms as potential predictors of encephalitic alphavirus infection compared with those of other arbovirus infections occurring in the region. Our results highlight the challenges for the clinical diagnosis of alphavirus disease in endemic regions with overlapping circulation of multiple arboviruses.

Neurotropic viral infection and the ensuant immune response are a significant cause of morbidity and mortality worldwide, which can range in severity from mild to permanent central nervous system (CNS) damage and death. Encephalitic alphaviruses include Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV; Alphavirus; Togaviridae). Injury to the CNS is an important determinant of poor outcome and tools to predict this outcome are lacking. Neurons are the primary target cells of encephalitic alphaviruses where cytopathology plays a major role in CNS dysfunction. Proteins are released following cell death, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) are neuromarkers of CNS tissue injury which could serve as biomarkers to assess injury severity, monitor disease progression, direct treatment, and as reliable endpoints to help develop novel medical countermeasures. Recent advances in the use of blood-based biomarkers for diagnosis of traumatic brain injury (TBI) which have FDA approved assays have provided a scientific foundation for expanding the biomarker technology to brain damage caused by other CNS pathologies like viral encephalitis. Here we evaluated the ability to detect these biomarkers for encephalitic alphaviruses, encephalitis of unknown origin, and hospitalized patients with severe coronavirus disease (COVID-19). Higher levels of specific biomarkers were detected in patients diagnosed with alphavirus or unknown encephalitis which may be useful in prognosis and treatment guidance of post viral disease. Collectively, our results suggest that these blood-based biomarkers may be a good indicator for brain injury resulting from viral infection.

BACKGROUND:Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort. METHODS:Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens. RESULTS:Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks. CONCLUSIONS:Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Early in the pandemic, in March of 2020, an outbreak of COVID-19 occurred aboard the aircraft carrier USS Theodore Roosevelt (CVN-71), during deployment in the Western Pacific. Out of the crew of 4,779 personnel, 1,331 service members were suspected or confirmed to be infected with SARS-CoV-2. The demographic, epidemiologic, and laboratory findings of service members from subsequent investigations have characterized the outbreak as widespread transmission of virus with relatively mild symptoms and asymptomatic infection among mostly young healthy adults. At the time, there was no available vaccination against COVID-19 and there was very limited knowledge regarding SARS-CoV-2 mutation, dispersal, and transmission patterns among service members in a shipboard environment. Since that time, other shipboard outbreaks from which data can be extracted have occurred, but these later shipboard outbreaks have occurred largely in settings where the majority of the crew were vaccinated, thereby limiting spread of the virus, shortening duration of the outbreaks, and minimizing evolution of the virus within those close quarters settings. On the other hand, since the outbreak on the CVN-71 occurred prior to widespread vaccination, it continued over the course of roughly two months, infecting more than 25% of the crew. In order to better understand genetic variability and potential transmission dynamics of COVID-19 in a shipboard environment of immunologically naïve, healthy individuals, we performed whole-genome sequencing and virus culture from eighteen COVID-19-positive swabs collected over the course of one week. Using the unique variants identified in those genomes, we detected seven discrete groups of individuals within the population aboard CVN-71 infected with viruses of distinct genomic signature. This is in stark contrast to a recent outbreak aboard another U.S. Navy ship with >98% vaccinated crew after a port visit in Reykjavik, Iceland, where the outbreak lasted only approximately 2 weeks and the virus was clonal. Taken together, these results demonstrate the utility of sequencing from complex clinical samples for molecular epidemiology and they also suggest that a high rate of vaccination among a population in close communities may greatly reduce spread, thereby restricting evolution of the virus.

The Rift Valley fever virus (RVFV) MP-12 vaccine is a promising human and veterinary vaccine. Although the vaccine elicited neutralizing antibody (nAb) in human volunteers, the minimal antibody titer that is needed to afford protection is unknown. Therefore, this study was conducted to determine the minimal nAb titer elicited by the RVFV MP-12 vaccine in human volunteers that protected mice against lethal RVFV challenge as a surrogate assessment of the protective efficacy of the vaccine. Among volunteers who were vaccinated with the MP-12 vaccine during a phase II trial, sera with antibody titers of 1:20 collected 5 years post-vaccination (PV), 1:40 titer collected 2 years PV, and 1:80 titer collected 1 year PV was passively transferred to groups of BALB/c mice. Blood samples were obtained 1 day after passive transfer to determine the RVFV neutralizing nAb titer before challenge with pathogenic RVFV (strain ZH501). Our results indicated that 1 day after passive transfer of the immune sera, an approximate 4-fold reduction in circulating nAb titers was detected in the mice. The presence of RVFV nAb titers in the range of 1:5 to 1:20 were generally protective (75-100% survival). These results suggested that circulating titers of 1:5 or higher offer a high degree of protection by MP-12-elicited antibody in human volunteers. Also, the findings highlighted the value of using the BALB/c mouse RVFV challengemodel as a surrogate for evaluating the protective nAb responses elicited byMP-12 and possible use for evaluating the efficacy of other RVFV vaccine candidates.

Little is known about severe acute respiratory syndrome coronavirus 2 "vaccine-breakthrough" infections (VBIs). Here we characterize 24 VBIs in predominantly young healthy persons. While none required hospitalization, a proportion endorsed severe symptoms and shed live virus as high as 4.13 x 10(3) plaque-forming units/mL. Infecting genotypes included both variant-of-concern (VOC) and non-VOC strains.

The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency's (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen that causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The development of an effective veterinary and human vaccine to protect against Rift Valley fever (RVF) disease remains a high priority. The live attenuated RVFV MP-12 is a promising vaccine candidate for the prevention of RVF in both human and domestic ruminants. The aim of this study was to determine the onset of protective immunity elicted in mice by a single dose of this vaccine. Groups of CD-1 mice were vaccinated intraperitoneally with RVFV MP-12 vaccine and challenged on days 2, 5, 6 and 7 post-vaccination (PV) with a lethal dose of virulent RVFV. The mice were observed once daily for terminal morbidity and blood samples were obtained from the retro-orbital sinus complex on days 23 and 28 PV of surviving mice to determine RVFV neutralizing antibody titers. In one test, 2 of 3 mice challenged on day 2 PV survived and all 3 mice challenged at days 5 and 7 PV also survived. A second test of 10 mice per group was performed, and half (5) of those challenged at day 2 PV survived while all (10) survived challenge at day 4 and 6 PV. All surviving animals develop antibody that ranged from 1:80 to 1:1,280 PV. In a separate experiment, RVFV MP-12 vaccinated CD-1 mice, but not challenged developed a low viremia for the first 3 days PV and neutralzing antibody was detected on days 5 through day 28 PV. These findings demonstrated that the RVFV MP-12 vaccine elicited a rapid protective immune response in mice as early as 2 days PV, thus further supporting the effectiveness of this vaccine candidate for preventing RVF among humans and domestic ruminants.

17 The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 18 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of 19 emerging variants, to trace the movement of variants, and to monitor effectiveness of 20 countermeasures. Additionally, determining the amount of viable virus present in clinical 21 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.16.21267862 doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. samples is helpful to better understand the impact these variants have on viral shedding. In this 22 study, we analyzed nasal swab samples collected between March 2020 and early November 2021 23 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries 24 stationed worldwide as a part of the Defense Health Agency’s (DHA) Global Emerging 25 Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse26 transcription PCR (qRT-PCR) positive samples were characterized by next-generation 27 sequencing and a subset was analyzed for isolation and quantification of viable virus. Not 28 surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. 29 military personnel beginning in July 2021 and primarily represents cases of vaccine 30 breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in 31 nasal swab samples from Delta variant cases when compared to cases involving other variants. 32 Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving 33 Delta as compared to unvaccinated personnel infected with other variants prior to the availability 34 of approved vaccines. This study provides important insight about the genomic and virological 35 characterization of SARS-CoV-2 isolates from a unique study population with a global presence. 36

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne RNA virus prevalent in Asia, Europe, and Africa, and can cause a hemorrhagic disease (CCHF) in humans with mortality rates as high as 60%. A general lack of both effective medical countermeasures and a comprehensive understanding of disease pathogenesis is partly driven by an historical lack of viable CCHF animal models. Recently, a cynomolgous macaque model of CCHF disease was developed. Here, we document the targeted transcriptomic response of non-human primates (NHP) to two different CCHFV strains; Afghan09-2990 and Kosova Hoti that both yielded a mild CCHF disease state. We utilized a targeted gene panel to elucidate the transcriptomic changes occurring in NHP whole blood during CCHFV infection; a first for any primate species. We show numerous upregulated genes starting at 1 day post-challenge through 14 days post-challenge. Early gene changes fell predominantly in the interferon stimulated gene family with later gene changes coinciding with an adaptive immune response to the virus. There are subtle differences between viral strains, namely duration of the differentially expressed gene response and biological pathways enriched. After recovery, NHPs showed no lasting transcriptomic changes at the end of sample collection.

The reinfection risk in individuals previously infected with SARS-CoV-2 is about a fifth of those never infected.1Hansen CH Michlmayr D Gubbels SM Mølbak K Ethelberg S Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.Lancet. 2021; 397: 1204-1212Summary Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar Whether reinfected individuals shed viable virus has been identified as an important question relevant to pandemic control.3Ledford H Coronavirus reinfections: three questions scientists are asking.Nature. 2020; 585: 168-169Crossref PubMed Scopus (39) Google Scholar In a prospective cohort study in The Lancet Respiratory Medicine,2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar we identified 19 cases of reinfection in people who at study entry were seropositive for both SARS-CoV-2 receptor binding domain and full-length spike protein, tested negative on three nasal swab PCR tests over a 2-week quarantine period, and subsequently developed a positive PCR test at least 2 weeks after leaving quarantine.2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar We have now investigated whether these SARS-CoV-2-reinfected individuals shed viable virus. Viral transport media was available from the first and some subsequent PCR-positive tests from 16 (84%) of 19 reinfected participants. Samples were cultured for SARS-CoV-2 in Vero E6 cells expressing TMPRSS24Matsuyama S Nao N Shirato K et al.Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.Proc Natl Acad Sci USA. 2020; 117: 7001-7003Crossref PubMed Scopus (852) Google Scholar in T25 cm2 flasks and monitored for cytopathic effect for 4 days followed by a second passage onto fresh cells to allow additional time for virus amplification. Samples were simultaneously titred by plaque assay in Vero E6/TMPRSS2 cells to quantify the level of detectable infectious virus. Viable virus was detected in only four (25%) of 16 participants, and only once in each, with titres ranging from 1·7 to 5·5 log10 plaque-forming units per mL. Serology and PCR cycle threshold (Ct) values using the US Food and Drug Administration-authorised Thermo Fisher TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MA, USA) were compared in those with viable virus detected (shedders) and those without viable virus detected (non-shedders; table; appendix p 2). At the time of positive viral culture, samples from shedders had lower Ct values. Although all participants had detectable SARS-CoV-2 spike IgG at study enrolment, none of the four shedders and only four (33%) of the 12 non-shedders had detectable serum neutralisation activity (limit of detection was 50% inhibitory dilution of 20) in serum at the time of enrolment into the study (methods as described2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Only one (25%) of the four who shed virus was symptomatic when viable virus was detected.TableBaseline serology, clinical features, and SARS-CoV-2 Ct levels in reinfected participants with samples cultured for viable virusVirus culture titreDifference*Difference in mean for the continuous variables and difference in percentage for binary variables.Positive (n=4)Negative (n=12)Baseline IgG S titre (log10)2·8 (0·2)2·9 (0·7)−0·08 (−0·88 to 0·72), p=0·834Baseline ID50 detected (>20)04 (33%)−33% (−87 to 21), p=0·207PCR positive >7 days3 (75%)4 (33%)42% (−20 to 100), p=0·166Symptomatic1 (25%)3 (25%)−0% (−57 to 57), p=1·000N gene Ct16·2 (4·0)31·9 (6·1)−15·64 (−22·68 to −8·60), p=0·0003S gene Ct17·8 (4·1)32·3 (6·2)−14·52 (−21·76 to −7·28), p=0·0007ORF1ab gene Ct16·8 (3·9)31·6 (6·1)−14·83 (−21·88 to −7·78), p=0·0005Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution.* Difference in mean for the continuous variables and difference in percentage for binary variables. Open table in a new tab Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution. All reinfections occurred before December, 2020, when the B.1.1.7 variant was first reported in the USA. Full-length viral genome was recovered from seven (44%) of 16 participants studied by viral transport medium culturing, including all four culture-positive individuals (methods previously described5Letizia AG Ramos I Obla A et al.SARS-CoV-2 transmission among marine recruits during quarantine.N Engl J Med. 2020; 383: 2407-2416Crossref PubMed Scopus (64) Google Scholar). Although the strains isolated all had the Asp614Gly spike protein mutation and two of the isolates had the Leu18Phe spike protein mutation, they did not have mutations associated with the B.1.1.7, P.1, B.1.351, or other variants of concern as currently defined by the US Centers for Disease Control and Prevention (appendix p 3). Limitations of our study are the use of virus culture as a proxy for capacity for transmission without direct study of transmission, the small sample size, the absence of specimens available for culturing from all reinfected participants, and the inability to compare potential for transmissibility between those who are infected for the first time and those who are reinfected. Overall, our findings suggest that about a quarter of young healthy individuals with subsequent SARS-CoV-2 reinfection shed viable virus. Some of these individuals were asymptomatic and could unknowingly transmit SARS-CoV-2 to others. All authors declare no competing interests. This study was supported by the Defense Health Agency and Defense Advanced Research Projects Agency. AGL, DRS, CG, DLW, HWC, RAL, SEL, JM, EN, CKP, ESA, MS, VAS, PS, and MT are military service members or government service employees. This work was prepared as part of their official duties. Title 17, US Code §105 provides that copyright protection under this title is not available for any work of the US Government. Title 17, US code §101 defines a US Government work as a work prepared by a military service member or employee of the US Government as part of that person's official duties. The views expressed in the article are those of the authors and do not necessarily express the official policy and position of the US Navy, the Department of Defense, the US Government, or the institutions affiliated with the authors. Download .pdf (.23 MB) Help with pdf files Supplementary appendix SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort studySeropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies. Full-Text PDF