Identification of two novel B-cell epitopes located on the spike protein of swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging alpha-coronavirus that causes diarrhea in piglets and results in serious economic losses. During SADS-CoV infection, the spike protein (S) serves as a crucial structural component of the virion, interacting with receptors and eliciting the production of neutralizing antibodies. Due to the potential risk of zoonotic transmission of SADS-CoV, the identification and screening of epitopes on the S glycoproteins will be crucial for development of sensitive and specific diagnostic tools. In this study, we immunized BALB/c mice with recombinant SADS-CoV S trimer protein and generated two S1-specific monoclonal antibodies (mAbs): 8D6 and 6E9, which recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 8D6 was mapped to 311NPDQRD316, the minimal fragment recognized by mAb 6E9 was mapped to 492ARFVDRL498. Homology analysis of the regions corresponding to 13 typical strains of different SADS-CoV subtypes showed high conservation of these two epitopes. These findings contribute to a deeper understanding of the structure of the SADS-CoV S protein, which is valuable for vaccine design and holds potential for developing diagnostic methods to detect SADS-CoV.

Comparative Analysis of Viral Load and Cytokines during SARS-CoV-2 Infection between Pregnant and Non-Pregnant Women.

To better understand the vulnerabilities of pregnant women during the COVID-19 pandemic, we conducted a comprehensive, retrospective cohort study to assess differences in immune responses to SARS-CoV-2 infection between pregnant and non-pregnant women. Nasopharyngeal swabs and serum specimens from 90 pregnant and 278 age-matched non-pregnant women were collected from 15 March 2020 to 23 July 2021 at NewYork-Presbyterian Queens Hospital in New York City. Multiplex reverse transcription polymerase chain reaction, neutralizing antibody, and cytokine array assays were used to assess the incidence, viral load, antibody titers and profiles, and examine cytokine expression patterns. Our results show a lower incidence of SARS-CoV-2 infection in pregnant women compared with non-pregnant women. Pregnant women infected with SARS-CoV-2 exhibited a substantially lower viral load. In addition, the levels of both anti-spike protein receptor-binding domain IgG neutralizing antibodies and anti-N Protein IgG were elevated in pregnant women. Finally, cytokine profiling revealed differential expression of leptin across cohorts. These findings suggest that pregnancy is associated with distinct immune and virological responses to SARS-CoV-2 infection, characterized by lower infection rates, substantially lower viral loads, and enhanced antibody production. Differential cytokine expression indicates unique immune modulation in pregnant women.

Correction: Liu et al. Cholesterol 25-Hydroxylase Suppresses Swine Acute Diarrhea Syndrome Coronavirus Infection by Blocking Spike Protein-Mediated Membrane Fusion. Viruses 2023, 15, 2406.

In the original publication [...].

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-ß) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-ß production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Cholesterol 25-Hydroxylase Suppresses Swine Acute Diarrhea Syndrome Coronavirus Infection by Blocking Spike Protein-Mediated Membrane Fusion.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging porcine intestinal coronavirus that can cause acute diarrhea, vomiting, rapid weight loss, and high mortality in newborn piglets. Cholesterol 25-hydroxylase (CH25H) is a molecular mediator of innate antiviral immunity and converts cholesterol to 25-hydroxycholesterol (25HC). Previous studies have reported that CH25H and 25HC have an antiviral effect against multiple viruses. However, the interplay between SADS-CoV infection and CH25H or 25HC is still uncertain. Here, we found that CH25H and its enzymatic product 25HC restrained SADS-CoV replication by blocking membrane fusion. Our results show that CH25H was upregulated by SADS-CoV infection in vitro and in vivo, and that it was an IFN-stimulated gene in porcine ileum epithelial cells. Moreover, CH25H and CH25H mutants lacking catalytic activity can inhibit SADS-CoV replication. Furthermore, 25HC significantly suppressed SADS-CoV infection by inhibiting virus entry. Notably, we confirmed that CH25H and 25HC blocked SADS-CoV spike protein-mediated membrane fusion. Our data provide a possible antiviral therapy against SADS-CoV and other conceivable emerging coronaviruses in the future.

Stress Distribution of Different Pedicle Screw Insertion Techniques Following Single-Segment TLIF: A Finite Element Analysis Study.

OBJECTIVES: At present, a variety of posterior lumbar internal fixation implantation methods have been developed, which makes it difficult for spine surgeons to choose. The stress distribution of the internal fixation system is one of the important indexes to evaluate these technologies. Common insertion technologies include Roy Camille, Magerl, Krag, AO, and Weinstein insertion techniques. This study aimed to compare the distribution of von Mises stresses in different screw fixation systems established by these insertion technologies. METHODS: Here, the three-dimensional finite element (FE) method was selected to evaluate the postoperative stress distribution of internal fixation. Following different pedicle screw insertion techniques, five single-segment transforaminal lumbar interbody fusion (TLIF) models were established after modeling and validation of the L1-S1 vertebrae FE model. RESULTS: By analyzing the data, we found that stress concentration phenomenon was in all the models. Additionally, Roy-Camille, Krag, AO, and Weinstein insertion techniques led to the great stress on lumbar vertebra, intervertebral disc, and screw-rod fixation systems. Therefore, we hope that the results can provide ideas for clinical work and development of pedicle screws in the future. It is worth noting that flexion, unaffected side lateral bending, and affected side axial rotation should be limited for the patients with cages implanted. CONCLUSIONS: Overall, our method obtained the results that Magerl insertion technique was the relatively safe approach for pedicle screw implantation due to its relatively dispersive stress in TLIF models.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Early Introduction and Community Transmission of SARS-CoV-2 Omicron Variant, New York, New York, USA.

The Omicron variant of SARS-CoV-2 has become dominant in most countries and has raised significant global health concerns. As a global commerce center, New York, New York, USA, constantly faces the risk for multiple variant introductions of SARS-CoV-2. To elucidate the introduction and transmission of the Omicron variant in the city of New York, we created a comprehensive genomic and epidemiologic analysis of 392 Omicron virus specimens collected during November 25-December 11, 2021. We found evidence of 4 independent introductions of Omicron subclades, including the Omicron subclade BA.1.1 with defining substitution of R346K in the spike protein. The continuous genetic divergence within each Omicron subclade revealed their local community transmission and co-circulation in New York, including both household and workplace transmissions supported by epidemiologic evidence. Our study highlights the urgent need for enhanced genomic surveillance and effective response planning for better prevention and management of emerging SARS-CoV-2 variants.

SARS-CoV-2 Continuous Genetic Divergence and Changes in Multiplex RT-PCR Detection Pattern on Positive Retesting Median 150 Days after Initial Infection.

Being in the epicenter of the COVID-19 pandemic, our lab tested 193,054 specimens for SARS-CoV-2 RNA by diagnostic multiplex reverse transcription polymerase chain reaction (mRT-PCR) starting in March 2020, of which 17,196 specimens resulted positive. To investigate the dynamics of virus molecular evolution and epidemiology, whole genome amplification (WGA) and Next Generation Sequencing (NGS) were performed on 9516 isolates. 7586 isolates with a high quality were further analyzed for the mutation frequency and spectrum. Lastly, we evaluated the utility of the mRT-PCR detection pattern among 26 reinfected patients with repeat positive testing three months after testing negative from the initial infection. Our results show a continuation of the genetic divergence in viral genomes. Furthermore, our results indicate that independent mutations in the primer and probe regions of the nucleocapsid gene amplicon and envelope gene amplicon accumulate over time. Some of these mutations correlate with the changes of detection pattern of viral targets of mRT-PCR. Our data highlight the significance of a continuous genetic divergence on a gene amplification-based assay, the value of the mRT-PCR detection pattern for complementing the clinical diagnosis of reinfection, and the potential for WGA and NGS to identify mutation hotspots throughout the entire viral genome to optimize the design of the PCR-based gene amplification assay.

Identification and epitope mapping of swine acute diarrhea syndrome coronavirus accessory protein NS7a via monoclonal antibodies.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric coronavirus that causes vomiting, severe diarrhea, dehydration and death in suckling piglets. NS7a is putative accessory protein that is predicted to be encoded by SADS-CoV, but still to be confirmed experimentally. In the present study, recombinant NS7a protein was expressed in a prokaryotic expression system and used as an antigen to prepare monoclonal antibodies (mAbs) specific to NS7a protein. We obtained two anti-NS7a mAbs, termed AH5 and EH3, that were shown by western blotting to react with the natural NS7a protein in Vero E6 cells infected with SADS-CoV. Using the produced mAbs, we observed by confocal microscopy that NS7a protein was expressed in the cytoplasm. Further studies revealed that the motif 31VNTWQEFA38 was the minimal unit of the linear B-cell epitope recognized by mAb AH5, and the motif 82FDLFERF88 was the minimal unit of the linear B-cell epitope recognized by mAb EH3. Alignment of amino acids showed that these two epitopes were highly conserved among different SADS-CoV strains and SADS-related coronaviruses from bats, but with one substitution in these two motifs in bat coronavirus HKU2. In summary, we generated and characterized two mAbs against SADS-CoV NS7a protein, and demonstrated NS7a expression in SADS-CoV-infected cells for the first time.

Direct-from-specimen microbial growth inhibition spectrums under antibiotic exposure and comparison to conventional antimicrobial susceptibility testing.

Increasing global travel and changes in the environment may escalate the frequency of contact with a natural host carrying an infection and, therefore, increase our chances of encountering microorganisms previously unknown to humans. During an emergency, the etiology of infection may be unknown at the time of patient treatment. The existing local or global Antimicrobial Stewardship Programs may not be fully prepared for emerging/re-emerging infectious disease outbreaks, especially if they are caused by an unknown organism, engineered bioterrorist attack, or rapidly evolving superbug. We demonstrate an antimicrobial efficacy profiling method that can be performed in hours directly from clinical urine specimens. The antimicrobial potency was determined by the level of microbial growth inhibition and compared to conventional antimicrobial susceptibility testing results. The oligonucleotide probe pairs on the sensors were designed to target Gram-negative bacteria, specifically Enterobacterales and Pseudomonas aeruginosa. A pilot study of 10 remnant clinical specimens from the Clinical Laboratory Improvement Amendments-certified labs of New York-Presbyterian Queens was conducted, and only one sample was not detected by the probes. The remaining nine samples agreed with reference AST methods (Vitek and broth microdilution), resulting in 100% categorical agreement. In a separate feasibility study, we evaluated a dual-kinetic response approach, in which we inoculated two antibiotic stripwells containing the same antimicrobial concentrations with clinical specimens at the original concentration (1x) and at a 10-fold dilution (0.1x) to cover a broader range of microbiological responses. The combined categorical susceptibility reporting of 12 contrived urine specimens was 100% for ciprofloxacin, gentamicin, and meropenem over a range of microbial loads from 105 to 108 CFU/mL.

Swine acute diarrhea syndrome coronavirus replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered enteric coronavirus. We have previously shown that the caspase-dependent FASL-mediated and mitochondrion-mediated apoptotic pathways play a central role in SADS-CoV-induced apoptosis, which facilitates viral replication. However, the roles of intracellular signaling pathways in SADS-CoV-mediated cell apoptosis and the relative advantages that such pathways confer on the host or virus remain largely unknown. In this study, we show that SADS-CoV induces the activation of ERK during infection, irrespective of viral biosynthesis. The knockdown or chemical inhibition of ERK1/2 significantly suppressed viral protein expression and viral progeny production. The inhibition of ERK activation also circumvented SADS-CoV-induced apoptosis. Taken together, these data suggest that ERK activation is important for SADS-CoV replication, and contributes to the virus-mediated changes in host cells. Our findings demonstrate the takeover of a particular host signaling mechanism by SADS-CoV and identify a potential approach to inhibiting viral spread.

Co-Infection with 4 Species of Mycobacteria Identified by Using Next-Generation Sequencing.

We identified co-infection with 4 species of mycobacteria in a woman in New York, New York, USA, by using next-generation sequencing. This procedure is useful for identifying co-infections with multiple mycobacteria, tracing the geographic origin of strains, investigating transmission dynamics in susceptible populations, and gaining insight into prevention and control.

Transportation protocols for accurate assessment of microbial burden classification using molecular methods.

Point-of-care testing is cost-effective, rapid, and could assist in avoiding hospital visits during a pandemic. However, they present some significant risks that current technologies cannot fully address. Skin flora contamination and insufficient specimen volume are two major limitations preventing self-collection microbiological testing outside of hospital settings. We are developing a hybrid testing procedure to bridge the laboratory test with patient-side specimen collection and transportation for molecular microbial classification of causative bacterial infection and early identification of microbial susceptibility profiles directly from whole blood or urine specimens collected patient-side by health care workers such as phlebotomists in nursing homes or family clinics. This feasibility study presents our initial development efforts, in which we tested various transportation conditions (tubes, temperature, duration) for direct-from-specimen viable pathogen detection to determine the ideal conditions that allowed for differentiation between contaminant and causative bacteria in urine specimens and optimal growth for low-concentration blood specimens after transportation. For direct-from-urine assays, the viable pathogen at the clinical cutoff of 105 CFU/mL was detected after transportation with molecular assays while contaminants (≤ 104 CFU/mL) were not. For direct-from-blood assays, contrived blood samples as low as 0.8 CFU/mL were reported positive after transportation without the need for blood culture.

Feasibility and potential significance of rapid in vitro qualitative phenotypic antimicrobial susceptibility testing of gram-negative bacilli with the ProMax system.

The emergence and evolution of antibiotic resistance has been accelerated due to the widespread use of antibiotics and a lack of timely diagnostic tests that guide therapeutic treatment with adequate sensitivity, specificity, and antimicrobial susceptibility testing (AST) accuracy. Automated AST instruments are extensively used in clinical microbiology labs and provide a streamlined workflow, simplifying susceptibility testing for pathogenic bacteria isolated from clinical samples. Although currently used commercial systems such as the Vitek2 and BD Phoenix can deliver results in substantially less time than conventional methods, their dependence on traditional AST inoculum concentrations and optical detection limit their speed somewhat. Herein, we describe the GeneFluidics ProMax lab automation system intended for a rapid 3.5-hour molecular AST from clinical isolates. The detection method described utilizes a higher starting inoculum concentration and automated molecular quantification of species-specific 16S rRNA through the use of an electrochemical sensor to assess microbiological responses to antibiotic exposure. A panel of clinical isolates consisting of species of gram-negative rods from the CDC AR bank and two hospitals, New York-Presbyterian Queens and Medical College of Wisconsin, were evaluated against ciprofloxacin, gentamicin, and meropenem in a series of reproducibility and clinical studies. The categorical agreement and reproducibility for Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, and Pseudomonas aeruginosa were 100% and 100% for ciprofloxacin, 98.7% and 100% for gentamicin and 98.5% and 98.5% for meropenem, respectively.

SARS-CoV-2 Viral Load Predicts Mortality in Patients with and without Cancer Who Are Hospitalized with COVID-19.

Patients with cancer may be at increased risk of severe coronavirus disease 2019 (COVID-19), but the role of viral load on this risk is unknown. We measured SARS-CoV-2 viral load using cycle threshold (CT) values from reverse-transcription polymerase chain reaction assays applied to nasopharyngeal swab specimens in 100 patients with cancer and 2,914 without cancer who were admitted to three New York City hospitals. Overall, the in-hospital mortality rate was 38.8% among patients with a high viral load, 24.1% among patients with a medium viral load, and 15.3% among patients with a low viral load (p < 0.001). Similar findings were observed in patients with cancer (high, 45.2% mortality; medium, 28.0%; low, 12.1%; p = 0.008). Patients with hematologic malignancies had higher median viral loads (CT = 25.0) than patients without cancer (CT = 29.2; p = 0.0039). SARS-CoV-2 viral load results may offer vital prognostic information for patients with and without cancer who are hospitalized with COVID-19.

Detection and Genetic Characterization of Community-Based SARS-CoV-2 Infections - New York City, March 2020.

To limit introduction of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), the United States restricted travel from China on February 2, 2020, and from Europe on March 13. To determine whether local transmission of SARS-CoV-2 could be detected, the New York City (NYC) Department of Health and Mental Hygiene (DOHMH) conducted deidentified sentinel surveillance at six NYC hospital emergency departments (EDs) during March 1-20. On March 8, while testing availability for SARS-CoV-2 was still limited, DOHMH announced sustained community transmission of SARS-CoV-2 (1). At this time, twenty-six NYC residents had confirmed COVID-19, and ED visits for influenza-like illness* increased, despite decreased influenza virus circulation.† The following week, on March 15, when only seven of the 56 (13%) patients with known exposure histories had exposure outside of NYC, the level of community SARS-CoV-2 transmission status was elevated from sustained community transmission to widespread community transmission (2). Through sentinel surveillance during March 1-20, DOHMH collected 544 specimens from patients with influenza-like symptoms (ILS)§ who had negative test results for influenza and, in some instances, other respiratory pathogens.¶ All 544 specimens were tested for SARS-CoV-2 at CDC; 36 (6.6%) tested positive. Using genetic sequencing, CDC determined that the sequences of most SARS-CoV-2-positive specimens resembled those circulating in Europe, suggesting probable introductions of SARS-CoV-2 from Europe, from other U.S. locations, and local introductions from within New York. These findings demonstrate that partnering with health care facilities and developing the systems needed for rapid implementation of sentinel surveillance, coupled with capacity for genetic sequencing before an outbreak, can help inform timely containment and mitigation strategies.

Clinical Performance of SARS-CoV-2 Molecular Tests.

Molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the gold standard for diagnosis of coronavirus disease 2019 (COVID-19), but the clinical performance of these tests is still poorly understood, particularly with regard to disease course, patient-specific factors, and viral shedding. From 10 March to 1 May 2020, NewYork-Presbyterian laboratories performed 27,377 SARS-CoV-2 molecular assays from 22,338 patients. Repeat testing was performed for 3,432 patients, of which 2,413 had initial negative and 802 had initial positive results. Repeat-tested patients were more likely to have severe disease and low viral loads. The negative predictive value of the first-day result among repeat-tested patients was 81.3% The clinical sensitivity of SARS-CoV-2 molecular assays was estimated between 58% and 96%, depending on the unknown number of false-negative results in single-tested patients. Conversion to negative was unlikely to occur before 15 to 20 days after initial testing or 20 to 30 days after the onset of symptoms, with 50% conversion occurring at 28 days after initial testing. Conversion from first-day negative to positive results increased linearly with each day of testing, reaching 25% probability in 20 days. Sixty patients fluctuated between positive and negative results over several weeks, suggesting that caution is needed when single-test results are acted upon. In summary, our study provides estimates of the clinical performance of SARS-CoV-2 molecular assays and suggests time frames for appropriate repeat testing, namely, 15 to 20 days after a positive test and the same day or next 2 days after a negative test for patients with high suspicion for COVID-19.

Bacteremia and Blood Culture Utilization during COVID-19 Surge in New York City.

A surge of patients with coronavirus disease 2019 (COVID-19) presenting to New York City hospitals in March 2020 led to a sharp increase in blood culture utilization, which overwhelmed the capacity of automated blood culture instruments. We sought to evaluate the utilization and diagnostic yield of blood cultures during the COVID-19 pandemic to determine prevalence and common etiologies of bacteremia and to inform a diagnostic approach to relieve blood culture overutilization. We performed a retrospective cohort analysis of 88,201 blood cultures from 28,011 patients at a multicenter network of hospitals within New York City to evaluate order volume, positivity rate, time to positivity, and etiologies of positive cultures in COVID-19. Ordering volume increased by 34.8% in the second half of March 2020 compared to the level in the first half of the month. The rate of bacteremia was significantly lower among COVID-19 patients (3.8%) than among COVID-19-negative patients (8.0%) and those not tested (7.1%) (P < 0.001). COVID-19 patients had a high proportion of organisms reflective of commensal skin microbiota, which, when excluded, reduced the bacteremia rate to 1.6%. More than 98% of all positive cultures were detected within 4 days of incubation. Bloodstream infections are very rare for COVID-19 patients, which supports the judicious use of blood cultures in the absence of compelling evidence for bacterial coinfection. Clear communication with ordering providers is necessary to prevent overutilization of blood cultures during patient surges, and laboratories should consider shortening the incubation period from 5 days to 4 days, if necessary, to free additional capacity.

First 12 Months of Life for Infants in New York City, New York, With Possible Congenital Zika Virus Exposure.

BACKGROUND: Our goal was to characterize the epidemiology and clinical significance of congenital Zika virus (ZIKV) exposure by prospectively following a cohort of infants with possible congenital exposure through their first year of life. METHODS: We included infants born in New York City between 2016 and 2017 who had or were born to a woman who had laboratory evidence of ZIKV infection during pregnancy. We conducted provider/patient interviews and reviewed medical records to collect information about the pregnant women and, for infants, clinical and neurodevelopmental status at birth and 2, 6, and 12 months of age. RESULTS: Of the 404 infants who met inclusion criteria, most (385 [95.3%]) appeared well, whereas 19 (4.7%) had a possible ZIKV-associated birth defect. Seven had congenital ZIKV syndrome, and 12 were microcephalic without other abnormalities. Although infants with congenital ZIKV syndrome manifested clinical and neurodevelopmental sequelae during their first year of life, all 12 infants with isolated microcephaly were normocephalic and appeared well by 2 months of age. Laboratory evidence of ZIKV was detected for 22 of the infants, including 7 (31.8%) with a birth defect. Among 148 infants without a birth defect and negative/no laboratory results on ZIKV testing, and for whom information was available at 1 year, 4 presented with a developmental delay. CONCLUSIONS: Among infants with possible congenital ZIKV exposure, a small proportion had possible ZIKV-associated findings at birth or at follow-up, or laboratory evidence of ZIKV. Identifying and monitoring infants with possible ZIKV exposure requires extensive efforts by providers and public health departments. Longitudinal studies using standardized clinical and developmental assessments are needed for infants after possible congenital ZIKV exposure.