Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

Conveyance Contact Investigation for Imported Middle East Respiratory Syndrome Cases, United States, May 2014.

In 2014, the Centers for Disease Control and Prevention conducted conveyance contact investigations for 2 Middle East respiratory syndrome cases imported into the United States, comprising all passengers and crew on 4 international and domestic flights and 1 bus. Of 655 contacts, 78% were interviewed; 33% had serologic testing. No secondary cases were identified.

Multifacility Outbreak of Middle East Respiratory Syndrome in Taif, Saudi Arabia.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is a novel respiratory pathogen first reported in 2012. During September 2014-January 2015, an outbreak of 38 cases of MERS was reported from 4 healthcare facilities in Taif, Saudi Arabia; 21 of the 38 case-patients died. Clinical and public health records showed that 13 patients were healthcare personnel (HCP). Fifteen patients, including 4 HCP, were associated with 1 dialysis unit. Three additional HCP in this dialysis unit had serologic evidence of MERS-CoV infection. Viral RNA was amplified from acute-phase serum specimens of 15 patients, and full spike gene-coding sequencing was obtained from 10 patients who formed a discrete cluster; sequences from specimens of 9 patients were closely related. Similar gene sequences among patients unlinked by time or location suggest unrecognized viral transmission. Circulation persisted in multiple healthcare settings over an extended period, underscoring the importance of strengthening MERS-CoV surveillance and infection-control practices.

Epidemiologic and laboratory features of a large outbreak of pertussis-like illnesses associated with cocirculating Bordetella holmesii and Bordetella pertussis--Ohio, 2010-2011.

BACKGROUND: During 9 May 2010-7 May 2011, an outbreak of pertussis-like illness (incidence, 80 cases per 100 000 persons) occurred in Franklin County, Ohio. The majority of cases were identified by IS481-directed polymerase chain reaction (PCR), which does not differentiate among Bordetella species. We sought to determine outbreak etiology and epidemiologic characteristics. METHODS: We obtained demographic, clinical, and vaccination-related data from the Ohio Disease Reporting System and Impact Statewide Immunization Information System. We tested sera from 14 patients for anti-pertussis toxin (PT) antibodies and used species-specific PCR on 298 nasopharyngeal specimens. RESULTS: Reported cases totaled 918. IS481 results were available for 10 serologically tested patients; 5 of 10 had discordant anti-PT antibody and IS481 results, suggestive of Bordetella holmesii, which lacks PT and harbors IS481. We identified specific Bordetella species in 164 of 298 specimens tested with multitarget PCR; B. holmesii and Bordetella pertussis were exclusively detected among 48 (29%) and 112 (68%), respectively; both were detected in 4 (2%). Among 48 patients with B. holmesii infections, 63% were aged 11-18 years, compared with 35% of 112 patients with B. pertussis infections (P = .001). Symptoms were similar among B. holmesii- and B. pertussis-infected patients. Adolescent pertussis ("Tdap") booster vaccinations were more effective against B. pertussis than B. holmesii (effectiveness: 67% and 36%, respectively; 95% confidence intervals, 38%-82% and -33% to 69%, respectively). CONCLUSIONS: We report the first documented mixed outbreak of B. pertussis and B. holmesii infections. Bordetella holmesii particularly affected adolescents. Although laboratory capacity limitations might inhibit routine use of multitarget PCR for clinical diagnosis, focused testing and enhanced surveillance might improve understanding the burden of B. holmesii infection.

Understanding COVID-19 Vaccine Hesitancy Among K-12 Staff, Parents, and Students: District of Columbia, February to April, 2022.

OBJECTIVE: Despite widespread availability of COVID-19 vaccines, millions of Americans have not received the recommended vaccine doses. In the District of Columbia (DC), COVID-19 vaccination rates are lowest among residents who are Non-Hispanic (NH) Black and among school-aged children. We assessed COVID-19 vaccine hesitancy among staff and parents of students in DC K-12 public and public charter schools. METHODS: We conducted a telephone-based survey from February 6 to April 16, 2022 to staff, students, and parents of students who participated in school-based COVID-19 screening testing. COVID-19-related survey items included: vaccination status, reasons for not getting vaccinated, perceived vaccine access, and trusted COVID-19 information sources. Utilizing time-to-event analyses, we evaluated differences across demographic groups. RESULTS: The interview response rate was 25.8% (308/1193). Most unvaccinated participants were NH Black and ages 5 to 11 years. Median time from vaccine eligibility to uptake was 236 days for NH Black participants vs. 10 days for NH White participants. Vaccine safety was the top concern among unvaccinated participants. Government and healthcare providers were the most trusted COVID-19 information sources. CONCLUSIONS: Differences in timing of vaccine uptake among respondents and greater vaccine hesitancy among NH Black participants compared to other racial/ethnic groups highlight a need for continued tailored outreach and communication using trusted sources to convey the importance, benefits, and safety of COVID-19 vaccination.

Population diversity among Bordetella pertussis isolates, United States, 1935-2009.

Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935-2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970-1990 (94%) but declined to ≈ 70% after 1991 and has remained constant. During 2006-2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.

Novel multitarget real-time PCR assay for rapid detection of Bordetella species in clinical specimens.

A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.

A computational genomics pipeline for prokaryotic sequencing projects.

MOTIVATION: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. RESULTS: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. AVAILABILITY AND IMPLEMENTATION: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

Effectiveness of adolescent and adult tetanus, reduced-dose diphtheria, and acellular pertussis vaccine against pertussis.

BACKGROUND: Pertussis is among the most poorly controlled bacterial vaccine-preventable diseases in the United States. In 2006, a tetanus, reduced-dose diphtheria, and acellular pertussis (Tdap) booster was recommended for adolescents and adults. Tdap vaccines were licensed on the basis of antibody response without vaccine effectiveness data. METHODS: From 30 September 2007 through 19 December 2007, a pertussis outbreak occurred at a nursery through twelfth grade school on St. Croix, US Virgin Islands. We screened all students for cough and collected clinical history, including Tdap receipt. Coughing students were offered diagnostic testing. We defined clinical case patients as students with cough 14 days in duration plus either whoop, paroxysms, or post-tussive vomiting, and we defined confirmed case patients as students with any cough with isolation of Bordetella pertussis or those with clinical cases and polymerase chain reaction or serological evidence of pertussis; other clinical cases were classified as probable. RESULTS: There were 51 confirmed or probable cases among 499 students (attack rate, 10%). Disease clustered in grades 6-12, with a peak attack rate of 38% among 10th graders. Of 266 students aged 11 years with complete data, 31 (12%) had received Tdap. Forty-one unvaccinated students (18%) had confirmed or probable pertussis, compared with 2 (6%) of the vaccinated students (relative risk, 2.9); vaccine effectiveness was 65.6% (95% confidence interval, -35.8% to 91.3%; P = .092). CONCLUSIONS: This first evaluation of Tdap vaccine effectiveness in the outbreak setting suggests that Tdap provides protection against pertussis. Increased coverage is needed to realize the full benefit of the vaccine program. Serological testing was an important tool for case identification and should be considered for inclusion in the Council of State and Territorial Epidemiologists case definition.

Pathology and pathogenesis of fatal Bordetella pertussis infection in infants.

BACKGROUND: Each year, Bordetella pertussis infection causes an estimated 294,000 deaths worldwide, primarily among young, nonvaccinated children. Approximately 90% of all deaths due to pertussis in the Unites States occur in young infants. These children often develop intractable pulmonary hypertension; however, the pathophysiologic mechanism responsible for this complication has not been well characterized, and there have been no detailed descriptions of the pathology of this disease since the 1940s. METHODS: Respiratory tissue samples obtained at autopsy from 15 infants aged

Real-time polymerase chain reaction detection of Bordetella pertussis DNA in acellular pertussis vaccines.

Using 2 real-time polymerase chain reaction (PCR) assays for Bordetella pertussis, 2 of 5 acellular pertussis vaccines were found to contain B. pertussis DNA. Because residual DNA in vaccines can cause environmental contamination, the administration of acellular pertussis vaccines to patients should be physically separated from the collection of patients' specimens for testing of B. pertussis DNA by real-time PCR.

Development and evaluation of dual-target real-time polymerase chain reaction assays to detect Bordetella spp.

Novel, highly specific, and sensitive real-time polymerase chain reaction (PCR) assays using 2 targets, insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1), were developed to detect Bordetella pertussis and to differentiate between relevant Bordetella spp. Sixty-four non-Bordetella isolates were negative by both assays, demonstrating the specificity of the assays. B. pertussis, Bordetella parapertussis, and Bordetella holmesii isolates were specifically identified using the assays. The lower limit of detection was less than 10 genomic equivalents per reaction for the IS481 and ptxS1 assays. These assays were evaluated using 145 human clinical specimens obtained during cough-illness outbreak investigations, and PCR results were compared with Bordetella spp. culture results. Twenty-seven (18.6%) specimens had late positive cycle threshold (Ct) values (35

Clinical evaluation and validation of laboratory methods for the diagnosis of Bordetella pertussis infection: Culture, polymerase chain reaction (PCR) and anti-pertussis toxin IgG serology (IgG-PT).

INTRODUCTION: The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests. METHODS: Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤ 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods-pertussis culture as the "gold standard," composite reference standard analysis (CRS), and latent class analysis (LCA). RESULTS: Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough. CONCLUSIONS: Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.

Molecular diagnosis of Nocardia farcinica from a cerebral abscess.

Histopathology and special stains of a brain biopsy specimen from a 42-year-old man revealed numerous gram-positive bacilli arranged in branching filaments, suggesting Nocardia infection. Antibiotic therapy with trimethoprim-sulfamethoxazole markedly decreased the abscess size, and the patient improved. DNA was analyzed from formalin-fixed sections of the cerebral abscess by a 16S ribosomal DNA polymerase chain reaction assay demonstrating the presence of either Nocardia farcinica or N otitidiscaviarum. A species-specific polymerase chain reaction assay confirmed N farcinica as the etiologic agent.

Morphologic, immunologic, and molecular methods to detect bacillus anthracis in formalin-fixed tissues.

Due to the importance of Bacillus anthracis as a cause of naturally occurring infection among humans and as an agent of bioterrorism, there is a vital need for rapid and specific assays, including immunohistochemistry (IHC) and polymerase chain reaction (PCR) assays, to detect the bacterium in formalin-fixed tissues. Colorimetric IHC assays were developed using a multistep indirect immunoalkaline phosphatase method with anti-B. anthracis cell wall (EAII-6G6-2-3) and anti-B. anthracis capsule (FDF-1B9) mAbs to detect B. anthracis antigens in formalin-fixed, paraffin-embedded bacterial cultures and tissues. B. anthracis antigens were localized, using both antibodies, in samples from B. anthracis-infected animals and humans. The colorimetric IHC assay with both antibodies was expedient in diagnosing the presence of B. anthracis in formalin-fixed, paraffin-embedded tissue from bioterrorism-associated cases of inhalational and cutaneous anthrax and from a case of naturally occurring cutaneous anthrax. Using the same antibodies, confocal microscopy demonstrated the structure of replicating B. anthracis in tissues. B. anthracis-specific primers were successfully used with PCR to amplify and detect B. anthracis sequences derived from formalin-fixed tissues of anthrax cases. In this study, morphologic, immunologic, and molecular assays were used to study and diagnose 22 veterinary and human anthrax cases.

Human cathelicidin, LL-37, inhibits respiratory syncytial virus infection in polarized airway epithelial cells.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract illness in young children worldwide. Treatment options for severe RSV disease remain limited and the development of therapeutic treatment strategies remains a priority. LL-37, a small cationic host defense peptide involved in anti-inflammatory and anti-bacterial responses, reduces replication of or infection by multiple viruses, including influenza virus, in vitro, and protects against lethal challenge with influenza virus in vivo. LL-37 also protects against RSV infection of HEp-2 cells in vitro; however, HEp-2 are not reflective of polarized airway epithelial cells and respond differently to RSV infection. An air-liquid interface (ALI) Calu-3 model that more closely mimics the human airway epithelium was established. Using this in vitro model, the effectiveness of LL-37 in preventing RSV infection and replication was examined. RESULTS: LL-37, when pre-incubated with virus prior to RSV infection (prophylactic), significantly reduced the level of viral genome detected in infected Calu-3 cells, and decreased chemokine expression associated with RSV infection in vitro. In contrast, therapeutic treatment of RSV-infected ALI Calu-3 at 24 h and 3 days post-infection had minimal impact on RSV infection. CONCLUSIONS: Differences in the efficacy of LL-37 at reducing RSV infection under prophylactic and therapeutic conditions may in part be ascribed to differences in the method of peptide exposure. However, the efficacy of LL-37 at reducing RSV infection under prophylactic conditions indicates that further studies examining the efficacy of LL-37 as a small peptide inhibitor of RSV are warranted.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.

Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan.

This article describes the pathological studies of fatal severe acute respiratory syndrome (SARS) in a 73-year-old man during an outbreak of SARS in Taiwan, 2003. Eight days before onset of symptoms, he visited a municipal hospital that was later identified as the epicenter of a large outbreak of SARS. On admission to National Taiwan University Hospital in Taipei, the patient experienced chest tightness, progressive dyspnea, and low-grade fever. His condition rapidly deteriorated with increasing respiratory difficulty, and he died 7 days after admission. The most prominent histopathologic finding was diffuse alveolar damage of the lung. Immunohistochemical and in situ hybridization assays demonstrated evidence of SARS-associated coronavirus (SARS-CoV) infection in various respiratory epithelial cells, predominantly type II pneumocytes, and in alveolar macrophages in the lung. Electron microscopic examination also revealed coronavirus particles in the pneumocytes, and their identity was confirmed as SARS-CoV by immunogold labeling electron microscopy. This report is the first to describe the cellular localization of SARS-CoV in human lung tissue by using a combination of immunohistochemistry, double-stain immunohistochemistry, in situ hybridization, electron microscopy, and immunogold labeling electron microscopy. These techniques represent valuable laboratory diagnostic modalities and provide insights into the pathogenesis of this emerging infection.

The pathology of rotavirus-associated deaths, using new molecular diagnostics.

Rotavirus, the most common cause of severe, dehydrating gastroenteritis among children worldwide, annually causes approximately 500,000 deaths among children aged <5 years. The primary site of rotavirus infection is the small intestine. Pathologic investigations of patients who died of rotavirus infection are limited to data from a few reported autopsies, and dehydration with electrolyte imbalance is believed to be the major cause of death. Several recent reports suggest that children who died during a rotavirus illness were viremic before death, because rotavirus was detected at several extraintestinal sites. We report 3 rotavirus-associated deaths among children, 2 of whom had evidence of rotavirus genome in extraintestinal tissues detected by use of novel molecular diagnostic methods. The part played by rotavirus in fatal cases is unclear and requires additional investigation of diarrhea-associated deaths, because a better understanding might alter the approach to treatment and the need for antiviral therapy.

Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine.

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.