Detection of IFN-γ Secretion in Blood Samples Collected Before and After Treatment of Varying Stages of Lyme Disease.

BACKGROUND: QuantiFERON enzyme-linked immunosorbent assay (ELISA; Qiagen) with Borrelia burgdorferi peptide antigens was previously shown to reliably detect interferon-γ (IFN-γ) in blood samples from adult patients with early Lyme disease and the response disappeared rapidly after treatment. We evaluated the response before and after appropriate antibiotic therapy in adolescent and adult subjects with more diverse stages of the illness. METHODS: Blood was obtained from patients with clinician-identified Lyme disease with constitutional complaints, erythema migrans, nerve palsy, cardiac abnormality, or arthritis before (n = 68) and 6 weeks (n = 46) and 6 months (n = 45) after therapy. The sera were tested for Lyme disease by standard 2-tiered testing (STTT) and anti-C6 antibodies by ELISA and the levels of IFN-γ in the blood samples were detected by QuantiFERON ELISA. RESULTS: A positive STTT result supported the clinical diagnosis of 37 (54%) subjects and anti-C6 antibodies were detected in 45 (66%) subjects, including 36 (97%) STTT-positive subjects, and the responses often persisted or expanded after antibiotic therapy. IFN-γ was detected in 49 (72%) subjects prior to treatment and the response most often significantly decreased 6 weeks (P = .007) or 6 months (P = .001) after treatment. CONCLUSIONS: The QuantiFERON ELISA reliably detected IFN-γ in blood samples from adult and adolescent patients with varying stages of Lyme disease and the response disappeared rapidly after treatment. Additional studies to more critically evaluate clinical utility as a laboratory test for diagnosis and confirmation of effective therapy are warranted.

Myocarditis in Multisystem Inflammatory Syndrome in Children Associated With Coronavirus Disease 2019.

Cardiac involvement as a complication of severe acute respiratory syndrome coronavirus 2 infection in children is a relatively new entity. We present our initial experience managing children with coronavirus disease 2019-related acute myocardial injury. The 3 patients presented here represent a spectrum of the cardiac involvement noted in children with coronavirus disease 2019-related multisystem inflammatory syndrome, including myocarditis presenting as cardiogenic shock or heart failure with biventricular dysfunction, valvulitis, coronary artery changes, and pericardial effusion.

Mycoplasma genitalium as a Cause of Pelvic Inflammatory Disease.

BACKGROUND: Mycoplasma genitalium is an increasingly recognized cause of pelvic inflammatory disease (PID). CASE: A 17-year-old female adolescent presented with chronic pelvic pain and dysmenorrhea. Test results for Chlamydia trachomatis and Neisseria gonorrhea were negative, and multiple radiologic test results were normal. The patient failed several empiric courses of therapy over 1 year. Laparoscopy revealed findings consistent with PID. Nucleic acid amplification test results were positive for M genitalium. SUMMARY AND CONCLUSION: M genitalium causing PID can be challenging to diagnose because of its often atypical presentation. Further epidemiological studies are needed to understand the burden of disease and to establish testing and treatment guidelines.

Biennial Upsurge and Molecular Epidemiology of Enterovirus D68 Infection in New York, USA, 2014 to 2018.

Enterovirus D68 (EV-D68) infection has been associated with outbreaks of severe respiratory illness and increased cases of nonpolio acute flaccid myelitis. The patterns of EV-D68 circulation and molecular epidemiology are not fully understood. In this study, nasopharyngeal (NP) specimens collected from patients in the Lower Hudson Valley, New York, from 2014 to 2018 were examined for rhinovirus/enterovirus (RhV/EV) by the FilmArray respiratory panel. Selected RhV/EV-positive NP specimens were analyzed using two EV-D68-specific real-time RT-PCR assays, Sanger sequencing and metatranscriptomic next-generation sequencing. A total of 2,398 NP specimens were examined. EV-D68 was detected in 348 patients with NP specimens collected in 2014 (n = 94), 2015 (n = 0), 2016 (n = 160), 2017 (n = 5), and 2018 (n = 89), demonstrating a biennial upsurge of EV-D68 infection in the study area. Ninety-one complete or nearly complete EV-D68 genome sequences were obtained. Genomic analysis of these EV-D68 strains revealed dynamics and evolution of circulating EV-D68 strains since 2014. The dominant EV-D68 strains causing the 2014 outbreak belonged to subclade B1, with a few belonging to subclade B2. New EV-D68 subclade B3 strains emerged in 2016 and continued in circulation in 2018. Clade D strains that are rarely detected in the United States also arose and spread in 2018. The establishment of distinct viral strains and their variable circulation patterns provide essential information for future surveillance, diagnosis, vaccine development, and prediction of EV-D68-associated disease prevalence and potential outbreaks.

Incidence and prevalence of common respiratory pathogens before and after implementation of the Cystic Fibrosis Foundation Infection Prevention and Control Guideline.

BACKGROUND: The 2013 Cystic Fibrosis Foundation's Infection Prevention and Control Guideline (CFF IP&C) was developed to reduce the risk of acquisition and transmission of respiratory pathogens in patients with cystic fibrosis (CF). OBJECTIVE: We hypothesised that the incidence of common CF respiratory pathogens would decrease at our centre after implementation of the guideline. METHODS: All patients with CF seen at our centre from August 2012 through August 2017 who had respiratory cultures were included. Patients were excluded from incidence analysis if they did not have at least one culture per year. Quarterly data were collected for one year before and three years after implementation of the guidelines to determine the incidence and prevalence of seven organisms commonly found in respiratory cultures of patients with CF. RESULTS: Quarterly and annual incidence and prevalence rates of common organisms did not change during the study period. DISCUSSION: There were no significant differences in the incidence or prevalence of common respiratory organisms in the first three years after implementation of the CF IP&C guideline. Long-term follow-up is needed to determine if changes occur over time.

A Longitudinal Epidemiology Study of Meningococcal Carriage in Students 13 to 25 Years Old in Quebec.

Neisseria meningitidis carriage data are necessary to inform serogroup B (NmB) immunization program implementation. This longitudinal study compared detection methods to measure N. meningitidis throat carriage prevalence in Quebec from November 2010 to December 2013 using cultured swab isolates and direct swab PCR from students in ninth grade (aged 13 to 15 years; n = 534) and eleventh grade/college entry (16 to 18 years; n = 363) and in university students in dormitories (18 to 25 years; n = 360) at 3 time points per group. Meningococcal and NmB carriage rates were lower in ninth- and eleventh-grade/college entry students than university students, regardless of methodology. Genotyping cultured isolates by PCR detected NmB and non-NmB in 2.1% and 7.3% of ninth-grade students, in 1.7% and 7.2% of eleventh-grade/college entry students, and in 7.5% and 21.9% of university students, respectively. NmB acquisition rates were 1.9, 0.7, and 3.3 per 1,000 person-months across respective age groups. Most NmB isolates (94.7%, 76.9%, and 86.8%, respectively) expressed subfamily A factor H binding-protein (fHBP) variants. The most common non-NmB serogroups were NmY (1.7%/1.1%) from ninth grade and eleventh grade/college entry and NmW (2.8%) from university students. Genomic analyses detected disease-associated sequence types in carriage isolates, and carriage could persist for months. This is the largest longitudinal carriage study in Canada and the first to report fHBP variants in NmB carriage isolates in healthy Canadians. These data contribute to identification of the optimal window for NmB vaccination in precollege adolescents and provide a baseline for investigating NmB vaccination effects on carriage in this population.IMPORTANCE Disease caused by Neisseria meningitidis is associated with serious complications and a high fatality rate. Asymptomatic individuals can harbor the bacterium in the throat, a state known as "carriage," which can lead to person-to-person spread of the pathogen. This study examined N. meningitidis carriage from 2010 to 2013 among 2 groups in the Quebec City region: ninth-grade students (aged 13 to 15 years), who were also followed in their last year of high school (eleventh grade/college entry; 16 to 18 years), and university students (18 to 25 years); both groups have been shown in some other geographic regions to have high rates of carriage. This study demonstrated that N. meningitidis carriage rates were higher among university students in dormitories than ninth-grade and eleventh-grade/college entry students. Understanding carriage rates in these age groups leads to better strategies to control N. meningitidis by targeting vaccination to those responsible for transmission within the population.

Comparison of the Severity of Respiratory Disease in Children Testing Positive for Enterovirus D68 and Human Rhinovirus.

OBJECTIVE: To compare the characteristics and severity of respiratory disease in children testing positive for enterovirus D68 (EV-D68) and for human rhinovirus (RhV). STUDY DESIGN: A retrospective single center study of children presenting with acute respiratory symptoms and positive polymerase chain reaction for RhV/EV from September 1, 2014 through October 31, 2014 was performed. Specimens were subsequently tested specifically for EV-D68 and specimens identified as RhV were subtyped when possible into RhV-A, RhV-B, and RhV-C species. Clinical manifestations in patients with EV-D68 were compared with those with non-EV-D68, RhV, and RhV-C. RESULTS: Of the 173 patients included in the analysis, 72 tested positive for EV-D68, 61 for RhV, and 30 for RhV-C. There were significantly fewer infants in the EV-D68 group. Patients with EV-D68 were more likely than those without EV-D68, and specifically with RhV-C, to have fever and wheezing. Patients with EV-D68 received more magnesium sulfate for respiratory distress not responding adequately to repeated doses of inhaled albuterol. Hospitalized patients with EV-D68 received more bronchodilator therapy than patients with RhV. Patients with EV-D68 were more likely to be admitted to the intensive care unit and were older than patients without EV-D68. There was no difference in length of overall hospitalization or time in the pediatric intensive care unit. CONCLUSIONS: Children with EV-D68 appeared to have more severe respiratory disease on admission than children with RhV as evidenced by higher rates of fever, wheezing, bronchodilator use and pediatric intensive care unit admission. Despite the initial difference in severity, no significant difference in length of stay was found suggesting that patients with EV-D68 recovered as quickly as other groups.

Congenital Babesiosis After Maternal Infection With Borrelia burgdorferi and Babesia microti.

We describe the cases of 2 infants with congenital babesiosis born to mothers with prepartum Lyme disease and subclinical Babesia microti infection. The infants both developed anemia, neutropenia, and thrombocytopenia, and 1 infant required red blood cell transfusion. Both infants recovered with treatment. Additional studies are warranted to define the optimal management strategy for pregnant women with early Lyme disease in geographic areas in which B microti infection is endemic.

Enterovirus D68 Subclade B3 Strain Circulating and Causing an Outbreak in the United States in 2016.

In 2014 the United States experienced a nationwide outbreak of Enterovirus D68 (EV-D68) infection. There were no confirmed cases of EV-D68 in 2015 and CDC was only aware of limited sporadic EV-D68 detection in the US in 2016. In this report, we analyzed 749 nasopharyngeal (NP) specimens collected in 2015 and 2016 from patients in the Lower Hudson Valley, New York using a previously validated EV-D68-specific rRT-PCR assay. EV-D68 was detected in none of 199 NP specimens collected in 2015, and in one of 108 (0.9%) samples from January to May and 159 of 442 (36.0%) samples from July to October 2016. Complete EV-D68 genome sequences from 22 patients in 2016 were obtained using a metagenomic next-generation sequencing assay. Comparative genome analysis confirmed that a new EV-D68 strain belonging to subclade B3, with 3.2-4.8% divergence in nucleotide from subclade B1 strains identified during the 2014 US outbreak, was circulating in the US in 2016 and caused an outbreak in the Lower Hudson Valley, New York with 160 laboratory-confirmed cases. Our data highlight the genetic variability and capacity in causing outbreak by diverse EV-D68 strains, and the necessity of awareness and more surveillance on their active circulation worldwide.

Complete Genome Sequences of Nine Enterovirus D68 Strains from Patients of the Lower Hudson Valley, New York, 2016.

Complete genome sequences of nine enterovirus D68 (EV-D68) strains from patients in New York were obtained in 2016 by metagenomic next-generation sequencing. Comparative genomic analysis suggests that a new subclade B3, with ~4.5% nucleotide divergence from subclade B1 strains causing the 2014 outbreak, is circulating in the United States in 2016.

Assessing next-generation sequencing and 4 bioinformatics tools for detection of Enterovirus D68 and other respiratory viruses in clinical samples.

We used 4 different bioinformatics algorithms to evaluate the application of a metagenomic shot-gun sequencing method in detection of Enterovirus D68 and other respiratory viruses in clinical specimens. Our data supported that next-generation sequencing, combined with improved bioinformatics tools, is practically feasible and useful for clinical diagnosis of viral infections.

Whole-Genome Sequence Analysis Reveals the Enterovirus D68 Isolates during the United States 2014 Outbreak Mainly Belong to a Novel Clade.

In the late summer and the fall of 2014, the United States experienced an unprecedented outbreak of enterovirus D68 (EV-D68) infections. During the outbreak, we collected nasopharyngeal swab specimens from patients in the Lower Hudson Valley of New York. Here, we conduct a retrospective study on the genomic diversity of EV-D68 strains. We first employ a metagenomic shotgun sequencing protocol on a total of 93 clinical samples, including 21 negative controls, the results of which allow assembly of 20 EV-D68 genomes, six complete and 14 near-complete. We then investigate their genetic relationships, along with additional 20 EV-D68 strains having whole-genome sequences publicly available. Our comparative genomic analysis uncovers that the majority (26/29) of EV-D68 strains circulating in the 2014 outbreak differ significantly from prior ones, have a main feature of three variables, C1817T, C3277A, and A4020G, and belong to a new clade. C3277A causes amino acid substitution T860N in the protease 2A(pro) cleavage site between VP1 and 2A, whereas A4020G causes S1108G in a 3C(pro) cleavage site between 2B and 2C. The two functional mutations may alter the proteases' cleavage efficiency, leading to increased rate of viral replication and transmission. These provide insights into the evolution of epidemic EV-D68 strains.

Neutropenia in Congenital and Adult Babesiosis.

OBJECTIVES: Anemia and thrombocytopenia are expected hematologic abnormalities in patients with acute babesiosis, whereas neutropenia (defined as an absolute neutrophil count of ≤1,800 neutrophils/µL for adults and <1,200 neutrophils/µL for infants) is not usually considered a feature of this infection. We studied the frequency with which neutropenia occurs in congenital and adult cases of babesiosis. METHODS: The frequency of neutropenia in cases of congenital babesiosis was determined based on a literature review and on the findings in an unreported case. The frequency of neutropenia in adult patients was assessed based on a review of the medical records of 51 patients who were diagnosed with babesiosis between 2010 and 2013 at two medical centers in the Northeastern United States. RESULTS: Four (80%; 95% confidence interval [CI], 36%-98%) of five infants with congenital babesiosis whose neutrophil count was reported were neutropenic. Among 51 adult cases with babesiosis, 11 (22%; 95% CI, 12%-35%) were neutropenic on clinical presentation, and seven others developed neutropenia over the next 1 to 21 days. Thus, a total of 18 (35%; 95% CI, 24%-49%) of the adult patients with babesiosis had neutropenia. CONCLUSIONS: Neutropenia appears to be a common finding in infants with congenital babesiosis and is also observed not infrequently in adults with this infection.

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014.

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5' untranslated region (5'-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

Effect of rotavirus vaccine on reducing acute gastroenteritis in a large outpatient pediatric network.

OBJECTIVES: To measure the effect of rotavirus vaccine (RVV) on acute gastroenteritis (AGE) managed by primary care physicians in the first 2 rotatvirus seasons following the introduction of RVV. DESIGN: Retrospective cohort study. SETTING: Practice-based network composed of 33 ambulatory pediatric practices in Philadelphia, Pennsylvania. PARTICIPANTS: All children born from February 22, 2006 (date of RVV licensure), through February 29, 2008, and who received care at any network site. MAIN EXPOSURE: Receipt of RVV. OUTCOME MEASURES: The primary outcomes were AGE-related office visits, telephone calls, and episodes (composite outcome consisting of all visits and calls within a 10-day period). RESULTS: Rates of AGE visits in the 2 rotavirus seasons following the introduction of RVV steadily decreased from 3.0 AGE visits per 100 total office visits in the 2005 season to 1.8 in the 2008 season. In 2007, vaccinations were administered to 9351 of 13 951 vaccine-eligible children (67.0%), and in 2008, they were administered to 9958 of 10 728 (92.8%). Among RVV-immunized children in 2007, AGE calls and episodes were significantly reduced with vaccine effectiveness of 53% and 46%, respectively. No significant difference was seen between RVV-immunized and RVV-nonimmunized children for any outcome in 2008. CONCLUSIONS: Rotavirus vaccine was associated with a significant reduction in outpatient AGE calls and episodes among immunized children in our network in 2007. Despite a reduction in winter AGE rates in the network, no difference was detected between RVV-immunized and RVV-nonimmunized children for any outcome in 2008. Further study is needed to understand the lack of vaccine effect in 2008.

Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates.

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-ß induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-ß induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.

Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates.

The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.

Outbreak of vancomycin-resistant enterococcus colonization among pediatric oncology patients.

OBJECTIVE: To detect the burden of vancomycin-resistant Enterococcus (VRE) colonization among pediatric oncology patients and to determine risk factors for VRE acquisition. DESIGN: Retrospective case-control study. SETTING: The Children's Hospital of Philadelphia. PATIENTS: Pediatric oncology patients hospitalized from June 2006 through December 2007. METHODS: Prevalence surveys revealed an increased VRE burden among pediatric oncology patients. For the case-control study, the 16 case patients were pediatric oncology patients who had 1 stool sample negative for VRE at screening before having a stool sample positive for VRE at screening; the 62 control patients had 2 consecutive screenings in which stool samples were negative for VRE. Case and control patients were matched on the duration of the interval between screens. Analyses were performed to determine the association between multiple exposures and VRE acquisition. RESULTS: The prevalence survey revealed that 5 (9.6%) of 52 patients had unsuspected VRE colonization at the time of hospitalization. Multivariate analysis identified a lack of empirical contact precautions (odds ratio [OR], 17.16 [95% confidence interval {CI}, 1.49-198.21]; P= .02) and the presence of a gastrointestinal device (OR, 4.03 [95% CI, 1.04-15.56]; P= .04) as significant risk factors for acquisition of VRE. Observations in the interventional radiology department revealed that staff could not access the portions of the electronic medical record in which isolation precautions were documented. Simple interventions that granted access and that trained interventional radiology staff to review the need for precautions, coupled with enhanced infection control practices, interrupted ongoing transmission and reduced the proportion of VRE screens that were positive to 15 (1.2%) of 1,270. CONCLUSIONS: Inadequate communication with regard to infection control precautions can increase the risk of transmission of epidemiologically important organisms, particularly when patients receive care at multiple clinic locations. Adherence to infection control practices across the spectrum of care may limit the spread of resistant organisms.

Methicillin-resistant Staphylococcus aureus as a cause of extensive retropharyngeal abscess in two infants.

We report 2 cases of extensive methicillin-resistant Staphylococcus aureus (MRSA) retropharyngeal abscesses in young infants. In 1 case, the abscess was associated with a reactive thrombosis of the jugular vein. Based on the existing literature and the rapid emergence of MRSA skin and soft tissue infections, it is possible that similar severe infections will occur with increasing frequency in young infants.

Recombinant human parainfluenza virus type 2 vaccine candidates containing a 3' genomic promoter mutation and L polymerase mutations are attenuated and protective in non-human primates.

Previously, we identified several attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and genetically stabilized those mutations using reverse genetics [Nolan SM, Surman S, Amaro-Carambot E, Collins PL, Murphy BR, Skiadopoulos MH. Live-attenuated intranasal parainfluenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses. Vaccine 2005;39(23):4765-74]. Here we describe the discovery of an attenuating mutation at nucleotide 15 (15(T-->C)) in the 3' genomic promoter that was also present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (Delta1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15(T-->C) mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the Delta1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/460A/948L and rV94(15C)/948L/Delta1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans.