Low rates of endocarditis in healthcare-associated Staphylococcus aureus bacteremia suggest that echocardiography might not always be required.

Healthcare-associated Staphylococcus aureus bacteremia (HA-SAB) is an increasingly frequently observed complication of medical treatment. Current guidelines recommend evaluation with echocardiography and preferably transesophageal echocardiography for the exclusion of infectious endocarditis (IE). We performed a retrospective analysis of all patients with HA-SAB between 1 January 2007 and 31 July 2012. Patients were divided into those with a high degree of clinical suspicion of IE (prosthetic intracardiac device, hemodialysis or positive blood cultures for 4 days or more) or those with a low degree of clinical suspicion of IE (absence of high-risk features based on previous literature as strong indicators of endocarditis). Three hundred and fifty-eight patients with HA-SAB were evaluated to determine the prevalence of IE, including 298 (83 %) who had echocardiography. Fourteen patients (4 %) had a final diagnosis of IE after echocardiography. In the group with a high degree of clinical suspicion 11 out of 84 patients (13 %) had IE. In the group with a low degree of clinical suspicion group 3 out 274 patients (1.1 %) had IE. HA-SAB has a low rate of IE, especially in the absence of high-risk features such as prolonged bacteremia, intracardiac prosthetic devices, and hemodialysis. Echocardiographic imaging in this low-risk population of patients is rarely helpful and may generally be avoided, although careful clinical follow-up is warranted. Patients with HA-SAB who have mechanical valves, intracardiac devices, prolonged bacteremia or dialysis dependency have a high incidence of IE and should be evaluated thoroughly using echocardiography.

Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Mutations in PIK3CD can cause hyper IgM syndrome (HIGM) associated with increased cancer susceptibility.

Autosomal dominant gain of function mutations in the gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, CMV and/or EBV viremia and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. Here we describe three patients in two families who were diagnosed with HIGM at a young age and were recently found to carry heterozygous mutations in PIK3CD. These patients had an abnormal circulating B cell distribution featuring a preponderance of early transitional (T1) B cells and plasmablasts. When stimulated in vitro, PIK3CD mutated B cells were able to secrete class-switched immunoglobulins. This finding implies that the patients' elevated serum IgM levels were unlikely a product of an intrinsic B cell functional inability to class switch. All three patients developed malignant lymphoproliferative syndromes that were not associated with EBV. Thus, we identified a novel subset of patients with PIK3CD mutations associated with HIGM, despite indications of preserved in vitro B cell class switch recombination, as well as susceptibility to non-EBV-associated malignancies.

Absolute myocardial blood flow determination using real-time myocardial contrast echocardiography during adenosine stress: comparison with single-photon emission computed tomography.

OBJECTIVE: To assess the feasibility and diagnostic accuracy of real-time myocardial contrast echocardiography (MCE)-derived absolute myocardial blood flow for detection of myocardial perfusion abnormalities compared with simultaneous technetium 99 m sestamibi single-photon emission computed tomography (SPECT). DESIGN: Prospective study. SETTING: Tertiary-care medical institution. PATIENTS: 79 patients with known or suspected coronary artery disease. INTERVENTIONS: Simultaneous SPECT and real-time MCE during adenosine stress. MAIN OUTCOME MEASURES: Absolute myocardial blood flow (MBF, ml/min/g), microbubble velocity (beta, min(-1)), and reserve values. Endpoints included sensitivity, specificity, positive likelihood ratio (LR+) or negative likelihood ratio (LR-) and area under the curve (AUC) of the receiver operating characteristic curve. RESULTS: Reserve measurements were feasible in 975 of 1343 segments (73%); of these, 130 segments (13%) were abnormal by SPECT. MCE perfusion parameters clearly distinguished abnormal from normal segments for beta reserve (1.13 (0.99) vs 2.22 (1.36), p<0.001) and MBF reserve (1.80 (2.29) vs 3.69 (2.79), p<0.001). The beta reserve cut-off of 1.60 provided the following: AUC, 0.787; sensitivity, 82%; specificity, 66%; LR+, 2.40; and LR-, 0.28. The MBF reserve cut-off of 1.90 provided the following: AUC, 0.779; sensitivity, 73%; specificity, 72%; LR+, 2.69; and LR-, 0.37. MBF reserve had an AUC of 0.773 for the left anterior descending coronary artery, 0.885 for the left circumflex coronary artery and 0.739 for the right coronary artery. CONCLUSIONS: Real-time MCE-derived absolute MBF, beta, and reserve values are feasible and accurate for detecting myocardial perfusion abnormalities as defined by SPECT.

Relationship between myocardial perfusion and dysfunction in diabetic cardiomyopathy: a study of quantitative contrast echocardiography and strain rate imaging.

OBJECTIVE: To use quantitative myocardial contrast echocardiography (MCE) and strain rate imaging (SRI) to assess the role of microvascular disease in subclinical diabetic cardiomyopathy. METHODS: Stress MCE and SRI were performed in 48 patients (22 with type II diabetes mellitus (DM) and 26 controls), all with normal left ventricular systolic function and no obstructive coronary disease by quantitative coronary angiography. Real-time MCE was acquired in three apical views at rest and after combined dipyridamole-exercise stress. Myocardial blood flow (MBF) was quantified in the 10 mid- and apical cardiac segments at rest and after stress. Resting peak systolic strain rate (SR) and peak systolic strain (epsilon) were calculated in the same 10 myocardial segments. RESULTS: The DM and control groups were matched for age, sex and other risk factors, including hypertension. The DM group had higher body mass index and left ventricular mass index. Quantitative SRI analysis was possible in all patients and quantitative MCE in 46 (96%). The mean epsilon, SR and MBF reserve were all significantly lower in the DM group than in controls, with diabetes the only independent predictor of each parameter. No correlation was seen between MBF and SR (r = -0.01, p = 0.54) or between MBF and epsilon (r = -0.20, p = 0.20). CONCLUSIONS: Quantitative MCE shows that patients with diabetes but no evidence of obstructive coronary artery disease have impaired MBF reserve, but abnormal transmural flow and subclinical longitudinal myocardial dysfunction are not related.

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21(high)- and CD21(low)-expressing B cells, the CD21(low) fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21(high) fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21(low) B cell population but not in the CD21(high) fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

Suppression of HIV replication in the resting CD4+ T cell reservoir by autologous CD8+ T cells: implications for the development of therapeutic strategies.

CD8+ T cell-mediated antiviral activity against HIV has been described consistently in infected individuals; however, the role of this activity in controlling replication of HIV in the latently infected, resting CD4+ T cell reservoir is unclear. By using an ex vivo system, we show that replication of HIV in this viral reservoir is effectively suppressed in coculture by autologous CD8+ T cells in long-term nonprogressors (LTNPs) and in patients whose viremia was controlled by highly active antiretroviral therapy (HAART), but not in therapy-naive patients who had substantial levels of plasma viremia. This antiviral activity was largely independent of cytotoxic CD8+ T lymphocytes (CTL). When the role of soluble CD8+ T cell-derived factors was examined, we found that CC-chemokines played a major role in inhibition of viral replication in the latent viral reservoir in some LTNPs and patients receiving HAART, but not in chronically infected patients who were not receiving antiretroviral therapy. Potent antiviral activity, independent of CC-chemokines, was found mainly in patients in whom HAART was initiated shortly after the acute phase of HIV infection. These results indicate that CD8(+) T cells provide potent suppressive activity against HIV replication in the latent viral reservoir via direct cellular contact in patients who are naturally LTNPs or in those who are treated with HAART. Furthermore, the profound antiviral activity exerted by non-CC-chemokine soluble factors in infected patients who began HAART early in HIV infection suggests that preservation of this HIV-suppressive mechanism by early initiation of therapy may play an important role in the containment of viral replication in infected patients following interruption of therapy.

Detection of HIV DNA and RNA using PCR.

This extensively updated unit describes the detection of HIV DNA and RNA using PCR using two basic techniques for quantifying the levels of viral DNA and RNA in infected cells. The schemes for both techniques include isolation of nucleic acids, PCR reactions and detection of amplified products using Southern blotting.

B cells of HIV-1-infected patients bind virions through CD21-complement interactions and transmit infectious virus to activated T cells.

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4(+) cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.

CD40-Mediated induction of CD4 and CXCR4 on B lymphocytes correlates with restricted susceptibility to human immunodeficiency virus type 1 infection: potential role of B lymphocytes as a viral reservoir.

Human immunodeficiency virus type 1 (HIV-1) replicates primarily in lymphoid tissues where it has ready access to activated immune competent cells. We used one of the major pathways of immune activation, namely, CD40-CD40L interactions, to study the infectability of B lymphocytes isolated from peripheral blood mononuclear cells. Highly enriched populations of B lymphocytes generated in the presence of interleukin-4 and oligomeric soluble CD40L upregulated costimulatory and activation markers, as well as HIV-1 receptors CD4 and CXCR4, but not CCR5. By using single-round competent luciferase viruses complemented with either amphotropic or HIV-derived envelopes, we found a direct correlation between upregulation of HIV-1 receptors and the susceptibility of the B lymphocytes to infection with dual-tropic and T-tropic strains of HIV-1; in contrast, cells were resistant to M-tropic strains of HIV-1. HIV-1 envelope-mediated infection was completely abolished with either an anti-CD4 monoclonal antibody or a peptide known to directly block CXCR4 usage and partially blocked with stromal cell-derived factor 1, all of which had no effect on the entry of virus pseudotyped with amphotropic envelope. Full virus replication kinetics confirmed that infection depends on CXCR4 usage. Furthermore, productive cycles of virus replication occurred rapidly yet under most conditions, without the appearance of syncytia. Thus, an activated immunological environment may induce the expression of HIV-1 receptors on B lymphocytes, priming them for infection with selective strains of HIV-1 and allowing them to serve as a potential viral reservoir.

CC-chemokines enhance the replication of T-tropic strains of HIV-1 in CD4(+) T cells: role of signal transduction.

This study demonstrates that several CC-chemokines, including those that inhibit entry and replication of macrophage-tropic strains of HIV, increase the replication of T cell (T)-tropic strains in CD4(+) T cells. Enhancement of T-tropic HIV replication is observed at early stages of replication, requires signaling through inhibitory guanine nucleotide-binding regulatory (Gi) proteins, and is associated with increased cell surface colocalization of CD4 and the T-tropic HIV coreceptor CXCR4. These findings may further our understanding of the factors that influence the replication and spread of T-tropic strains of HIV in vivo and suggest that the use of cell signaling CC-chemokines as therapeutic agents for the purpose of limiting HIV replication in vivo should be approached with caution.

Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro.

Macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell expressed and secreted), which are the natural ligands of the CC-chemokine receptor CCR5, inhibit replication of MT-2- negative strains of HIV-1 by interfering with the ability of these strains to utilize CCR5 as a coreceptor for entry in CD4(+) cells. The present study investigates the capacity of natural killer (NK) cells isolated from HIV-infected individuals to produce CC-chemokines and to suppress HIV replication in autologous, endogenously infected cells as well as to block entry of MT-2-negative HIV into the CD4(+) T cell line PM-1. NK cells freshly isolated from HIV-infected individuals had a high number of mRNA copies for MIP-1alpha and RANTES. NK cells produced significant amounts of RANTES, MIP-1alpha, and MIP-1beta constitutively, in response to stimulation with IL-2 alone and when they were performing their characteristic lytic activity (K562 killing). After CD16 cross-linking and stimulation with IL-2 or IL-15 NK cells produced CC-chemokines to levels comparable to those produced by anti-CD3-stimulated CD8(+) T cells. Furthermore, CD16 cross-linked NK cells suppressed (49-97%) viral replication in cocultures of autologous CD8/NK-depleted PBMC to a degree similar to that of PHA or anti-CD3-stimulated CD8(+) T cells. In 50% of patients tested, NK-mediated HIV suppression could be abrogated by neutralizing antibodies to MIP-1alpha, MIP-1beta and RANTES; in contrast, CD8(+) T cell-mediated suppression was not significantly overcome upon neutralization of CC-chemokines. Supernatants derived from cultures of CD16 cross-linked NK cells stimulated with IL-2 or IL-15 dramatically inhibited entry of a MT-2-negative strain of HIV, BaL, in the CD4(+)CCR5(+) PM-1 T cell line. These data suggest that activated NK cells may be an important source of CC-chemokines in vivo and may suppress HIV replication by CC-chemokine-mediated mechanisms in addition to classic NK-mediated lytic mechanisms.

CD4 deletion mutants evaluated for human immunodeficiency virus type 1 infectivity in a highly efficient system of expression and detection based on LTR-dependent reporter gene activation.

The human CD4 glycoprotein is thought to be involved at several stages of the infection process with the human immunodeficiency virus type 1. To pursue this line of investigation with CD4 deletion mutants, we combined a system of high transient cell-surface expression of the target molecule with an assay of HIV-1 infectivity based on induction of LTR-linked luciferase activity. The approach was also designed to distinguish between defects in gp120 binding and postbinding events. Optimal assay conditions were established with wild-type CD4 and the previously characterized CD4 mutant, d367-371. New deletions of CD4 domains D3 and D4 were then designed from a rat model of the D3D4 atomic coordinates with the concern of maintaining overall structural integrity. While all CD4 mutants were found to be defective towards HIV, it was demonstrated that the mutations affected different stages of the entry process. These data indicate that the system is well suited for studying the intricacy of molecular interactions involving HIV envelope glycoproteins and its receptors.

Postbinding events mediated by human immunodeficiency virus type 1 are sensitive to modifications in the D4-transmembrane linker region of CD4.

Evidence from both structural and functional studies of the CD4 molecule suggests that several domains, including the transmembrane (TM) domain and the adjoining extracellular region (D4-TM linker), contribute to the post-gp12O-binding events leading to human immunodeficiency virus-mediated membrane fusion. To investigate such a role in syncytium formation and cell-free infectivity, we generated several deletion and substitution mutations in the TM and D4-TM linker regions of the CD4 molecule. We found that while the TM domain of CD4 was dispensable for cell-cell and virus-cell interactions, modifications in the D4-TM linker led to perturbations in both processes. Deletion of the five amino acid residues linking D4 to the TM domain resulted in a delayed and reduced capacity to form syncytia, whereas replacement of the residues with the heterologous sequence from the CD8 molecule restored the kinetic profile to wild-type CD4 levels. On the other hand, both mutants of the CD4 D4-TM linker demonstrated delayed cell-free human immunodeficiency virus type 1 infectivity profiles. The defective fusion capacity may be linked to structural perturbations identified with anti-CD4 monoclonal antibodies in the D1-D2 interface and D3 domain of the deletion mutant yet absent in D1 and D4. While all cells were found to bind comparable levels of gp120, both D4-TM linker mutants appeared to induce a decrease in the V3 loop exposure of bound gp120. This underexposure may explain the delays in cell-free infectivities observed for both of these mutants. Together, these findings confirm a role for regions of the CD4 molecule located outside D1 in post-gp120-binding events and suggest that the D4-TM interface contributes to the conformational changes that direct the fusion process.

PATH: a work sampling-based approach to ergonomic job analysis for construction and other non-repetitive work.

A high prevalence and incidence of work-related musculoskeletal disorders have been reported in construction work. Unlike industrial production-line activity, construction work, as well as work in many other occupations (e.g. agriculture, mining), is non-repetitive in nature; job tasks are non-cyclic, or consist of long or irregular cycles. PATH (Posture, Activity, Tools and Handling), a work sampling-based approach, was developed to characterize the ergonomic hazards of construction and other non-repetitive work. The posture codes in the PATH method are based on the Ovako Work Posture Analysing System (OWAS), with other codes included for describing worker activity, tool use, loads handled and grasp type. For heavy highway construction, observations are stratified by construction stage and operation, using a taxonomy developed specifically for this purpose. Observers can code the physical characteristics of the job reliably after about 30 h of training. A pilot study of six construction laborers during four road construction operations suggests that laborers spend large proportions of time in nonneutral trunk postures and spend approximately 20% of their time performing manual material handling tasks. These results demonstrate how the PATH method can be used to identify specific construction operations and tasks that are ergonomically hazardous.

Expression of HIV env gene in a human T cell line for a rapid and quantifiable cell fusion assay.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins present at the surface of infected cells are known to mediate fusion with CD4-positive target cells. In this study we have developed a novel Env-expressing cell line for investigating the fusion process in a biologically significant system. Cell surface expression of the HIV-1 env gene, isolated from the highly fusogenic strain SF33, was obtained in the CD4-negative T cell line A2.01. To render the system versatile and efficient, HIV-1 regulatory proteins Tat and Rev were supplied in trans. The presence of Env at the cell surface was shown by cytofluorometry and immunofluorescence and precursor processing of gp160 to gp120/gp41 was demonstrated by Western blot. The fusion capacity of A2.01-Env cells was assessed by coculture with CD4-positive T lymphocytes or the fusion indicator cell line, HeLa-CD4-LTR-beta-Gal. By coincubation with CD4-positive T cells such as SupT1, A2.01-Env cells were observed to mediate rapidly numerous well-defined syncytia in a reproducible fashion. By expressing Tat, they also had the capacity to trans-activate the LTR-linked reporter beta-Gal gene following fusion with HeLa-CD4-LTR-beta-Gal cells. The fusion-inhibiting anti-CD4 monoclonal antibodies Q425 and Q428 were used to block specifically Env-mediated fusion with CD4-positive cells and to demonstrate application of this system to the search for potential fusion-blocking agents. Our system thus offers a biologically significant model for studying fusion events with the advantages of being rapid, reproducible and versatile.

Emerging participatory approaches to ergonomic interventions in the construction industry.

The Central Artery/Third Harbor Tunnel (CA/T) construction project in Boston is the largest and most costly highway construction project ever undertaken in the United States. The Construction Occupational Health Project (COHP) of the Work Environment Department at the University of Massachusetts-Lowell was established to conduct exposure assessment and to develop, introduce, and evaluate interventions in the areas of ergonomics and industrial hygiene on the CA/T project. For both political and practical reasons, COHP is using a participatory approach to intervention in the construction industry. The research process is employing participation at all the levels of the construction hierarchy in the form of various advisory groups. These advisory groups have been formed from existing joint labor-management advisory committees and are presently engaged in two participatory intervention activities: (1) evaluations of intervention ideas, and (2) comparisons of safety systems.

Comparison of the p125 coding region of bovine viral diarrhea viruses.

The p125 (p54/p80) coding region of two cytopathic (CP) strains (Oregon and Singer) and two noncytopathic (NCP) strains (NY-1 and Draper) of bovine viral diarrhea virus (BVDV) were amplified by the polymerase chain reaction, cloned and sequenced. The sequence data confirmed that the two CP strains do not possess any insertion or deletion in their p125 gene as observed in many other CP strains. In the p80, which showed a high amino acid sequence homology among all strains, no amino acid substitution should could be found which distinguished these CP strains from the NCP strains NY-1 and SD-1. Many amino acid substitutions were found in p54 but their individual importance in the CP phenotype is not clear since critical domains of p54 have not yet been experimentally defined. The p54 protein is much less conserved than p80, and sequence homology, as well as dendrogram analysis, permitted us to distinguish two genotypic groups of BVDV (Ia and Ib). The mean homology between strains of these two groups was 77.3/80.4% for the nucleic acid/amino acid sequences while it was 88.0/88.8% and 91.6/93.3% within groups Ia and Ib, respectively. Furthermore, we found that the p125 sequence of our NY-1 strain showed only 92% sequence homology with the partial p80 gene reported for NY-1 but 99.8% homology with another partial sequence of the p125 gene of NY-1 reported elsewhere. These observations underscored the difficulty of maintaining a specific BVDV strain, especially the NCP biotype, in cell cultures.