Maintenance of surrogate light chain expression induces developmental delay in early B cell compartment.

The production of a mature B cell requires passage through a number of developmental checkpoints. The pre-BCR plays a critical role in passage through the pro-B cell/pre-B cell checkpoint, and thus plays a central role in regulating the differentiation of a B cell. Due to the significance of this receptor, it is imperative that pre-BCR expression and function are precisely regulated. In this study, we have investigated a system in which the regulation of the pre-BCR is altered. We have found that continued expression of components of the pre-BCR (lambda5) resulted in a delay in the kinetics of B cell maturation. Pro-B cells from normal mouse bone marrow retrovirally infected with lambda5 exhibited a delay in differentiation. As compared with wild-type cells at the same time point, there is a reduction in the presence of cell surface markers that indicate developmental progression, and there is a 6- to 16-fold decrease in the production of Ig-positive cells in B cell maturation assays. The capacity to alter B cell progression by modifying and extending pre-BCR expression argues that the receptor and its associated signals play a unique role in directing developmental outcomes.

Validation of a microsphere-based immunoassay for detection of anti-West Nile virus and anti-St. Louis encephalitis virus immunoglobulin m antibodies.

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis.

Second human case of Cache Valley virus disease.

We document the second known case of Cache Valley virus disease in a human. Cache Valley virus disease is rarely diagnosed in North America, in part because laboratories rarely test for it. Its true incidence, effect on public health, and full clinical spectrum remain to be determined.

Nucleic acid amplification assays for detection of La Crosse virus RNA.

We report the development of nucleic acid sequence-based amplification (NASBA) and quantitative real-time reverse transcription (RT)-PCR assays for the detection of La Crosse (LAC) virus in field-collected vector mosquito samples and human clinical samples. The sensitivities of these assays were compared to that of a standard plaque assay in Vero cells. The NASBA and quantitative real-time RT-PCR assays demonstrated sensitivities greater than that of the standard plaque assay. The specificities of these assays were determined by testing a battery of reference strain viruses, including representative strains of LAC virus and other arthropod-borne viruses. Additionally, these assays were used to detect LAC viral RNA in mosquito pool samples and human brain tissue samples and yielded results within less than 4 h. The NASBA and quantitative real-time RT-PCR assays detect LAC viral RNA in a sensitive, specific, and rapid manner; these findings support the use of these assays in surveillance and diagnostic laboratory systems.

St. Louis encephalitis in early infancy.

We describe a case of St. Louis encephalitis in a 19-day-old infant who presented with fever and seizure activity. To our knowledge, this is the youngest case of St. Louis encephalitis ever reported.

Evaluation of a diagnostic algorithm using immunoglobulin M enzyme-linked immunosorbent assay to differentiate human West Nile Virus and St. Louis Encephalitis virus infections during the 2002 West Nile Virus epidemic in the United States.

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was > or =1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.

Occlusive vasculitis in a patient with concomitant West Nile virus infection.

PURPOSE: To describe a patient with occlusive, retinal vasculitis and concomitant, confirmed, acute West Nile virus (WNV) infection. DESIGN: Observational case report. METHODS: Main outcome measures included comprehensive ophthalmic examination with fluorescein angiography, color photography, and serologic testing for WNV and St. Louis encephalitis (SLE) virus including plaque reduction neutralization testing (PRNT). RESULTS: A 46-year-old woman developed a sudden decrease in vision in her left eye 2 weeks after confirmed WNV infection and demonstrated multiple, small, patchy areas of retinal edema with scattered microaneurysms. Fluorescein angiography showed multiple branch artery occlusions with extensive nonperfusion. Serologic titers for WNV were positive for acute infection. Plaque reduction neutralization testing confirmed WNV infection and excluded St. Louis encephalitis virus infection. Other etiologies of occlusive vasculitis were not present. CONCLUSIONS: Occlusive, retinal vasculitis may occur in the setting of acute WNV infection.

Selection of Ig mu heavy chains by complementarity-determining region 3 length and amino acid composition.

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring V(H)81X and J(H)4 gene segments from J(H)(+/-) and lambda5(-/-)mice. V(H)81X-J(H)4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.

Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination.

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.

Persistence of virus-reactive serum immunoglobulin m antibody in confirmed west nile virus encephalitis cases.

Twenty-nine laboratory-confirmed West Nile virus (WNV encephalitis patients were bled serially so that WNV-reactive immunoglobulin (Ig) M activity could be determined. Of those patients bled, 7 (60%) of 12 had anti-WNV IgM at approximately 500 days after onset. Clinicians should be cautious when interpreting serologic results from early season WNV IgM-positive patients.

Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs.

Use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States.

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.