Alison J. Johnson

Epidemic West Nile encephalitis, New York, 1999: results of a household-based seroepidemiological survey

BACKGROUND In the summer of 1999, West Nile virus was recognised in the western hemisphere for the first time when it caused an epidemic of encephalitis and meningitis in the metropolitan area of New York City, NY, USA. Intensive hospital-based surveillance identified 59 cases, including seven deaths in the region. We did a household-based seroepidemiological survey to assess more clearly the public-health impact of the epidemic, its range of illness, and risk factors associated with infection. METHODS We used cluster sampling to select a representative sample of households in an area of about 7.3 km(2) at the outbreak epicentre. All individuals aged 5 years or older were eligible for interviews and phlebotomy. Serum samples were tested for IgM and IgG antibodies specific for West Nile virus. FINDINGS 677 individuals from 459 households participated. 19 were seropositive (weighted seroprevalence 2.6% [95% CI 1.2-4.1). Six (32%) of the seropositive individuals reported a recent febrile illness compared with 70 of 648 (11%) seronegative participants (difference 21% [0-47]). A febrile syndrome with fatigue, headache, myalgia, and arthralgia was highly associated with seropositivity (prevalence ratio 7.4 [1.5-36.6]). By extrapolation from the 59 diagnosed meningoencephalitis cases, we conservatively estimated that the New York outbreak consisted of 8200 (range 3500-13000) West Nile viral infections, including about 1700 febrile infections. INTERPRETATION During the 1999 West Nile virus outbreak, thousands of symptomless and symptomatic West Nile viral infections probably occurred, with fewer than 1% resulting in severe neurological disease.

Diminished Proliferation of Human Immunodeficiency Virus-Specific CD4+ T Cells Is Associated with Diminished Interleukin-2 (IL-2) Production and Is Recovered by Exogenous IL-2

ABSTRACT Virus-specific CD4+ T-cell function is thought to play a central role in induction and maintenance of effective CD8+ T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4+ T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4+ T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-γ in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer+ or total-Gag-specific CD4+ T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4+ T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4+ T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.

Use of Immunoglobulin M Cross-Reactions in Differential Diagnosis of Human Flaviviral Encephalitis Infections in the United States

ABSTRACT 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.

Human Immunodeficiency Virus Viremia Induces Plasmacytoid Dendritic Cell Activation In Vivo and Diminished Alpha Interferon Production In Vitro

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection has been associated with perturbations of plasmacytoid dendritic cells (PDC), including diminished frequencies in the peripheral blood and reduced production of type I interferons (IFNs) in response to in vitro stimulation. However, recent data suggest a paradoxical increase in production of type 1 interferons in vivo in HIV-infected patients compared to uninfected controls. Using a flow cytometric assay to detect IFN-α-producing cells within unseparated peripheral blood mononuclear cells, we observed that short-term interruptions of antiretroviral therapy are sufficient to result in significantly reduced IFN-α production by PDC in vitro in response to CpG A ligands or inactivated HIV particles. The primary cause of diminished IFN-α production was reduced responsiveness of PDC to de novo stimulation, not diminished per cell IFN-α production or migration of cells to lymphoid organs. Real-time PCR analysis of purified PDC from patients prior to and during treatment interruptions revealed that active HIV-1 replication is associated with upregulation of type I IFN-stimulated gene expression. Treatment of hepatitis C virus-infected patients with IFN-α2b and ribavirin for hepatitis C virus infection resulted in a profound suppression of de novo IFN-α production in response to CpG A or inactivated HIV particles, similar to the response observed in HIV-infected patients. Together, these results suggest that diminished production of type I interferons in vitro by PDC from HIV-1-infected patients may not represent diminished interferon production in vivo. Rather, diminished function in vitro is likely a consequence of prior activation via type I interferons or HIV virions in vivo.

Changes in Paracrine Interleukin-2 Requirement, CCR7 Expression, Frequency, and Cytokine Secretion of Human Immunodeficiency Virus-Specific CD4+ T Cells Are a Consequence of Antigen Load

ABSTRACT Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7− CD45RA− phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses.

Duplex Microsphere-Based Immunoassay for Detection of Anti-West Nile Virus and Anti-St. Louis Encephalitis Virus Immunoglobulin M Antibodies

ABSTRACT West Nile (WN) virus was introduced into the United States in 1999, when the first human cases of WN fever and encephalitis appeared in New York City. From there, the virus has spread throughout North America, in some areas cocirculating with the related flavivirus St. Louis encephalitis (SLE) virus. Public health laboratories currently use an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) as a primary test for human serodiagnosis, followed by a confirmatory plaque-reduction neutralization test (PRNT). The MAC-ELISAs take 2 days to perform; therefore there is a need for a more rapid test. This report describes a duplex microsphere-based immunoassay (MIA) that shortens the test processing time to about 4.5 h. The assay employs two sets of microspheres coupled to a single flavivirus group-reactive antibody, which are used to capture the WN and SLE viral antigens independently. Immunoglobulin G-depleted serum is concurrently assayed for IgM antibodies to each of the viral antigens. The results are standardized and classified by using quadratic discriminant analysis so that a single result, anti-WN IgM-positive, anti-SLE IgM-positive, negative, or nonspecific, can be determined. The duplex MIA results compared favorably to those of the plaque-reduction neutralization test and MAC-ELISA. The assay proved to be reproducible, produced accurate classifications as to the infecting virus, and was specific.

Diminished Production of Monocyte Proinflammatory Cytokines during Human Immunodeficiency Virus Viremia Is Mediated by Type I Interferons

ABSTRACT The effect of human immunodeficiency virus (HIV) infection and high-level HIV replication on the function of monocytes was investigated. HIV-positive patients had elevated levels of spontaneous production of some or all of the monocyte proinflammatory cytokines measured (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor alpha [TNF-α]) compared to uninfected controls. In patients on therapy with high frequencies of monocytes producing proinflammatory cytokines, this frequency was diminished in the context of viremia during an interruption of therapy. Diminished production of proinflammatory cytokines during viremia was restored by culture with autologous CD4+ T cells or monocytes from an on-therapy time point or lipopolysaccharide (LPS). Microarray analysis demonstrated that diminished monocyte production of proinflammatory cytokines was correlated with elevated type I interferon-stimulated gene transcripts. The addition of exogenous alpha 2A interferon diminished the spontaneous production of IL-1β, IL-6, and TNF-α but did not affect responses to LPS, recapitulating the changes observed for HIV-viremic patients. These results suggest that monocyte function is diminished during high-level HIV viremia and that this effect is mediated by chronic stimulation by type I interferons. This effect on monocytes during viremia may play a role in diminished innate or adaptive immune system functions in HIV-infected patients. In addition, the restoration of these functions may also play a role in some immune reconstitution syndromes observed during initiation of therapy.

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

ABSTRACT 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 ≥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.

Detection of Anti-West Nile Virus Immunoglobulin M in Chicken Serum by an Enzyme-Linked Immunosorbent Assay

ABSTRACT The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.