HSV IgG antibody inhibits virus detection in CSF.

BACKGROUND: Before PCR testing of cerebrospinal fluid (CSF), laboratory diagnosis of herpes encephalitis (HSE) was based on virus isolation from brain biopsy. Viral isolation from CSF has limited clinical value due to low virus recovery; the cause for which has not been demonstrated. OBJECTIVE: To investigate the role of anti-HSV antibodies on recovery of HSV from CSF via cell culture. STUDY DESIGN: HSV-positive CSF samples were evaluated for their ability to neutralize HSV in cell culture. The presence of HSV-specific IgG and IgM antibodies were analyzed using HSV-infected cells. To identify whether HSV-specific IgG is the cause of viral inhibition, IgG was removed using anti-human IgG magnetic beads. Viral inhibition from CSF originating from asymptomatic patients was examined as a comparison. RESULTS: CSF from 13 patients with acute HSV CNS disease was analyzed. All displayed high levels of viral neutralization to both HSV-1 and HSV-2 regardless of the infecting subtype. Interestingly, all the CSF samples stained strongly for anti-IgG antibody but none for anti-IgM antibody. Removal of IgG from CSF eliminated the viral inhibitory activity. Neutralizing IgG antibody was also found to be common in CSF of most patients, even in the absence of HSV disease. CONCLUSIONS: Viral specific IgG is the major determinant of viral inhibition in CSF and prevents virus recovery in cell culture. In CSF from HSE un-infected patients, viral inhibitory IgG originates from circulating serum antibody and is commonly present in CSF. However, this inhibitory IgG is not protective for the development of HSV disease.

Human coronaviruses are uncommon in patients with gastrointestinal illness.

BACKGROUND: Coronaviruses infect numerous animal species causing a variety of illnesses including respiratory, neurologic and enteric disease. Human coronaviruses (HCoV) are mainly associated with respiratory tract disease but have been implicated in enteric disease. OBJECTIVES: To investigate the frequency of coronaviruses in stool samples from children and adults with gastrointestinal illness by RT-PCR. STUDY DESIGN: Clinical samples submitted for infectious diarrhea testing were collected from December 2007 through March 2008. RNA extraction and RT-PCR was performed for stools negative for Clostridium difficile using primer sets against HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. Clinical data from samples positive for coronaviruses were reviewed and recorded. RESULTS: Samples from 479 patients were collected including 151 pediatric (< or = 18 years), and 328 adults (>18 years). Of these samples, 4 patients (1.3%, 2 adult; 2 pediatric) screened positive for the presence of a coronavirus. All detected coronaviruses were identified as HCoV-HKU1. No stools screened positive for either HCoV-229E, HCoV-NL63 or HCoV-OC43. All HCoV-HKU1 positive samples occurred between mid-January to mid-February. Clinical manifestations from HCoV-HKU1 positive patients included diarrhea, emesis and respiratory complaints. Three (75%) patients were admitted to the hospital with a median length of stay of 6 days. CONCLUSIONS: Coronaviruses as a group are not commonly identified in stool samples of patients presenting with gastrointestinal illness. HCoV-HKU1 can be identified in stool samples from children and adults with gastrointestinal disease, with most individuals having respiratory findings as well. No stool samples screened positive for HCoV-NL63, HCoV-229E, or HCoV-OC43.

Monoclonal antibodies to VP1 recognize a broad range of enteroviruses.

Enteroviruses (EVs) are common seasonal viruses that are associated with a variety of diseases. High-quality monoclonal antibodies (MAbs) are needed to improve the accuracy of EV diagnosis in clinical laboratories. In the present study, the full-length VP1 genes of poliovirus 1 (Polio 1) and coxsackievirus B3 (Cox B3) were cloned, and the encoded proteins were expressed and used as antigens in an attempt to raise broad-spectrum MAbs to EVs. Two pan-EV MAbs were isolated: one raised against Polio 1 VP1 and the other against Cox B3 VP1. The binding sites of both pan-EV MAbs were mapped to an amino acid sequence within a conserved region in the N terminus of Polio 1 VP1 by peptide and competition enzyme-linked immunosorbent assay. Two additional MAbs, an EV70-specific MAb and an EV71/Cox A16-bispecific MAb, developed against EV70 and 71 VP1 proteins, were pooled with the two pan-EV MAbs (pan-EV MAb mix) and tested for their sensitivity and specificity in the staining of various virus-infected cells. The pan-EV MAb mix detected all 40 prototype EVs tested and showed no cross-reactivity to 18 different non-EV human viruses. Compared with two commercially available EV tests, the pan-EV MAb mix exhibited higher specificity than one test and broader spectrum reactivity than the other. Thus, our study demonstrates that full-length Polio 1 VP1 and Cox B3 VP1 can serve as effective antigens for developing a pan-EV MAb and that the pan-EV MAb mix can be used for the laboratory diagnosis of a wide range of EV infections.

Excretion of human immunodeficiency virus type 1 through polarized epithelium by immunoglobulin A.

Human immunodeficiency virus (HIV) is transmitted primarily sexually across mucosal surfaces. After infection, HIV propagates initially in the lamina propria below the polarized epithelium and causes extensive destruction of mucosal T cells. Immunoglobulin A (IgA) antibodies, produced in the lamina propria and then transcytosed across the mucosal epithelium into the lumen, can be the first line of immune defense against HIV. Here, we used IgA monoclonal antibodies against HIV envelope proteins to investigate the abilities of polarized primate and human epithelial cells to excrete HIV virions from the basolateral to the apical surface via polymeric Ig receptor (pIgR)-mediated binding and the internalization of HIV-IgA immune complexes. African green monkey kidney cells expressing pIgR demonstrated HIV excretion that was dependent on the IgA concentration and the exposure time. Matched IgG antibodies with the same variable regions as the IgA antibodies and IgA antibodies to non-HIV antigens had no HIV excretory function. A mixture of two IgA anti-bodies against gp120 and gp41 showed a synergistic increase in the level of HIV excreted. The capacity for HIV excretion correlated with the ability of IgA antibodies to bind HIV and of the resulting immune complexes to bind pIgR. Consistent with the epithelial transcytosis of HIV-IgA immune complexes, the colocalization of HIV proteins and HIV-specific IgA was detected intracellularly by confocal microscopy. Our results suggest the potential of IgA antibodies to excrete HIV from mucosal lamina propria, thereby decreasing the viral burden, access to susceptible cells, and the chronic activation of the immune system.

Evidence of human bocavirus circulating in children and adults, Cleveland, Ohio.

BACKGROUND: Viral respiratory illness is a major cause of morbidity and mortality. The human bocavirus (HBoV) is a recently recognized parvovirus isolated from human respiratory secretions. OBJECTIVES: To define the clinical and epidemiologic characteristics in adult and pediatric patients with evidence of HBoV. STUDY DESIGN: From October 2005 through October 2006, we screened respiratory samples from children and adults negative for common respiratory pathogens for HBoV by PCR. Demographic and clinical characteristics were obtained from medical records of HBoV positive individuals. RESULTS: Of 2075 samples screened, 1826 (88.0%) represented distinct respiratory events: 1539 (84.3%) were pediatric (<18 years), and 273 (15.0%) adult (> or =18 years). Forty (2.2%) patients had HBoV: 36 (2.3%) children and 4 (1.5%) adults. HBoV positive children had history of prematurity (31.3%) and cardiac disease (18.8%). Adults had underlying pulmonary (100%) and cardiac (50%) disease. Twenty-seven children (84.4%) were hospitalized; 9 (28.1%) required intensive care. All adults were hospitalized; none required intensive care. Nosocomial acquisition likely occurred in 3 patients. CONCLUSIONS: HBoV circulates in Cleveland, OH, in children and adults with similar frequencies, and can warrant hospitalization and intensive care. Further study would clarify our understanding of this newly recognized human pathogen.

Calu-3/A-549 mixed cells as a replacement for primary rhesus monkey kidney cells for virus detection.

BACKGROUND: Primary kidney cells derived from rhesus macaques (pRhMK) are heavily relied upon for the detection and culture of a wide range of clinically relevant viruses. The use of these cultures is problematic due to the possible presence of endogenous viruses, the need to sacrifice a primate, and the inherent variance found in primary cultures. OBJECTIVE: To develop a continuous cell line or mixed cell co-culture that could replace dependence on pRhMK cells. STUDY DESIGN: Cells from the Calu-3 and A-549 cell lines were used to prepare mixed cell monolayers that were compared to pRhMK cells for their ability to detect respiratory viruses, measles, mumps, enteroviruses, and herpes viruses. Clinically derived and laboratory virus strains were used for these comparisons in culture plates or 16 mm tubes. RESULTS: Calu-3/A-549 cells are more sensitive than pRhMK for the detection of adenovirus, enteroviruses and herpes simplex virus and are about equally sensitive for the detection of other respiratory viruses, measles, mumps and varicella-zoster virus. CONCLUSIONS: Calu-3/A-549 cells are an equivalent or better alternative to pRhMK cells for the detection of many clinically relevant viruses.

Immunoglobulin A antibodies against internal HIV-1 proteins neutralize HIV-1 replication inside epithelial cells.

We show that intraepithelial cell neutralization of HIV by IgA antibodies to internal viral proteins can occur during antibody transcytosis from the basolateral to the apical surface. Polarized epithelial cells expressing the polymeric immunoglobulin receptor (pIgR) were transfected with HIV proviral DNA, and IgA was added to the basolateral side. Transcytosing IgA antibodies against Gag and RT significantly inhibited HIV replication as assessed by infection of HeLa-CD4-LTR/beta-Gal cells and direct p24 assay. Consistent with intracellular neutralization, colocalization of the internal virus proteins and their IgA antibodies was demonstrated by confocal microscopy. Thus, at least in the context of infections of polarized epithelia, antibody-mediated neutralization may not be restricted to viral surface antigens.

Enhancement of cytomegalovirus detection in mink lung cells using CMV Turbo.

BACKGROUND: Detection of HCMV from clinical specimens was a slow process until the development of shell vial method with staining for immediate early antigen (IEA). Culture though still considered insensitive is widely used for other than blood samples. A method to improve culture sensitivity is desirable. OBJECTIVES: TurboTreat, a CMV pretreatment medium from Diagnostic Hybrids Inc., was evaluated on Mv1Lu, R-Mix and MRC-5 cells for improved sensitivity for HCMV detection. STUDY DESIGN: Monolayers of Mv1Lu, R-Mix and MRC-5 cells in 48-well plates were treated with TurboTreat for overnight (o/n), 4 h or left untreated and then inoculated with previously positive HCMV specimens. After o/n incubation, cells were fixed, stained and positive cells counted. RESULTS: CMV TurboTreat enhanced detection 2-3-fold after 4 h treatment and 4-6-fold after o/n treatment in Mv1Lu and R-Mix cells and to a lesser extend in MRC-5 cells with clinical isolates of HCMV. With frozen clinical specimens, Mv1Lu cells treated o/n, 4 h or untreated detected 23, 21 and 15 positive specimens, respectively. R-Mix cells detected 19, 18, and 14 positives, respectively and MRC-5 cells detected 16, 15 and 15 positives, respectively. In no case was a positive detected in another cell line regardless of treatment that was not detected in Mv1Lu treated o/n. CONCLUSION: The o/n pretreatment with CMV TurboTreat on Mv1Lu cells is the optimum condition of treatment and significantly enhanced the detection of HCMV. Even 4 h pretreatment of Mv1Lu cells showed significant enhancement over untreated Mv1Lu, R-Mix and MRC-5 cells. Pretreatment of Mv1Lu cells with CMV TurboTreat for 4 h or longer increased the sensitivity of rapid HCMV detection.

Intraepithelial cell neutralization of HIV-1 replication by IgA.

HIV is transmitted sexually through mucosal surfaces where IgA Abs are the first line of immune defense. In this study, we used paired IgA and IgG mAbs against HIV gp160 to study intraepithelial cell neutralization and inhibition of HIV replication. African green monkey kidney cells, Vero C1008, polarizable epithelial cells transfected to express the polymeric Ig receptor (pIgR), were transfected with HIV proviral DNA, and intracellular neutralization mediated by the mAbs was assessed. D47A and D19A IgA, which neutralized HIV in a conventional assay, potently inhibited intracellular HIV replication as assessed by infecting HeLa-CD4-long terminal repeat/beta-galactosidase cells (human cervical carcinoma cell line) and CEMx174 cells (human T cell line) with apical supernatant, basolateral medium, and cell lysate from transfected cells. D47A also inhibited the production of virus as assessed by direct assay of p24. In contrast, D47 and D19 IgG, sharing the same V regions, but which were not transcytosed by the pIgR, did not inhibit intracellular HIV replication, nor did D47A and D19A IgA in pIgR- cells, incapable of transcytosing IgA. Confocal immunofluorescence microscopy showed prominent colocalization of HIV protein and D47A, in agreement with the intracellular neutralization data. D10A, which did not neutralize HIV in the conventional assay, and irrelevant IgA did not show intracellular neutralization or colocalization. Control studies with two kinds of conditioned medium confirmed that HIV neutralization had indeed occurred inside the cells. Thus, during its transcytosis through epithelial cells, HIV-specific IgA can neutralize HIV replication.

Cryopreserved cell monolayers for rapid detection of herpes simplex virus and influenza virus.

Cryopreserved cell monolayers are a new cell culture technology intended to ensure the availability of cells in the laboratory for virus detection. Two cryopreserved cell monolayers, ELVIS for the detection of herpes simplex virus (HSV) and R-Mix for the detection of influenza virus, were evaluated. The results indicated that fresh and cryopreserved cell monolayers are comparable in sensitivity for the detection of HSV and influenza virus. The cells retain the same level of sensitivity for up to 4 months at -80 degrees C.

Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model.

Three defense functions of immunoglobulin A (IgA), immune exclusion, intracellular neutralization, and virus excretion, were assessed in a measles virus model using polarized epithelial cells expressing the polymeric immunoglobulin receptor and monoclonal antibodies against the viral H and F envelope proteins and the internal N protein. Anti-H IgA was the most effective antibody at preventing infection via the apical surface, i.e., immune exclusion. This IgA was also the most effective at intraepithelial cell neutralization after infection at the apical surface and endocytosis of IgA at the basolateral surface, although an antibody against the internal N protein was also effective. In the intracellular neutralization experiments, confocal immunofluorescence microscopy showed prominent colocalization of anti-H IgA and H protein inside virus-infected cells, whereas colocalization of anti-F and F protein and of anti-N and N protein was much less, in agreement with the neutralization results. Combinations of IgA anti-H, anti-F, and anti-N showed no synergistic effects in intracellular neutralization. In the immune excretion experiments, virus immune complexes with either anti-H or anti-F IgA placed beneath polarized epithelial cells could be transported to the apical supernatant. Anti-F IgA, which was relatively poor at immune exclusion and intracellular neutralization, was the most robust at virus excretion. Thus, the studies collectively demonstrated three different antiviral functions of IgA in relation to epithelium and also suggested that the particular viral component with which a given IgA antibody reacts is an important determinant of the magnitude of the antiviral effect.

Engineered BGMK cells for sensitive and rapid detection of enteroviruses.

Decay-accelerating factor (DAF) has been reported to be a cellular receptor for several enteroviruses. Buffalo green monkey kidney (BGMK) cells expressing human DAF (BGMK-hDAF cells) showed increased susceptibility and sensitivity to several types of enteroviruses compared to wild-type BGMK cells. When 17 frozen positive clinical samples were tested, BGMK cells detected 8 and BGMK-hDAF cells detected 16. Since the CaCo-2 cell line has been documented to support the replication of most enteroviruses, CaCo-2 cells were mixed with BGMK-hDAF cells in order to increase the number of viruses detected. Thirty-four frozen clinical samples that previously had tested positive for enteroviruses were tested, and the following numbers were detected: 33 of 34 by CaCo-2/BGMK-hDAF cells, 29 of 34 by CaCo-2/BGMK cells, 28 of 34 by H292/RD (E-mix A) and A-549/BGMK (E-mix B) cells, and 26 of 34 by MRC-5 and pRhMK cells.

CV-1 and MRC-5 mixed cells for simultaneous detection of herpes simplex viruses and varicella zoster virus in skin lesions.

BACKGROUND: Culture for varicella zoster virus (VZV) is relatively insensitive. Herpes simplex viruses (HSV) culture methods, which rely on primary rabbit kidney (pRK), mink lung (Mv1Lu) or the ELVIS HSV culture system fail to detect VZV. Culture of atypical vesicular skin lesions should be able to detect both HSV and VZV. OBJECTIVES: In this study, we evaluated the sensitivity of a newly developed mixture of CV-1/MRC-5 cells for the concurrent detection of both HSV and VZV. STUDY DESIGN: The CV-1/MRC-5 mixed cells were compared with pRK cells and Mv1Lu cells for the detection of HSV and to MRC-5 and A-549 cells for the detection of VZV. Fresh clinical samples submitted for HSV culture, VZV culture, and/or direct immunofluorescent assay (DFA) as well as frozen clinical samples previously positive for VZV were used for these comparisons. RESULTS: This preliminary study suggest that CV-1/MRC-5 mixed cells are as sensitive as pRK and Mv1Lu cells for the detection of HSV and appear to be more sensitive than MRC-5 and A-549 cells for the detection of VZV. Although the sample size is small, pre-CPE staining with VZV specific monoclonal antibody (Mab) at day 2 post-inoculation may provide a rapid detection of VZV with these mixed cells, but not with MRC-5 or A549 cells. In addition, culture of VZV in mixed cells from fresh clinical specimens appears to be as sensitive as antigen detection by DFA. Finally, 1% of specimens from skin lesions submitted for HSV culture grew VZV, highlighting the importance of culturing for both VZV and HSV, particularly in the case of atypical lesions. CONCLUSION: CV-1/MRC-5 mixed cells are highly sensitive for the simultaneous culture of HSV and VZV. The ability to detect either HSV or VZV from skin lesions is important for patient management.