Yung T Huang

Case Western Reserve University School of Medicine, Cleveland, Ohio, United States

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Publications (22)80.97 Total impact

  • Stacy Weber Fening · Frank Esper · David Scholl · Yung T Huang ·
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    ABSTRACT: 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. To investigate the role of anti-HSV antibodies on recovery of HSV from CSF via cell culture. 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. 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. 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.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 07/2012; 55(2):164-7. DOI:10.1016/j.jcv.2012.07.002 · 3.02 Impact Factor
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    Frank Esper · Zhen Ou · Yung T Huang ·
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    ABSTRACT: 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. To investigate the frequency of coronaviruses in stool samples from children and adults with gastrointestinal illness by RT-PCR. 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. 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. 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.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 03/2010; 48(2):131-3. DOI:10.1016/j.jcv.2010.03.007 · 3.02 Impact Factor
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    Alison Wright · Michael E Lamm · Yung T Huang ·
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    ABSTRACT: 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.
    Journal of Virology 11/2008; 82(23):11526-35. DOI:10.1128/JVI.01111-08 · 4.44 Impact Factor
  • Brian D.W. Chow · Yung T Huang · Frank P Esper ·
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    ABSTRACT: 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. To define the clinical and epidemiologic characteristics in adult and pediatric patients with evidence of HBoV. 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. 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. 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.
    Journal of Clinical Virology 10/2008; 43(3):302-6. DOI:10.1016/j.jcv.2008.07.009 · 3.02 Impact Factor
  • Stacy Weber Fening · Joseph A Jollick · Yung T Huang ·
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    ABSTRACT: 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. To develop a continuous cell line or mixed cell co-culture that could replace dependence on pRhMK cells. 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. 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. Calu-3/A-549 cells are an equivalent or better alternative to pRhMK cells for the detection of many clinically relevant viruses.
    Journal of Clinical Virology 08/2008; 42(3):254-9. DOI:10.1016/j.jcv.2008.02.009 · 3.02 Impact Factor
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    Alison Wright · Huimin Yan · Michael E Lamm · Yung T Huang ·
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    ABSTRACT: 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.
    Virology 12/2006; 356(1-2):165-70. DOI:10.1016/j.virol.2006.08.006 · 3.32 Impact Factor
  • Wei Yang · Scott Hite · Yung T Huang ·
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    ABSTRACT: 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. 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. 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. 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. 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.
    Journal of Clinical Virology 11/2005; 34(2):125-8. DOI:10.1016/j.jcv.2005.02.008 · 3.02 Impact Factor
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    ABSTRACT: 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.
    The Journal of Immunology 05/2005; 174(8):4828-35. DOI:10.4049/jimmunol.174.8.4828 · 4.92 Impact Factor
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    Yung T Huang · Huimin Yan · Yan Sun · Joseph A Jollick · Heather Baird ·
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    ABSTRACT: 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.
    Journal of Clinical Microbiology 12/2002; 40(11):4301-3. DOI:10.1128/JCM.40.11.4301-4303.2002 · 3.99 Impact Factor
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    Huimin Yan · Michael E Lamm · Ewa Björling · Yung T Huang ·
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    ABSTRACT: 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.
    Journal of Virology 12/2002; 76(21):10972-9. DOI:10.1128/JVI.76.21.10972-10979.2002 · 4.44 Impact Factor
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    Yung T Huang · Paul Yam · Huimin Yan · Yan Sun ·
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    ABSTRACT: 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.
    Journal of Clinical Microbiology 03/2002; 40(2):366-71. DOI:10.1128/JCM.40.2.366-371.2002 · 3.99 Impact Factor
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    Yung T Huang · Scott Hite · Visar Duane · Huimin Yan ·
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    ABSTRACT: 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. 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. 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. 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. 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.
    Journal of Clinical Virology 03/2002; 24(1-2):37-43. DOI:10.1016/S1386-6532(01)00230-X · 3.02 Impact Factor
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    ABSTRACT: Simian immunodeficiency virus (SIV) infection of primates provides an important model for infection of humans by HIV. Since mucosal epithelium is likely to be an important portal of entry, we decided to study aspects of the interaction of SIV with epithelial cells. SIV was shown to produce virus efficiently in polarized epithelial cells (Vero C1008) transfected with SIVmac239 proviral DNA. The virus titer in the epithelial cell culture fluid reached 10(3) TCID50/ml at day 3 posttransfection. Initially after transfected epithelial cells were plated on a permeable membrane, virus budded at both the apical and the basolateral domains. However, after the cells formed a tight monolayer, 95-100% of the virus particles budded basolaterally, as assessed by release of p27 antigen into the fluid above and below the monolayer. This finding was confirmed by electron microscopy, which showed that the mature virus budded basolaterally in polarized cells. After introduction of the CD4 gene into Vero cells by a retrovirus vector, polarizable cells were able to be infected by cell-free SIVmac239 virus. The virus titer reached 10(4) TCID50/ml in culture fluid and virions also budded basolaterally, the same as the virus from transfected cells. Two viruses (SIVmac1A11 and SIVmac251) that contain truncated TMgp28 instead of TMgp41 also budded basolaterally. Furthermore, we found that HIV-1 with full-length or truncated TMgp41 also budded basolaterally.
    Virology 05/1999; 257(1):24-34. DOI:10.1006/viro.1999.9637 · 3.32 Impact Factor
  • Brian M. Turchek · Yung T. Huang ·
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    ABSTRACT: Herpes simplex virus (HSV) is a common pathogen with two serotypes: HSV-1 and HSV-2. HSV infection does not pose much of a threat to an immunocompetent host but to an immunocompromised host or a neonate the infection can be fatal. The Enzyme-Linked Virus Inducible System (ELVIS) employs a genetically altered baby hamster kidney (BHK) cell line that allows for the rapid overnight detection of HSV but also includes an immunofluorescent stain for the simultaneous detection and typing of HSV-1 and HSV-2. To evaluate the ELVIS HSV ID/Typing System in comparison with HSV identification and typing in primary rabbit kidney (PRK) cells grown in shell vials. Over a period of 6 weeks, 130 specimens were submitted to the diagnostic virology laboratory and cultured for the presence of HSV. Two PRK shell vials and one ELVIS BHK shell vial were inoculated with patient specimen. PRK shell vials were observed for cytopathic effect (CPE) for up to 4 days. When CPE was observed the PRK shell vials were fixed and one shell vial was stained with HSV-1 monoclonal antibody (Mab) and the other was stained with HSV-2 Mab. The coverslips were observed under the fluorescent microscope for specific apple-green fluorescence. The BHK shell vials were incubated overnight, fixed, and stained with galactopyranoside (X-Gal). If blue cells were present, the specimen was positive for HSV. The coverslip was then observed under the fluorescent microscope for the presence of specific apple-green fluorescence, indicating HSV-2. If no specific apple-green stain was observed, the coverslip was stained with a fluorescent conjugated goat anti-mouse IgG to determine the presence of HSV-1. Of the 130 specimens, PRK shell vials detected 43 positive HSV; 30 were HSV-2 and 13 were HSV-1. The ELVIS BHK shell vials detected 42 positive HSV; 30 were HSV-2 and 12 were HSV-1. One low titer specimen was not identified as being HSV positive. Two specimens were not directly typed by the ELVIS system. One specimen had only one blue cell present and did not show specific staining for either HSV-1 or HSV-2. The other specimen had only five blue cells present and only one fluorescent cell present that was difficult to type. As suggested by the manufacturer's instructions, both specimens that were not directly typed were re-grown overnight from their supernatants and were correctly identified and typed. The ELVIS HSV ID/Typing System is a rapid, highly specific and sensitive method of overnight HSV detection and typing.
    Journal of Clinical Virology 02/1999; 12(1):65-9. DOI:10.1016/S0928-0197(98)00066-X · 3.02 Impact Factor
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    ABSTRACT: IgA is thought to provide three levels of anti-viral protection in the respiratory and other mucous membranes. First, IgA antibodies can complex with free virions, preventing their adhesion to the epithelium. Second, since IgA is actively transported by the polymeric immunoglobulin receptor (pIgR) through epithelial cells into the mucosal secretions, IgA may be able to interrupt virus production within infected epithelial cells by binding to newly synthesized viral proteins. Finally, since mucosal immunoglobulins are produced by plasma cells in the lamina propria, IgA antibodies, via the pIgR, can potentially shuttle viral antigens released from epithelial cells back into the mucosal secretions.
    Seminars in Virology 08/1996; 7(4):285-292. DOI:10.1006/smvy.1996.0035
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    ABSTRACT: The concept of the Collaborative Mucosal Immunization Research Group for AIDS (CMIG) was originally conceived by the AIDS Vaccine Branch, National Institute of Allergy and Infectious Diseases (NIAID) in order to provide support for a cooperative research environment for the development of mucosal immunity to AIDS. We have purposely organized five groups of investigators at five different locations to determine how effective mucosal immunity to AIDS can be optimally approached. CMIG recognizes that both rectal (homosexual) as well as vaginal (heterosexual) infections with HIV are two of the major ways that AIDS currently disseminates through the human population. Thus, we have chosen the SIV model of infection of rhesus macaques, but more importantly the CMIG have joined two of our five components in order to use the significant expertise developed for mucosal transmission of SIV and immunity. Thus, we have brought the extensive expertise with vaginal and rectal immunization and immunity to spread [Drs. Chris Miller and Marta Marthas, California Regional Primate Research Center (CRPRC), Davis and Drs. Thomas Lehner and Martin Cranage, United Medical and Dental School Guy's Hospital, London and the Centre for Applied Microbiology and Research (Guy's/CAMR)]. Two additional components were added in order to perform mucosal immune response studies required to develop and to optimize a mucosal vaccine. First, extensive CD4+ T helper (Th) cell (e.g., Th1 and Th2) and CD8+ T cell subset studies are a major effort of the coordinating group at the University of Alabama at Birmingham (Drs. Hiroshi Kiyono and Jerry R. McGhee). This component is closely interacting with both the CRPRC and Guy's/CAMR components in terms of SIV-specific CD4+ and CD8+ T cell subset responses. For example, SIV-specific CTL responses are jointly examined using different techniques by CRPRC, Guy's/CAMR and UAB investigators. Further, it is also important to examine a balance between SIV-specific and Th1 and Th2 cell responses following mucosal immunization since the Th cell-derived cytokines are essential for the induction of appropriate antigen-specific mucosal immune responses. This issue is currently being extensively examined by the CMIG effort and a summary of our findings is discussed in this review. A major question in mucosal immunity involves the functions of secretory IgA (S-IgA) antibodies and this area is of particular importance in rectal and reproductive tract immunity. A novel and exciting in vitro epithelial cell assay system is used to study how effectively S-IgA neutralizes SIV infection (Drs. John Huang, John Nedrud and Michael Lamm, Case Western Reserve, Cleveland). A clear advantage of this CMIG effort is the unique expertise in design of mucosal delivery systems for an AIDS vaccine. We are using state-of-the-art recombinant bacteria, i.e.. rSalmonella and rVibrios for mucosal immunization [Drs. Yichen Lu and Bryan Roberts, Virus Research Institute (VRI), Boston]. In addition, we are also testing other mucosal delivery systems including DNA vaccine, microspheres, cholera toxin (CT) and CT-B, recombinant poliovirus, and immune complexes. These studies represent the first efforts to induce not only Th cell mediated S-IgA responses, but also CTL responses to SIV in primates immunized with different mucosal vector delivery systems. In order to focus our effort for the induction of SIV-specific immune responses following mucosal immunization, investigators from the CMIG are attempting to understand the induction and regulation of antigen-specific immune responses in rhesus macaques mucosally immunized with different preparations of SIV vaccines.
    Advanced Drug Delivery Reviews 12/1995; 18(1-18):23-52. DOI:10.1016/0169-409X(95)00049-D · 15.04 Impact Factor
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    ABSTRACT: Cleavage activation of the Sendai virus (Fushimi strain) fusion (F) protein was analyzed by site-directed mutagenesis of the amino acids proximal to the highly conserved fusion peptide. In addition, the functional properties of the wild-type and mutant proteins were examined to determine their ability to elicit the formation of syncytia when co-expressed with the hemagglutinin-neuraminidase (HN) glycoprotein. Viral genes were expressed from recombinant T7 transcription vectors (pT7/T3 plasmids) containing F or HN genes, after transfection into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). The wild-type F protein sequence (112VPQSRF) which contains a monobasic cleavage activation site was altered to include a tribasic, 112VPRKRF (mB3), or a pentabasic sequence, 112RRRKRF (mB5) adjacent to the fusion peptide. Although addition of basic residues to the normal protein sequence resulted in enhanced cleavage activation of the mB3 and mB5 proteins, only the mB5 protein was able to induce syncytia formation in CV-1 or HeLa T4 cells. Further analysis by the introduction of acidic residues upstream of the cleavage activation site was performed to determine whether increased hydrophilicity of the surrounding residues might contribute to cleavage activation. The mutants examined, mAcB1 (104NDDEENAGVPQSRF), mAcB3 (104NDDEENAGVPRKRF), and mAcB5 (104NDDEENAGRRRKRF) all contained DEE in replacement for the wild-type TTQ sequence (104NDTTQNAGVPQSRF). Analysis showed that only mAcB3 was efficiently cleaved by the endogenous cellular proteases, while mAcB1 was minimally cleaved, and mAcB5 not at all. Consequently, only the mAcB3 mutant was able to support fusion of CV-1 or HeLa T4 cells when co-expressed with HN.
    Virus Research 05/1995; 36(1):15-35. DOI:10.1016/0168-1702(94)00102-I · 2.32 Impact Factor
  • Yung T. Huang · Rita R. Romito · Milita Panin ·
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    ABSTRACT: The RNA species synthesized in HPIV-2 infected CV-1 cells were identified and characterized. The largest RNA of approximately 5.5 x 10(6) in molecular weight (MW) based on electrophoretic mobility, was identified as the genomic RNA. The other small RNA species of MWs 2.4 x 10(6), 1.1 x 10(6), 0.77 x 10(6), 0.68 x 10(6) and 0.5 x 10(6) were identified as mRNAs. The five smallest RNAs were also synthesized in vitro and comigrated with RNAs synthesized in virus-infected cells. mRNAs synthesized both in vitro and in virus-infected cells were translated in vitro. NP, P, M and V proteins synthesized in vitro comigrated, when analyzed by SDS-PAGE, with the authentic proteins synthesized in virus-infected cells. Additionally, peptide mapping showed that the NP, P and M proteins synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Analysis of the proteins from virions identified L, HN, NP, F (F1, F2), P, M and V proteins as virion structural proteins. Electrophoretic mobility of reduced and nonreduced F proteins was found to be different due to the conformational changes conferred by disulfide bonds.
    Virus Research 03/1995; 35(2):181-92. DOI:10.1016/0168-1702(94)00095-T · 2.32 Impact Factor
  • Robert C. Huang · Milita Panin · Rita R. Romito · Yung T. Huang ·
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    ABSTRACT: The effect of 6-diazo-5-oxo-L-norleucine (L-DON), a glutamine analog, on RSV replication was studied. At a concentration of 0.01 mM L-DON, 99% of RSV replication in treated CV-1 cells was inhibited. At this concentration of L-DON, the level of cellular protein synthesis was identical to untreated control cells. Trypan blue staining revealed that all the cells remained viable even at concentrations of L-DON as high as 10 mM. In addition, L-DON added as late as 24 h post infection can effectively suppress viral replication. Analysis of viral mRNA levels by Northern blot revealed that secondary transcription and subsequent steps in the virus life cycle were inhibited. Immunoprecipitation of viral proteins from drug treated or untreated cultures showed that synthesis of all viral proteins was drastically reduced by L-DON, with a slightly greater inhibition of viral glycoproteins. Furthermore, immunofluorescent staining showed that drug treated cells expressed both F and N proteins and that F was inserted into the membrane as the native F protein.
    Antiviral Research 01/1995; 25(3-4):269-79. DOI:10.1016/0166-3542(94)90009-4 · 3.94 Impact Factor
  • Yung T. Huang · Rita R. Romito · Bishnu P. De · Amiya K. Banerjee ·
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    ABSTRACT: An in vitro transcription system for human respiratory syncytial virus (RSV) is described. Purified viral nucleocapsid (RNP) isolated from virus-infected cells was shown to support transcription of all 10 genes encoded by the virus as determined by Northern blot hybridization. The mRNAs synthesized were polyadenylated and comigrated with the corresponding mRNAs synthesized in virus-infected cells when analyzed in agarose-urea gel electrophoresis. The in vitro-synthesized mRNAs are functional as determined by their capacity to synthesize protein in vitro. The transcriptional reaction was significantly stimulated by the uninfected host cell lysate, indicating a requirement of host factor(s) in mRNA synthesis. Preliminary results suggest that cellular actin is involved in this process.
    Virology 05/1993; 193(2):862-7. DOI:10.1006/viro.1993.1195 · 3.32 Impact Factor

Publication Stats

354 Citations
80.97 Total Impact Points


  • 1991-2012
    • Case Western Reserve University School of Medicine
      • • Department of Pathology
      • • Department of Pediatrics
      Cleveland, Ohio, United States
  • 1991-2008
    • Case Western Reserve University
      • • Institute of Pathology
      • • Department of Oral Pathology
      Cleveland, Ohio, United States
  • 2002
    • Karolinska Institutet
      Solna, Stockholm, Sweden