Comparison of Quantitation of Cytomegalovirus DNA by Real-Time PCR in Whole Blood with the Cytomegalovirus Antigenemia Assay

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.

Epstein-Barr virus latent genes

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.

M gene analysis of canine coronavirus strains detected in Korea

The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported / Evaluación de los patrones de neutralización de las únicas cinco cepas argentinas descritas de arteritis viral equina

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.

Phylogenetic characterization of genes encoding for glycoprotein 5 and membrane protein of PRRSV isolate HH08

A porcine reproductive and respiratory syndrome virus (PRRSV) was obtained from clinic samples. Genes 5 and 6 encoding for the viral glycoprotein 5 and a membrane protein of the PRRSV designated as HH08 were amplified by reverse transcription-PCR. These sequences were compared with reference sequences derived from different geographical locations. The results indicated that the virus belongs to the North American type rather than European. Comparative analyses of the genetic diversity between the PRRSV isolate HH08 and other Chinese as well as foreign reference strains of PRRSV were discussed based on the sequence comparison and the topology of phylogenetic trees constructed in this study.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Arsenic trioxide reduces the invasive and metastatic properties of nasopharyngeal carcinoma cells in vitro

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 æM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 ± 3.9 percent in HNE1-LMP1 cells vs 37.89 ± 4.9 percent in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 ± 3.5 vs 65.87 ± 5.9 percent), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 ± 3.7 and 27.91 ± 2.1 percent), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 ± 3.9 vs 29.19 ± 6.27 percent) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.

Búsqueda de resistencia a amantadina en cepas de virus influenza A aisladas en Santiago de Chile, entre los años 2001 y 2002 / Search of amantadine-resistance in influenza A strains isolated in Santiago, Chile, 2001-2002

Amantadina es un fármaco eficaz para el tratamiento y prevención de influenza A. Su mecanismo de acción es inhibir la proteína M2. Su uso por períodos prolongados puede generar resistencia, la cual ocurre por mutaciones en el gen que codifica para la proteína M2. La mutación más frecuentemente encontrada se ubica en la posición 31. El uso de técnicas de biología molecular permite detectar estas mutaciones. Los objetivos fueron determinar la existencia de resistencia a amantadina en cepas de virus influenza A aisladas entre los años 2001 y 2002 en un laboratorio de virología en Santiago de Chile, y validar un nuevo método de biología molecular para reconocer cepas resistentes. Para ello se utilizó metodología de RPC y análisis de tamaño de fragmentos de restricción. En 31 cepas procesadas no se observó la presencia de cambios en la posición 31. Estos hallazgos sugieren que la resistencia a amantadina es muy baja o está ausente en nuestro medio. Esto podría explicarse por un limitado uso de este fármaco en esta población. El método descrito puede servir de base para un monitoreo prospectivo de resistencia, que pueda ser de utilidad al médico clínico.

Immunogenicity of baculovirus expressed recombinant proteins of Japanese encephalitis virus in mice

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E + prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.

Nasopharyngeal carcinoma in Indonesia has a low prevalence of the 30-base pair deletion of Epstein-Barr virus latent membrane protein 1.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), one of the highest incidence of tumors in Indonesia. EBV infection is ubiquitous around the world, but NPC occurs with a remarkable geographic distribution. This phenomenon suggests that there are subtypes of EBV, some of which may have greater tumorigenic potential. The latent membrane protein 1 (LMP 1) gene encoded by EBV is tumorigenic due to its ability to transform rodent fibroblast. It was originally shown that the LMP 1 gene from NPC of Chinese patients harbors a deletion of 30-bp in the carboxyl terminal of the gene. However, the deletion is also present in healthy control and in other EBV-positive tumors. We examined the polymorphism of LMP 1 in 56 tumor biopsies of Indonesian patients with NPC and identified low prevalence of the 30-bp deletion of LMP 1. Sequence analysis showed unique mutations of LMP 1 which suggests that strain-specific variations of EBV are found in Indonesia. The low frequency of 30-bp deletion in the country with high prevalence of NPC indicates that the deletion may represent a geographic polymorphism rather than a predisposing factor in the development of NPC.

Targeted RNA recombination of the membrane and nucleocapsid protein genes between mouse hepatitis virus and bovine coronavirus

The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from bovine coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of bovine coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study bovine coronavirus genetics towards the construction of an infectious cDNA clone.

Gene cloning of the human cytomegalovirus (HCMV) antigen reactive with the serum from a HCMV-infected patient

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.