Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling

Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR((R)) Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout beta-actin and elongation factor 1alpha (EF-1alpha) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 degrees C for IPNV NS, trout beta-actin, and EF-1alpha genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 10(7) RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin could be used for the detection and quantification of IPNV. The development of a non-lethal detection method will be useful for the detection of IPNV and potentially other viruses of finfish in farmed and wild fish.     

Proteolytic activity of the cowpea mosaic virus encoded 24K protein synthesized in Escherichia coli

The function of the 24-kilodalton (24K) protein encoded by cowpea mosaic virus (CPMV) has been studied by constructing a bacterial expression plasmid that contained a cloned chimeric segment consisting of partial DNA copies of CPMV M-RNA (including sequences coding for both capsid proteins) and B-RNA (including sequences coding for the 24K protein). Viral sequences were transcribed from the phage T7 promoter phi 10 of plasmid pT7-6 using T7-RNA polymerase expressed from plasmid pGP1-2 present in the same cells. Upon inducing the synthesis of T7-RNA polymerase several new polypeptides that contained CPMV-specific sequences were expressed, as demonstrated by immunoprecipitation and immunoblotting. Furthermore a proteolytic activity was detected in induced cells which cleaved the viral protein sequences specifically at two glutamine-glycine sites. One of the cleavage products represented capsid protein VP23. The proteolytic activity was absent when an 87-bp deletion was introduced in the coding region for the 24K protein, indicating that this protein represented the protease involved in the proteolytic processing at those specific sites.     

Optimal purification method for Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of vector administration

Herpes simplex virus (HSV)-1 amplicon vectors could be packaged in the presence of replication-competent helper virus or in a helper virus-free system. In the latter system, cytotoxicity due to the expression of de novo viral gene expression is greatly reduced due to the absence of helper virus. However, the titers produced are relatively low in the range of 10(7) and 10(8)TU/ml after sucrose gradient concentration. This may become a limitation to certain gene transfer applications, such as brain disorder studies since the volume of vectors that could be administered is restricted. In contrast, amplicon viral vectors of high titers can be easily generated in the presence of helper viruses. Despite the potential cytotoxicity caused by the presence of helper virus in the latter method of viral packaging, studies involving vector targeting would still require the complementing function of helper virus for the generation of recombinant HSV-1 amplicon vectors with modified viral envelopes. In view of this, the optimal method of purifying Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of viral vector administration is examined. Parameters such as the ratio of amplicon versus helper viruses, the percentage of viral lost, and the extent of liver cytotoxicity induced by these viral vectors purified using different methods were investigated. In addition, the maximum recombinant HSV-1 viral dosage was also determined in vivo. Taken together, these findings may be of importance to the efficient production of contaminant-free HSV-1 amplicon viral vectors required for animal and human studies.     

Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9

Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10^?1 TCID₅₀/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.     

Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon

A real-time polymerase chain reaction (PCR) assay utilizing a molecular beacon for the quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea (LYCIV), was developed, which involved the amplification of a 122bp DNA fragment from a conserved region of LYCIV ATPase gene. The specific probe consisting of two short arm and a central loop sequences complementary to the target amplicon was characterized with respect to its efficiency of quenching (E(ff)), and signal to background ratio by spectrofluorometric analysis of its hybridization with the complementary oligonucleotide target. The positive control plasmid pFHT-ATPase containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (C(T)) value and logarithmic positive plasmid concentration were close to one (r(2)=0.998) and the detection limit of the assay was 70 copies of positive plasmid/assay. The specificity of this real-time PCR was also demonstrated by using the genomic DNA templates from the healthy fish, white spot syndrome baculovirus (WSSV), and epizootic heamatopietic necrosis virus (EHNV), respectively. The coefficient of variation (CV) of the assay ranged from 1.16 to 4.42%, depending on the concentration of the positive plasmid. The quantitative detection of different tissues from LYCIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (6.86 x 10(6) and 4.62 x 10(6) viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. These results suggested that the real-time PCR assay reported here could be used for rapid, sensitive, and quantitative detection of LYCIV infection.     

HSV-1 amplicon peptide display vector

There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33-176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCdelta2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH amplicons carry a GFP transgene and the gCdelta2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1 x 10(5) Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2 x 10(5) amplicon vector transducing units (t.u.)/ml and 1 x 10(7) virus t.u./ml. As the amplicon plasmids also contain the neomycin resistance gene (neo(r)), long term vector producer cell lines were created using G418 selection. This amplicon system provides means to rapidly and efficiently generate HSV-1 amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neo(r) transgenes for marker and selection functions.     

Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens

It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.     

Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis

Both the viral titer and the genotype significantly determine clinical outcomes and responses to antiviral treatment in chronic hepatitis B virus (HBV) infection. A method was developed for large-scale A-to-G genotyping with simultaneous viral quantification. The assay was run on a LightCycler instrument using hybridization probes. The genotype was determined from the melting points of the probes in a two-step manner. Set 1 amplicons differentiated genotypes B, E, and F from A, C, D, and G and simultaneously quantified viremia by real-time PCR. Melting curve analysis using the set 2-1 amplicon or the set 2-2 amplicon reaction mixture was then used to differentiate these genotype groups into single genotypes. HBV DNA quantification was consistent with that of the Amplicor assay and linear in a range from 10(2) to 10(13) copies/ml. By comparison with the restriction fragment length polymorphism method, 92.3% of 441 samples were accurately genotyped by the current assay. The method should be useful for genotyping and quantification of HBV DNA in areas where all genotypes exist.     

Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression

We developed approaches using antisense RNA to inhibit hepatitis C virus (HCV) RNA translation and HCV core protein expression. An HCV genotype 1b cDNA comprising nt 1-1321 or a fusion construct consisting of HCV (nt 1-584) and luciferase cDNAs were inserted downstream of T7 and CMV promoter sequences and used to generate HCV RNA target molecules. Such constructs will produce HCV core or HCV coreluciferase fusion proteins in vitro or within transfected cells. Seven different antisense RNA constructs were designed to target the highly conserved 5' region of HCV RNA at nt positions 1-402. For in vitro experiments, synthesized HCV RNA target sequences and antisense RNAs were mixed at various molar ratios and subsequently translated in a rabbit reticulocyte lysate system. In cell culture studies, the HCV core-luciferase fusion cDNA was co-transfected with antisense RNA-producing constructs into human hepatocellular carcinoma (HCC) cells. Luciferase activity in cell lysates was measured to determine quantitatively antiviral effects within the cell. It was found that translation of HCV RNAs was efficiently inhibited by antisense RNA in vitro. The specificity of this inhibition was confirmed using control target RNA sequences or nonrelevant antisense RNA constructs. Co-transfection studies demonstrated that antisense RNA inhibited HCV core-luciferase fusion protein expression by 41-57% in HuH-7 HCC cells. These studies indicate that antisense RNA will find viral target RNA sequences in HuH-7 cells and inhibit HCV RNA translation. More important, these studies have defined critical viral RNA target sequences susceptible to antisense inhibitory effects within the cell.     

Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection

Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based real-time quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vbeta families and 13 Jbeta segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNgamma-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10(-5) to 10(-6) antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by >or=2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNgamma-producing T cell clones using as few as 2 x 10(6) PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases.     

A macrophage fusion assay for rapid screening of cloned HIV-1 Env using dual recombinant vaccinia viruses expressing distinct RNA polymerases.

HIV-1 cell tropism is determined initially at the level of fusion mediated by the viral envelope glycoprotein (Env). Cell-cell fusion assays are employed widely to study Env-mediated fusion, and generally require transfection of target cells with a reporter plasmid that is activated upon fusion with Env-expressing effector cells. Macrophages are an important target for HIV-1, but fusion studies using primary macrophages are limited by their resistance to transfection. An assay described previously used recombinant vaccinia virus to express T7 polymerase in macrophages, and effector cells transfected with a T7-driven reporter plasmid and infected with recombinant vaccinia virus expressing Env. However, this requires a recombinant vaccinia virus for each Env. We developed a method to study fusion using primary macrophages and HIV-1 env plasmid clones under control of the T7 promoter. Macrophages were infected with a recombinant vaccinia virus expressing the SP6 RNA polymerase. Effector 293T cells were infected with a recombinant vaccinia virus expressing T7 polymerase, and co-transfected with T7-driven env plasmids and an SP6-driven reporter gene plasmid. Cell-cell fusion mediated by T7-driven Env results in SP6-driven reporter gene transactivation. This approach is suitable for rapid analysis of multiple primary isolate, chimeric, or mutant env genes cloned into plasmid vectors.     

Functional capacity of immunoglobulin G preparations and the F(ab'')2 split product.

Five immunoglobulin G preparations, including one 5S F(ab')2 split product, were compared for activity against common bacterial, viral, and protozoan pathogens. Standard assays were used to quantitate antibodies to tetanus, diphtheria, cytomegalovirus, herpes simplex virus types 1 and 2, rubella virus, and Toxoplasma gondii. Opsonization and killing of bacteria were examined by chemiluminescence methods using Streptococcus pneumoniae types 5, 12F, and 14 and Staphylococcus aureus. Antibodies to the viral pathogens and T. gondii were not detectable for the 5S immunoglobulin even at high concentrations (50 mg/ml) but were present in all 7S preparations at immunoglobulin concentrations of 10 mg/ml. Relatively lower activities for tetanus and diphtheria antibody were also seen with the F(ab')2 product. Opsonizing capacity against all pneumococcal serotypes and Staphylococcus aureus was lowest for the 5S product and highest for the commercially available intravenous immunoglobulin product that is purified by using a pH 4.25 formulation. These data do not support potential clinical usefulness of immunoglobulin G split products and suggest wide variations of specific antibody among commercial intravenous immunoglobulin preparations.     

Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load.

Epstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.