Development of a multiple internal control for clinical diagnostic real-time amplification assays

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.     

Growth control of influenza A virus by M1 protein: analysis of transfectant viruses carrying the chimeric M gene.

Analysis of fast-growing reassortants (AWM viruses) of influenza A virus produced by mixed infection with a fast-growing WSN strain and a slowly growing Aichi strain indicated that the M gene plays a role in the regulation of virus growth rate at an early step of infection (J. Yasuda, T. Toyoda, M. Nakayama, and A. Ishihama, Arch. Virol. 133:283-294, 1993). To determine which of the two M gene products, M1 or M2, is responsible for the growth rate control, one recombinant WSN virus (CWA) clone possessing a chimeric M gene (WSN M1-Aichi M2) was generated by using an improved reverse genetics and transfection system. The recombinant CWA virus retained the phenotype of both large plaque formation and early onset of virus growth. This indicates that the WSN M1 protein is responsible for rapid virus growth.     

Use of a restriction enzyme-digested PCR product as substrate for helicase assays

DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated.     

Attenuation of human influenza a viruses

The attenuation of two human influenza A viruses has been carried out, using the selection of inhibitor-resistant strains and multiple passages at low temperatures. A virus related to A2/Tokyo/3/67 was obtained in an inhibitor-resistant form. When this was compared with the inhibitor-sensitive strain in a volunteer trial it was relatively non-pathogenic. The second virus, A2/Hongkong/1/68, was subjected to much longer treatment, but nevertheless remained slightly sensitive to serum inhibitor. When given to volunteers it was less pathogenic than before but attenuation was incomplete. A2/Hongkong/1/68 was also modified by passage at low temperatures. Many of these passages are apparently necessary for full attenuation.All attenuated viruses were infective and antigenic.     

Serum metabolomics as a novel diagnostic approach for pancreatic cancer

Pancreatic cancer is one of the leading causes of cancer-related death, and there is currently little hope of a cure because there are no effective biomarkers for its early detection. Therefore, the search for novel biomarkers that would allow the early detection of pancreatic cancer is ongoing. In this study, the differences between the metabolomes of pancreatic cancer patients with Stage III, Stage IVa, or Stage IVb disease (n = 20) and healthy volunteers (n = 9) were evaluated by metabolomics, which is the endpoint of the Omics cascade and therefore the last step in the cascade before the phenotype. In our experimental conditions using gas chromatography mass spectrometry (GC/MS), a total of 60 metabolites were detected in serum, and the levels of 18 of the 60 metabolites were significantly changed in pancreatic cancer patients compared with those in healthy volunteers. Then, Principal Component Analysis (PCA), which is a basic form of Multiple Classification Analysis, was performed, and the PCA scores plots based on the 60 metabolites highlighted the metabolomic differences between the pancreatic cancer patients and healthy volunteers. The differences between different stages of pancreatic cancer were also assessed by Partial Least Squares Discriminant Analysis (PLS-DA), which is one of Multiple Classification Analysis, and we found that it was possible to discriminate among the Stage III, Stage IVa, and Stage IVb groups. In addition, values of the 9 metabolites in 1 Stage I pancreatic cancer patient were similar to those obtained from the Stage III, Stage IVa, and Stage IVb pancreatic cancer patients. Our findings will aid the discovery of novel biomarkers that allow the early detection of pancreatic cancer by metabolomic approaches.     

Recombinant chimeric antibody hCAb as a novel anti-human colorectal carcinoma agent

A new chimeric IgG1 antibody hCAb which could be specifically directed against a cell surface-associated glycoprotein of colorectal cancer cells was prepared by genetic engineering technology in our lab. In this study, we explored the potential therapeutic mechanisms and described the evaluation of hCAb directed against colorectal cancer. The standard 51Cr release assay showed that like many other clinically validated IgG1 monoclonal antibodies, hCAb primarily acts by antibody-dependent cell-mediated cytotoxicity (ADCC). The maximal cell lysis of ADCC induced by hCAb was over 50% in the presence of peripheral blood mononuclear cells (PBMCs). Moreover, in vivo studies showed potent antitumor effects in nude mice with SW480 and Hce-8693 tumor xenografts. The treatment with hCAb induced a dramatic reduction (over 70%) in tumor volume in comparison to untreated control group. Furthermore, during the period of treatment, the animals treated by hCAb did not show signs of wasting or other visible signs of toxicity. No obvious tissue damage in vital organs was detected. The chimeric antibody hCAb may be a promising candidate in the treatment of human colorectal cancer. This study can provide a reference for the potential application of hCAb in clinical trial.     

Development of multiplex rt-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.     

Plasma metabonomics as a novel diagnostic approach for major depressive disorder

Major depressive disorder (MDD) is a socially detrimental psychiatric disorder, contributing to increased healthcare expenditures and suicide rates. However, no empirical laboratory-based tests are available to support the diagnosis of MDD. In this study, a NMR-based plasma metabonomic method for the diagnosis of MDD was tested. Proton nuclear magnetic resonance ((1)H NMR) spectra of plasma sampled from first-episode drug-na??ve depressed patients (n = 58) and healthy controls (n = 42) were recorded and analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). The OPLS-DA score plots of the spectra demonstrated that the depressed patient group was significantly distinguishable from the healthy control group. Moreover, the method accurately diagnosed blinded samples (n = 26) in an independent replication cohort with a sensitivity and specificity of 92.8% and 83.3%, respectively. Taken together, NMR-based plasma metabonomics may offer an accurate empirical laboratory-based method applicable to the diagnosis of MDD.     

Use of glutaraldehyde-fixed Escherichia coli cells as a convenient support for radioimmune assays.

Escherichia coli were activated by incubation with 1% glutaraldehyde, and the activated bacteria were then conjugated by incubation with IgG. About 66,000 molecules of IgG were bound per cell. The Escherichia coli-antibody conjugates were then used in competitive radioimmune assays. When a specific IgG fraction was used to prepare the conjugate, each cell bound about 16,000 molecules of antigen. With an antigen which had a specific activity of about 300 cpm/ng (or about 4.8 × 10⁴ cpm/pmol), the assay was linear over a range of 40–300 ng/ml (0.3–2.0 pmol/ml). About 100 samples were assayed per day.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

Matrix gene of influenza a viruses isolated from wild aquatic birds: ecology and emergence of influenza a viruses

Wild aquatic birds are the primary reservoir of influenza A viruses, but little is known about the viruses' gene pool in wild birds. Therefore, we investigated the ecology and emergence of influenza viruses by conducting phylogenetic analysis of 70 matrix (M) genes of influenza viruses isolated from shorebirds and gulls in the Delaware Bay region and from ducks in Alberta, Canada, during >18 years of surveillance. In our analysis, we included 61 published M genes of isolates from various hosts. We showed that M genes of Canadian duck viruses and those of shorebird and gull viruses in the Delaware Bay shared ancestors with the M genes of North American poultry viruses. We found that North American and Eurasian avian-like lineages are divided into sublineages, indicating that multiple branches of virus evolution may be maintained in wild aquatic birds. The presence of non-H13 gull viruses in the gull-like lineage and of H13 gull viruses in other avian lineages suggested that gulls' M genes do not preferentially associate with the H13 subtype or segregate into a distinct lineage. Some North American avian influenza viruses contained M genes closely related to those of Eurasian avian viruses. Therefore, there may be interregional mixing of the two clades. Reassortment of shorebird M and HA genes was evident, but there was no correlation among the HA or NA subtype, M gene sequence, and isolation time. Overall, these results support the hypothesis that influenza viruses in wild waterfowl contain distinguishable lineages of M genes.     

Sialic acid species as a determinant of the host range of influenza A viruses

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.     

Antigenic and genetic characterization of a novel hemagglutinin subtype of influenza A viruses from gulls.

Influenza A virus isolates from ring-billed, Franklin, blackback, and herring gulls in the United States possess a hemagglutinin (HA) distinct from the 12 reference HA subtypes. Serological assays (hemagglutination inhibition and double-immunodiffusion) with specific antisera to reference strains and to a representative gull isolate showed that the HA of the gull virus was not antigenically related to that of any known subtype. The gull virus did not replicate in ducks or chickens but did replicate in ferrets. Comparison of the nucleotide sequences (and deduced amino acid sequences) of the 3' 20% of the HA genes of these viruses indicates that the gull viruses represent a genetically distinct group. We propose that this HA, which has been detected only in gull isolates thus far, be called the H13 subtype.     

Use of an immunofluorescence micromethod for determining the antigens of the influenza and tick-borne encephalitis viruses

The diagnostic possibilities of several variants of the solid-phase immunofluorescent micromethod (requiring 100-200 microliter of the reaction mixture), intended for the determination of influenza virus and tick-borne encephalitis virus antigens in material obtained from patients and ticks, have been studied. A high degree of correlation between the results obtained by the methods under investigation and the control methods has been established.     

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct two influenza A viruses, GP2/BIP-NA and HGP2/BIP-NA, which contained bicistronic neuraminidase (NA) genes. The mRNAs derived from the bicistronic NA genes have two different open reading frames (ORFs). The first ORF encodes a foreign polypeptide (GP2 or HGP2) containing amino acid sequences derived from the gp41 protein of human immunodeficiency virus type 1. The second ORF encodes the NA protein; its translation is achieved via an internal ribosomal entry site which is derived from the 5' noncoding region of the human immunoglobulin heavy-chain-binding protein mRNA. The GP2 (79 amino acids) and HGP2 (91 amino acids) polypeptides are expressed in cells infected with the corresponding transfectant virus. The HGP2 polypeptide, which contains transmembrane and cytoplasmic domains identical to those of the hemagglutinin (HA) protein of influenza A/WSN/33 virus, is packaged into virus particles. This novel influenza virus system involving an internal ribosomal entry site element may afford a way to express a variety of foreign genes in mammalian cells.