Rapid diagnosis of Naja atra snakebites.

BACKGROUND: The clinical diagnosis of snakebites is critical and necessary in many parts of the world, especially in Southeastern Asia, where venomous snakebites are a burden on public health. It is difficult to define or recognize the species of venomous snake because of the overlapping clinical manifestations of envenomations. A quick and reliable method for identifying the snake species is necessary. We designed and tested a strip of lateral flow system for the diagnosis of cobra snake bites in Taiwan. METHODS: We developed a kit based on an immunochromatographic method for rapid detection of cobra (Naja atra) venom in human serum. The test and control lines composed of 1 mg/ml polyclonal duck antivenom and 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solutions, respectively, were coated on nitrocellulose strips. Colloidal gold was conjugated with rabbit polyclonal anti-cobra venom antibodies. From July 2007 to December 2012, we used the kit to test serum from snakebite patients and to examine the agreement between our rapid test and the currently used sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Our kit was able to detect cobra venom in serum samples in 20 minutes with a detection limit of 5 ng/ml. An absence of cross-reactivity with other non-cobra venoms from Taiwan was noted in vitro. A total of 88 snakebite patients (34 cobra and 54 other non-cobra) were tested. The sensitivity of the strips based on the ELISA results was 83.3% and the specificity was 100%. There was a strong agreement between the results of the ELISA and immunochromatographic strips (κ = 0.868). DISCUSSION AND CONCLUSIONS: This data indicates that an immunochromatographic strip might be suitable for cobra venom detection and could be used as a quick diagnostic tool in cases of N. atra snakebite.

A seroepidemiology study of varicella among children aged 0-12 years in Taiwan.

The epidemiology pattern of varicella appears to vary among regions with different climates, population densities, and degrees of development. This study investigated the age-specific varicella zoster virus (VZV) seroprevalence in children aged 0 to 12 years in Taiwan and compared these seroprevalences between free and private vaccination areas. Residual sera were collected from 13 hospitals with 1,401 valid samples. Immunoglobulin G antibodies to VZV were measured by enzyme-linked immunosorbent assay. Parents of 656 children answered questions about the varicella incidence and varicella vaccination history of their children. In the 8-12 year-olds, the seroprevance ranged between 88.0-93.8% in northern, central, and eastern, while it was only 76.1% in southern Taiwan. The seroprevalence of children 0-5 years old were significantly different between free and private vaccination areas. Seropositive children who reported no history of varicella or receiving varicella vaccine accounted for 26.1-59.3% of the total positive cases. Our findings suggest the possible effects of climate, geographical conditions, and lifestyle on the seroepidemiology of VZV in Taiwan. The efforts of implementing a varicella vaccination program in Taiwan should focus on reaching high levels of coverage.

Diagnosis and genetic analysis of Japanese encephalitis virus infected in horses.

Nervous disorders were found in two horses and verified as aseptic encephalitis by necropsy in the summer of 2000. To investigate agents that affected the horses, diagnostic procedures involving virus isolation, neutralization test and reverse transcription-polymerase chain reaction (RT-PCR) were performed. We intracranially inoculated litters of suckling mice with tissues suspected of containing aseptic encephalitis, including cerebrum, cerebellum, brain stem, thalamus, and cerebrospinal fluids; the mice were then observed for 14 days. Neutralizing antibodies against Japanese encephalitis (JE) viruses were present in the cerebrospinal fluid of the horses in titers of 10. Sequences of 500 nucleotides of the premembrane gene of JE virus, synthesized by RT-PCR, from both the cerebrum and cerebellum were determined. The phylogenetic analysis based on sequences of the premembrane gene revealed a relationship with the JE virus. The divergences at the nucleotide level of 1.2-5.7% and at the amino acid level of 0-4.3% were conserved with other JE strains. The results demonstrated that the pathogens causing equine encephalitis were JE viruses. The strains were closely related to Taiwanese isolates.

Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells.

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020 Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the alpha chain of fibrinogen preferentially and cleaved the beta chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.

Suppression of immune response and protective immunity to a Japanese encephalitis virus DNA vaccine by coadministration of an IL-12-expressing plasmid.

IL-12 plays a central role in both innate and acquired immunity and has been demonstrated to potentiate the protective immunity in several experimental vaccines. However, in this study, we show that IL-12 can be detrimental to the immune responses elicited by a plasmid DNA vaccine. Coadministration of the IL-12-expressing plasmid (pIL-12) significantly suppressed the protective immunity elicited by a plasmid DNA vaccine (pE) encoding the envelope protein of Japanese encephalitis virus. This suppressive effect was associated with marked reduction of specific T cell proliferation and Ab responses. A single dose of pIL-12 treatment with plasmid pE in initial priming resulted in significant immune suppression to subsequent pE booster immunization. The pIL-12-mediated immune suppression was dose dependent and evident only when the IL-12 gene was injected either before or coincident with the pE DNA vaccine. Finally, using IFN-gamma gene-disrupted mice, we showed that the suppressive activity of the IL-12 plasmid was dependent upon endogenous production of IFN-gamma. These results demonstrate that coexpression of the IL-12 gene can sometimes produce untoward effects to immune responses, and thus its application as a vaccine adjuvant should be carefully evaluated.

Differential expression and geographic variation of the venom phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus.

To investigate the geographic variations in venoms of two medically important pitvipers, we have purified and characterized the phospholipases A2 (PLA2s) from the pooled venoms of Calloselasma rhodostoma from Malaysia, Thailand, Indonesia, and Vietnam, as well as the individual venom of Trimeresurus mucrosquamatus collected from both North and South Taiwan. Enzymatic and pharmacological activities of the purified PLA2s were also investigated. The complete amino acid sequences of the purified PLA2s were determined by sequencing the corresponding cDNAs from the venom gland and shown to be consistent with their molecular weight data and the N-terminal sequences. All the geographic venom samples of C. rhodostoma contain a major noncatalytic basic PLA2-homolog and two or three acidic PLA2s in different proportions. These acidic PLA2s contain Glu6-substitutions and show distinct inhibiting specificities toward the platelets from human and rabbit. We also found that the T. mucrosquamatus venoms from North Taiwan but not those from South Taiwan contain an Arg6-PLA2 designated as TmPL-III. Its amino acid sequence is reported for the first time. This enzyme is structurally almost identical to the low- or nonexpressed Arg6-PLA2 from C. rhodostoma venom gland, and thus appears to be a regressing venom component in both of the Asian pitvipers.

Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines.

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.

Genetic characterization of the mRNAs encoding alpha-bungarotoxin: isoforms and RNA editing in Bungarus multicinctus gland cells.

The mRNA encoding alpha-bungarotoxin (alpha-Butx) was prepared from the venom glands of Bungarus multicinctus by Cap-finder cDNA synthesis. The sequences of the 3'- and 5'-flanking regions including a signal peptide of alpha-Butx were almost identical with those of Elapidae and Hydrophiidae toxins, suggesting that they may have the same origin. Sixteen polymorphic mRNA sequences of alpha-Butx were detected from B.multicinctus gland cells. Analysis of the genomic DNA of alpha-Butx indicated that the polymorphic mRNA originated from one DNA sequence. Most of the mutations in alpha-Butx mRNA were silent and the hot-spot variations occurred at 78, 107, 129, 198 and 201 nt in alpha-Butx mRNA. Ten distinct protein sequences of alpha-Butx could be deduced from the polymorphic mRNA and one of the isoforms has already been isolated. Since alpha-Butx DNA is a single copy in the genome, the RNA polymorphism might result from post-transcriptional editing. These results indicate that the authentic alpha-Butx is in fact derived from edited mRNAs. RNA editing may contribute a common mechanism toward the diversity of alpha-neurotoxins in snake glands.

Antigenic characterization of nine wild-type Taiwanese isolates of Japanese encephalitis virus as compared with two vaccine strains.

The antigenic properties of nine wild-type Japanese encephalitis viruses isolated in Taiwan during 1990 1994 were investigated by comparison with two inactivated vaccine strains (Beijing-1, Nakayama-NIH). All of the nine Taiwanese isolates were found to induce higher cytopathology in Vero cells but showed similar mouse virulence as the two vaccine strains. Antigenic characterization using six E protein-specific monoclonal antibodies shows two of the nine wild-type isolates (i.e. CH1949 and CH2195) presented different antigenic properties of hemagglutination inhibition and plaque reduction neutralization. The E-protein gene nucleotide sequences of CH1949 and CH2195 were determined and compared with other published sequences of the two vaccine strains and other 19 Asian/Taiwanese isolates. Phylogenetic tree analysis indicates these two wild-type Taiwanese isolates are more distant from the two vaccine strains.

Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence.

Two different plaque variants of Japanese encephalitis virus were selected from a wild-type Taiwanese isolate using Vero cells. One variant was found to exhibit small plaque morphology with retarded virus replication kinetics in Vero cells, and was demonstrated to be resistant to monoclonal antibody (mAb) E3.3 neutralization. The other variant showed large plaque morphology, was sensitive to mAb E3.3 neutralization, and manifested reduced virulence in mice on both intracranial and intraperitoneal inoculations. These two variants propagated in Vero cells retained high levels of infectivity but had relatively low HA titers as compared with the parent strain. The envelope sequences of these two variants showed four amino acid differences at residues E-85 (Glu/Arg), E-306 (Glu/Gly), E-331 (Ser/Arg), and E-387 (Met/Arg). Our results indicated the neutralizing epitope of Japanese encephalitis virus did not overlap with virus virulence determinant.

Large-scale Vero cell culture on microcarriers in a twenty-liter stirred tank fermentor.

In biotechnology, animal cell culture is an important process for the production of many biologicals such as vaccines, monoclonal antibodies, or other recombinant products. Among many established continuous cell lines, Vero cells can be maintained in many passages in cultures without inducing tumorigenicity and have been recommended by World Health Organization for the production of human biologicals. Owing to its anchorage-dependent growth characteristics, Vero cells can be grown on microcarrier in a suspension vessel where microcarrier provides the culture system with a high culture surface to volume ratio. In this paper we compared the growth kinetics of Vero cells on Cytodex 1 microcarrier in a 20-liter fermentor vs. 100 ml spinner flask culture. The kinetics of Vero cell growth in the 20-liter fermentor was similar to the results obtained from small spinner flask culture, as determined by cell specific growth rate or corresponding doubling time. The approximately 150-fold increase in culture vessel volume did not compromise the growth kinetics of Vero cells, suggesting the system is applicable for large stirred-tank fermentor cultures.

[Study on the stability of Japanese encephalitis vaccine--development of freeze-dry dosage form].

In order to prolong shelf-life and improve the quality of the vaccine product, not only an effective stabilizer but also a more proper dosage form has been sought. The stability of a Japanese encephalitis (JE) vaccine produced from mouse brain along with a variety of stabilizers and lyophilization protocols was evaluated. Without any stabilizers added, almost 90% of the antigenicity would vanish after freeze-drying process. Comparative studies of various compounds, including carbohydrates, amino acids, peptides and medium 199, on both antigenicity preservation and thermostability of the vaccine were carried out. The results indicated that the best reconstituted vaccines were prepared with two stabilizer formulations, sucrose and sucrose/gelatin. They were further examined by accelerated stability test at room and higher temperatures. The sucrose-added lyophilized vaccine can retain its original antigenicity for more than 60 days both at 37 degrees C and 45 degrees C. We conclude the thermostability efficiency of each of the stabilizers tested is as that follows: sucrose > sucrose/gelatin > gelatin/medium > gelatin.

[A simplified emulsification method for antigens with oil adjuvants].

In order to enhance the immune response, people usually add adjuvant to the antigen in immunization. Freund's adjuvant is one of the most used. It can prolong the duration of the antigen staying in the body of the animal, and through continuous stimulation of the antigen the production of antibody is increased. This paper describes a few points in facilitating the emulsification of the antigen and making it more effective. The process can be summarized as follows: (1) Force a small amount of adjuvant from syringe B by pushing it into syringe A filled with antigen solution to mingle with the latter. (2) Push the same volume of the mix from syringe A back to syringe B slowly. (3) Repeat the above mixing process until the mixed portion has become milky white. (4) Gradually increase the volume by small amounts and each time do the same mixing until final completion. After immunization with the mixture, potent sera were always obtained. It has become a routine in this laboratory to use such mixture in its immunization tasks.

Safety and immunoresponse study of intravesical instillation of Taipei-NIPM bacillus Calmette-Guérin.

The intravesical instillation of bacillus Calmette-Guérin (BCG) has proved to be an effective modality for prophylaxis of recurrent superficial bladder cancer and treatment of carcinoma in situ. The domestic BCG, named Taipei-NIPM, which is produced by the National Institute of Preventive Medicine, has been used as an anti-TBC vaccine in Taiwan for decades. In this study, we have investigated the safety and immune response in animals after intravesical BCG treatment to test its feasibility in future clinical application. Weekly instillation of BCG (1 mg/ml, 5-8 x 10(7) CFU/mg) for 6 instillations could induce lymphocytic infiltration, submucosal fibrosis, and granulomatous reaction after 4 weeks with mild decrease of bladder weight and high incidence of hematopyuria (75%). No changes in appetite, body weight, and splenic weight were noticed in the chronically treated rats. A delayed hypersensitivity test through foot pad swelling measurement reached 80% positive rates at 2 weeks. Cystometric study revealed a mild decrease in bladder capacity (33.3%) at 2 weeks and of contractile pressure of the urinary bladder in rats receiving BCG instillations. In addition, increase in lymphocytic infiltration by natural killer (NK) cells against YAC-1 cells could be detected after BCG treatment and this was related to dosage of BCG. The urothelial damage by cauterization and the number of instillations could also enhance NK cell activity. Low toxicity and high safety of intravesical instillation of the domestic BCG are demonstrated by this pilot animal study. This information on local and systemic immune responses can provide a solid base for future efficacy of BCG immunotherapy in bladder cancer patients using this domestic strain BCG.

[Preparation of toxoid from Taiwan cobra (Naja naja atra) venom].

Potency of sixty antitoxic unit was reached after two immunizations in 2-week intervals of rabbits and horses with 10-25 mg Taiwan cobra (Naja naja atra) venom which was detoxified by 0.125% glutaraldehyde. Now this procedure has become a routine antivenine-producing method by which snake bivalent neurotropic antivenine is produced. The stability test showed that Taiwan cobra toxoid kept at 37 degrees C for 40 days, the antigenicity increased by 24% and toxicity decreased by 10% as compared to the toxoid maintained at 4 degrees C.

Effects of venom proteases on peptide chromogenic substrates and bovine prothrombin.

Eighteen proteases were isolated from six hemorrhagic venoms of snakes belonging to the families of Crotalidae and Viperidae. According to their actions, they are classified as thrombin-like enzymes, alpha-fibrinogenases, beta-fibrinogenases, Factor X activator, prothrombin activator, hemorrhagins and esterases. Thrombin-like enzymes, beta-fibrinogenases, hemorrhagins and esterase hydrolyzed Phe-Pip-Arg-pNA (S-2238, substrate for thrombin) more strongly than CBZ-Ile-Glu-Gly-Arg-pNA (S-2222, substrate for Factor Xa), CBZ-Phe-Val-Arg-pNA (B-7632) or CBZ-Pro-Phe-Arg-pNA (B-2133). Thrombin-like enzymes, beta-fibrinogenase and esterase hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine ethyl ester. S-2238 is the most susceptible chromogenic substrate for most venom proteases. Thrombin-like enzymes degraded prothrombin molecule progressively down to prethrombin 2 while alpha- and beta-fibrinogenases degraded it only to prethrombin 1. Factor X activator of Vipera russelli venom and esterase of T. mucrosquamatus venom did not have any effect on prothrombin. Thus, the effects of venom proteases on prothrombin are not parallel to their amidolytic or esterolytic effects.

Protective murine monoclonal antibodies to tetanus toxin.

Monoclonal antibodies against tetanus toxin were generated by fusion of mouse NS-1 myeloma cells with spleen cells from BALB/C mice immunized with tetanus toxoid. Twenty seven hybridomas against tetanus toxin were obtained. Six hybridoma clones, designated as 1A6B12, 1H7D9, 3A8G9, 3A9F2, 3F9H9, 4A6D11 were selected for further studies. All of them were IgG1, k chain and bound specifically to tetanus toxin and toxoid. All six clones were injected intraperitoneally into pristane-primed BALB/C mice. Antibodies with titer up to 10(6) were obtained in the ascites. Results obtained from in vivo neutralization test showed that 1A6B12, 3A8G9, 3F9H9, 4A6D11 mAbs did have neutralizing activities against tetanus toxin. Monoclonal antibody 4A6D11 had the strongest neutralizing activity. 4A6D11 were purified from ascites by DEAE-52 ion exchange chromatography. Comparing to U.S.A. standard antitetanus toxin antiserum, 50 micrograms purified 4A6D11 mAb had 1 international unit neutralizing activity. The purified 4A6D11 mAb was also coupled to cyanogen bromide-activated sepharose to make an affinity column. Pure tetanus toxin can be obtained by passing crude tetanus toxin through this column and eluting the adsorbed toxin with 4M urea. Large scale purified tetanus toxin could be obtained by this method.

[The detoxification of Naja naja atra venom and preparation of potent antivenin].

The 99.2% toxicity of Naja naja atra venom can be detoxified by treatment with 0.25% GA (glutaraldehyde) solution at pH 6.8 and still remains its antigenicity. Using the GA treated venom incorporated with Freund's complete adjuvant as immunogen, the titer of immune horse sera can be enhanced rapidly. The modified immunization method not only shortened the period of immunization (from 180 to 60 days), but also increased the potency of immune sera (from 75 to 170 units). The method also diminished the mortality rate of the horse during the immunization period (from 37 to 0%) and increased the antibody production rate (from 20 to 100%), as compared to Tanaka's method. With the present method, significant economic effects can be achieved. The neutralization antibody titer of Naja naja atra antivenin could be elevated 3.55 times through purification of the antivenin with the pepsin digestion method. The antivenin recovery rate using the pepsin digestion method was about 55.44%. The solubility of lyophilized antivenin was significantly improved by the addition of 2% glycine. In addition to an increase of antivenin potency and purity, the problem of an inadequate production rate has also been resolved. Now lyophilized antivenin can be supplied even to remote areas, thus providing excellent opportunities for clinical application. This Institute has already adopted this new immunization schedule in lieu of the old Tanaka method.

[Clinical evaluation of a domestically prepared enzyme immunoassay kit for the detection of hepatitis B surface antigen].

National Institute of Preventive Medicine (NIPM) has succeeded in the development of an enzyme immunoassay (EIA) kit for detection of hepatitis B surface antigen. The sandwich principle was used for the test. Guinea pig anti-HBs IgG was used for coating microtiter plates and Horseradish peroxidase was conjugated with goat specific anti-HBs. Its stability is longer than 4 months. The lowest detectable dose is 0.7 ng/ml or better for subtype ad of HBsAg tested with Hepatitis Sensitivity Panel, Bureau of Drug and Food, Department of Health. The regression curve was determined by testing 66 samples with Auszyme II (EIA Kit. Abbott Lab.) and NIPM Kit, while Auszyme II used as a reference kit. The two EIA kits correlated well with a coefficient of determination (r2) of 0.86. Evaluation on 1,157 patients' and officers' serum samples in Tri-Service General Hospital showed that the positive rate was 24.7% (286/1,157) by RIA (Clinical Assays, Travenol Lab., USA) and that of NIPM EIA Kit was 24.4% (282/1,157). There was no statistical significance in terms of positive rate (p greater than 0.05). The positive rates of 534 blood donors are 22.1% (118/534) and 21.9% (117/534) respectively. Another evaluation on 974 serum samples in National Taiwan University Hospital showed that the positive rate was 27.2% (265/974) by Ausria II-125 (RIA. Abbott Lab.) and that of NIPM EIA Kit was 27.4% (267/974). The undetectable rate and false positive rate of NIPM EIA Kit were 0.41% (4/974) and 0.62% (6/974) respectively. In comparison with four other kind of commercial EIA Kits, the results of NIPM EIA Kit were satisfactory also. We have scaled up the reagent preparations for the kit, except the antibody coated microtiter plate preparation. At the end of March 1985 we will supply 850 EIA kits for the Development Center for Biotechnology for their hepatitis B vaccine production program.