Partially purified DEN3 virus was used as antigen in a sensitive dot enzyme immunoassay (DEIA) for the detection of antibodies to flavivirus antigens. We describe here the method used to prepare and optimise the antigen-bearing nitrocellulose membranes and present the results obtained from screening 20 acute phase sera from patients shown to have had recent dengue infections by the haemagglutination inhibition (HI) test. Sixteen pairs of acute and convalescent sera from dengue-negative patients had no detectable antibody to dengue virus by HI. These were shown to have no antibody detectable by DEIA. Sera positive for dengue antibodies by HI had DEIA titers ranging from 10 to several thousand times greater than the titers detected by HI.
A dot enzyme immunoassay (DEIA) was used to determine the levels of antibody to dengue 3 virus in the acute and convalescent sera of febrile patients with a clinical diagnosis of dengue fever or dengue haemorrhagic fever. The antibody titres were compared with titres determined by the haemagglutination inhibition (HI) test. The results of the study showed that, besides being more simple to perform, the DEIA is in order of magnitude more sensitive than the HI test. Furthermore, the data suggest that it is possible to use a single dilution as a cutoff point to predict with reasonable accuracy, if a patient has had a recent dengue infection. The DEIA test for antibodies to dengue virus is an appropriate technology highly suitable for rapid diagnosis and surveillance in developing countries.
The Australian, heat-resistant, a virulent V4 strain of Newcastle disease (ND) virus was selected for further heat resistance to give a variant designated V4-UPM. V4-UPM was sprayed on to food pellets which were fed to chickens in amounts calculated to give about 10(6) EID50 per chicken. Chickens vaccinated only once by feeding developed no haemagglutination-inhibition (HI) antibodies and were not protected against challenge with a viscerotropic velogenic strain of ND virus. Chickens given food pellet vaccine at 3 and 6 weeks of age developed HI antibodies and were substantially protected against parenteral and contact challenge with virulent ND virus. Similar protection was achieved when the V4-UPM vaccine was given intranasally on two occasions or when the vaccine virus was allowed to spread by contact from intranasally vaccinated chickens to nonvaccinated chickens. Heat resistant ND vaccine incorporated in food pellets may provide a method for protecting village chickens against ND in tropical countries.
Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.
Dengue fever/Dengue haemorrhagic fever (DF/DHF) has been a public health problem in Malaysia with an endemic level of about 7 per 100,000 population per year. In 1990, Malaysia experienced its most severe outbreak of DF/DHF with a record total of 5,590 cases referred to the Division of Virology, Institute for Medical Research (IMR). Of these, 1,880 were confirmed serologically to be DF/DHF. The conventional serological procedure, the Haemagglutination Inhibition (HI) test, for the diagnosis of DF/DHF is cumbersome and causes delay in diagnosis. Another problem associated with the HI test has been that it has often been difficult to obtain a second convalescent serum sample for an accurate diagnosis. This has raised an urgent need to establish a "rapid" test for diagnosis of DF/DHF. As such the authors recently carried out an evaluation of a newly available commercial rapid test, namely, the Dengue Blot Assay (Diagnostic Biotechnology Singapore Pte Ltd). The test is intended for use in laboratory confirmation of dengue virus infection. The evaluation was to determine if the test could be utilised as a routine laboratory test and to establish its sensitivity and specificity. Over 400 samples were tested against the Dengue Blot Assay. Results were checked against an in-house Dengue IgM ELISA and HI assay. Preliminary results indicate that the sensitivity and specificity of the Dengue Blot is satisfactory. Our results also indicate that the Dengue Blot has a useful role to play in a routine laboratory especially since it provides rapid results on single serum samples thereby reducing the workload in a busy diagnostic laboratory.
The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.
A dot enzyme immunoassay for determination of antibodies to Japanese encephalitis virus was designed for use as a field technique for the surveillance of Japanese encephalitis virus activity among domestic pigs. The test was compared with the neutralization test and the hemagglutination inhibition test and found to be more sensitive than the hemagglutination inhibition test and comparable to the neutralization test in sensitivity but more simple to perform than either the neutralization or the hemagglutination inhibition tests. An IgM capture ELISA for the determination of JEV specific porcine IgM was also utilized to determine current infection rates in pigs. The tests which do not involve the determination of specific IgM are better used for testing sentinel animals for providing clues as to the rate of transmission of JEV among pigs. IgM tests determining acute infection are less likely to be useful unless animals are tested very frequently or if a great number of animals are tested at any one time.
The potential of RT-PCR to rapidly diagnose dengue infections from both acute and convalescent phase patients' sera was evaluated. The RNA extraction method involved binding of the viral RNA to silica particles in the presence of high concentration of guanidine thiocyanate. The protocol that was established was sensitive enough to detect 40 plaque forming units per 100 microliter of serum and results could be obtained within one day. Results from this study indicate that clinical samples should be collected in the early acute phase of illness when anti-dengue antibodies were undetectable or of low titres to ensure a more reliable diagnosis.
Involvement of the central nervous system in dengue fever and dengue hemorrhagic fever has always been thought to be secondary to vasculitis with resultant fluid extravasation, cerebral edema, hypoperfusion, hyponatremia, liver failure, and/or renal failure. Thus, the condition has been referred to as dengue encephalopathy. Encephalitis or direct involvement of the brain by the virus was thought to be unlikely. This paper reports on six children who were seen over a period of two years presenting on the second or third day of illness with dengue encephalitis. The diagnosis was based upon a clinical picture of encephalitis and confirmed by cerebrospinal fluid (CSF) microscopy and electroencephalography changes. All six cases were confirmed dengue infections. Dengue 3 virus was isolated from the CSF of four cases and in one case, dengue 2 was detected by the polymerase chain reaction in both the CSF and blood. In the sixth case, virologic evidence was negative but dengue immunoglobulin M was detected in the CSF and blood. Since the onset of encephalitis appears early in the course of illness coinciding with the viremic phase, we postulate that the virus crosses the blood-brain barrier and directly invades the brain causing encephalitis. This study provides strong evidence that dengue 2 and 3 viruses have neurovirulent properties and behave similarly to other members of the Flaviviridae.
Rapid diagnosis of dengue infection is essential to patient management and disease control. The development of a rapid (5 min) immunochromatographic test and a 2 h commercial capture enzyme linked immunosorbent assay (ELISA) for anti-dengue IgM and IgG antibodies may lead to more rapid and accurate testing in peripheral health settings and diagnostic laboratories.
The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.
Trivalent inactivated influenza vaccine (TIV) is reformulated annually to contain representative strains of 2 influenza A subtypes (H1N1 and H3N2) and 1 B lineage (Yamagata or Victoria). We describe a sentinel surveillance approach to link influenza variant detection with component-specific vaccine effectiveness (VE) estimation.
This study evaluated whether MF59-adjuvanted subunit trivalent influenza vaccine for the 2003/04 winter season (A/Moscow/10/99, H3N2; A/New Caledonia/20/99, H1N1; B/Hong Kong/330/01) would confer protection against mismatched and frequently co-circulating variants of influenza B/Victoria- and B/Yamagata-like virus strains. Haemagglutination inhibiting (HI) antibodies were measured in middle-aged and elderly volunteers against the homologous B/Victoria-like vaccine strain (B/Hong Kong/330/01) and against mismatched B/Victoria-like (B/Malaysia/2506/04) and B/Yamagata-like (B/Singapore/379/99 and B/Shanghai/361/02) strains. Immunization induced significant increases in the amounts of HI antibodies against all influenza B strains under investigation. However, the responses against the heterologous B/Shanghai/361/02 virus did not reach the desirable values of seroprotection. An age-dependent decline of the responses was found for B/Victoria-like antigens, but not for B/Yamagata-like strains. Although further studies are needed, our data support the recommendation of including influenza B viruses of the B/Victoria and B/Yamagata lineages in the future influenza vaccine preparations.
Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.
Influenza vaccine provides protection against infection with matched strains, and this protection correlates with serum antibody titres. In addition to antibodies, influenza-specific CD8+ T-lymphocyte responses are important in decreasing disease severity and facilitating viral clearance. Because this response is directed at internal, relatively conserved antigens, it affords some cross-protection within a given subtype of influenza virus. With the possibility of a broader A(H1N1) Mexico outbreak in the fall of 2009, it appeared worthwhile studying the degree of cellular immune response-mediated cross-reactivity among influenza virus isolates. The composition of the 2006-2007 influenza vaccine included the A/New Caledonia/20/1999 strain (comprising a virus that has been circulating, and was included in vaccine preparations, for 6-7 years) and two strains not previously included (Wisconsin and Malaysia). This combination afforded us the opportunity to determine the degree of cross-reactive cellular immunity after exposure to new viral strains. We analysed the antibody responses and the phenotype and function of the T cell response to vaccine components. The results obtained show that antibody responses to A/New-Caledonia were already high and vaccination did not increase antibody or cytotoxic T lymphocyte responses. These data suggest that repeated exposure to the same influenza stain results in limited boosting of humoral and cellular immune responses.
Early definitive diagnosis of dengue virus infection may help in the timely management of dengue virus infection. We evaluated the Standard Diagnostics (SD, South Korea) dengue virus nonstructural protein NS1 antigen enzyme-linked immunosorbent assay (SD dengue NS1 Ag ELISA) for the detection of dengue virus NS1 antigen in patients' sera, using a total of 399 serum samples in a comparison with real-time reverse transcription (RT)-PCR, an in-house IgM capture (MAC)-ELISA, and a hemagglutination inhibition (HI) assay. Of the 320 dengue sera, 205 (64%) tested positive for NS1 antigen compared to 300 (93.75%) by either MAC-ELISA or RT-PCR, 161 (50.31%) by RT-PCR, and 226 (70.36%) by MAC-ELISA only. The assay was able to detect NS1 antigen in convalescent-phase sera until day 14 of infection. The NS1 detection rate is inversely proportional while the IgM detection rate is directly proportional to the presence of IgG antibodies. The overall sensitivity and specificity of the SD dengue NS1 Ag ELISA in the detection of "confirmed dengue virus" sera are 76.76% and 98.31%, respectively. This suggests that the SD kit is highly specific and sensitive for the detection of NS1 antigen. However, caution is needed when the kit is used as a single assay, as detection in samples that contained the virus was only about 81.97%. Combining this assay with an IgM and/or IgG assay will increase the sensitivity of detection, especially in areas with a higher prevalence of secondary dengue virus infections.
The study evaluated the immunogenicity and efficacy of a trivalent subunit MF59-adjuvanted influenza vaccine (A/Wisconsin/67/05 (H3N2), A/Solomon Islands/3/06 (H1N1) and B/Malaysia/2506/04) in preventing serologically diagnosed infections in a group of 67 institutionalized elderly volunteers during 2007/2008 winter, characterized by co-circulation of drifted A/H3N2, A/H1N1 and B influenza viruses. Influenza vaccination induced a significant increase in the amounts of hemagglutination inhibiting antibodies, both against the vaccine and the epidemic drifted strains. However, vaccination did not prevent the circulation of the new drifted influenza B virus (B/Florida/4/06-like), belonging to the B/Yamagata/16/88-lineage, antigenically and genetically distinct from B/Victoria/2/87-lineage viruses from which the vaccine B strain was derived.
The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p<0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.
A series of plasmids containing the HSP70 gene of Mycobacterium tuberculosis fused to the hemagglutinin (H5) gene of H5N1 avian influenza virus (AIV) (H5-HSP70 (heat shock protein 70) vaccine) or individual H5 gene (H5 vaccine) or HSP70 gene (HSP70 vaccine) were constructed based on the plasmid pcDNA3.1. Expression of H5 gene in Vero cells in vitro and in chickens in vivo was confirmed following their transfection and immunization with H5 or H5-HSP70 vaccines. Controls consisted of HSP70 vaccine, empty plasmid pcDNA3.1 and co-administered H5 and HSP70 vaccines. H5-HSP70 vaccine produced in chicken higher hemagglutination inhibition (HI) antibody titer than H5 vaccine. However, the increase was not statistically significant. We have demonstrated for the first time that the H5 DNA vaccine with fused HSP70 gene may produce an enhanced induction of humoral immune response to AIV in chickens.
Data on the immunogenicity of the influenza vaccine in children after liver transplantation are sparse. Our study aims to evaluate the response of such patients to the trivalent influenza vaccine, administered by different protocols in 2 influenza seasons.