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Pigs experimentally inoculated with bovine herpesvirus-1 or equine herpesvirus-1 developed mild clinical disease signs. Regression of clinical disease was accompanied by development of specific virus-neutralizing antibodies. These antibodies did not react positively with pseudorabies antigens in the serum-virus neutralization test, an indirect radioimmunoassay, or a microimmunodiffusion test.
Nineteen street rabies virus strains, isolated in Egypt from humans (two), dogs (nine), cats (two), farm animals (two), gerbils (three), and a jackal were antigenically analyzed. The Pasteur strain used for the preparation of human rabies vaccine, the Flury high and low egg passage stains (HEP, LEP) used for animal vaccines, and the challenge virus standard (CVS) strain were also assayed. All were examined by the indirect fluorescent antibody test, using a panel of 20 monoclonal antibodies against the nucleocapsid of rabies and rabies-related viruses. The rabies isolates demonstrated patterns of reactivity with the antinucleocapsid panel different from those of the Pasteur, HEP, and CVS strains. Representative human, dog, and rodent isolates were analyzed by neutralization tests in mice, with a second panel of 19 monoclonal antibodies against rabies and Mokola envelope glycoproteins. With this panel, the isolates demonstrated patterns of reactivity different from the vaccine strains. These data indicate antigenic variation between wild virus and vaccine strains.
A Western blot method (WB) was adapted for rapid screening of antibodies against coxsackie virus B1-B6 in sera from patients with newly diagnosed insulin-dependent diabetes mellitus, myocarditis or febrile syndrome of suspected coxsackie viral aetiology. The use of a mixture of all 6 coxsackie virus B serotypes as the common antigen permitted a very rapid and inexpensive detection of antibody-positive sera for preliminary diagnosis and further detailed assay. Comparison of the results with those obtained in parallel run virus-neutralization tests showed a higher sensitivity and comparable specificity of WB.
A theoretically new neutralization method was developed for rapid and quantitative detection of virus neutralizing antibodies. The method involves the specific measurement of viral antigens produced by unneutralized virus by antiviral immune serum and peroxidase-labelled complement Clq (P*-Clq). After incubation of 37 degrees C for 18 h for amplification of herpes simplex virus (HSV) as a model, to detect unneutralized HSV, the amounts of HSV antigens produced in cells are measured by OD reading of enzymatic activities of P*-Clq bound to the HSV antigen-antibody complex formed after addition of anti-HSV probe serum and P*-Clq. Results showed that the OD reading of bound P*-Clq was proportional to the input m.o.i. of HSV and that neutralization of HSV with immune serum or human sera resulted in significant reduction of HSV-specific OD readings, depending on the antibody titers of serum samples used. The neutralizing antibody activity can be expressed as a percentage (NT%) of the quantity of neutralized virus, and the entire assay procedure can be completed within 24 h. This new method can replace the conventional neutralization test.
Bluetongue virus (BTV) is an arthropod-borne orbivirus that infects sheep, wild ruminants and occasionally cattle. Detection and specific identification of BTV is a multistep process. The first step involves the isolation of the virus from the animal's blood or other tissues, followed by inoculation of embryonating chicken eggs (ECE). After the virus has been amplified in ECE, it is passaged into BHK-21 cell culture for subsequent replication and identification. The virus is then amplified further and identified in microtiter plates by the immunoperoxidase assay using a group specific monoclonal antibody. Finally, the viral isolate is typed by a virus neutralization test.
White-tailed deer (Odocoileus virginianus) were infected experimentally with two strains of peste des petits ruminants virus. The response varied from fatal consequence to subclinical infection. The clinical signs and gross lesions were similar to those in goats. Virus was recovered from all the infected deer, and survivors developed specific antibodies demonstrated by complement fixation and virus neutralization tests. Survivors also resisted challenge with virulent rinderpest virus that was lethal to a control deer.
The paper presents the results of a long-term comparative study of the infection of Ixodes tick imagoes with the pathogen of tularemia by using the biological assay and antibody neutralization test. The infection rates determined by both methods were found to be identical or close. If there was a difference in the rates in some years, it was not statistically significant. A statistically significant difference could not be found in the infection rates for ticks and in the dependence on their species composition though the level of infection in representatives of one species greatly differed from that in others. The serological study of Ixodes tick imagoes, aimed at determining their infection with a tularemia pathogen, yielded the results which are comparable with those of the biological assay and may be used in epizootological studies without using the biological test or by significantly limiting its use.
A combinatorial human antibody Fab pComb3H library, generated from splenic lymphocytes of a Puumala hantavirus (PUUV) immune individual, was selected against PUUV using the phage display technique. Panning was carried out with antigens immobilized by MAbs directed to the two PUUV envelope glycoproteins G1 and G2. Thirteen Fabs, with reactivity directed to PUUV and specifically the G2 protein, as assessed by immunofluorescence and ELISA respectively, were isolated in crude preparations. By a focus reduction neutralization test (FRNT), four of the 13 crude Fab preparations exhibited type-specific neutralization of PUUV (strain Sotkamo) with 44-54% reduction in the number of foci. After affinity purification, the four Fab clones exhibited 50% focus reduction of PUUV at concentrations below 2 microg/ml. Sequencing of the heavy and light chain complementarity determining regions (CDR) 1-3 showed that the four selected clones were identical within the antibody binding regions. In inhibition tests with the PUUV G2-specific MAbs, 4G2 and 1C9, a new epitope important for neutralization, designated as G2-a3, was defined. This epitope, overlapping partially the neutralizing epitope recognized by the human MAb 1C9, seems to be unique for the PUUV serotype since none of the Fab clones neutralized any of the other hantaviruses tested.
Hemorrhagic fever with renal syndrome (HFRS) is a fulminant infectious disease characterized by fever, hemorrhage, renal impairment, and thrombocytopenia. Hantaviruses associated with this belong to different serotypes: Hantaan (HTN), Seoul (SEO), Dobrava/Belgrade (DOB), and Puumala (PUU). The first two, HTN and SEO, are endemic in China. To investigate the epidemiology of HFRS and virus transmission in China, we constructed prokaryotic plasmids encoding truncated recombinant HTN and SEO nucleocapsid proteins (NPs), which lacked 154 amino acid (aa), 99 aa, or 49 aa in the N-terminal region, respectively. After expression, the truncated rNPs were tested as serotyping antigens, particularly for use in the enzyme-linked immunosorbent assay (ELISA). In addition, 68 acute and 52 convalescent sera were collected from HFRS patients from Harbin, Lantian, and Kaifeng regions in China in 2004, which had hantavirus specific antibodies by IFA. A neutralization test was used to differentiate these, which showed that 73 were due to HTN infection, 33 to SEO infection, and 14 undetermined. By ELISA, the truncated rNPs, that lacked 99 (rNP100) or 49 (rNP50) N-terminal amino acids of the NPs of HTN and SEO, were able to differentiate HTNV and SEOV-specific immune sera, but the rNP155 could not. Particularly, the ELISAs based on the rNP50s had a result comparable to PRNT. Thus, the rNP50 is recommended as efficient serotyping antigen for hantavirus infection diagnosis by ELISA.
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.
An NK cell activity-resistant human lymphoid T cell line (CEM-NKr) expressing the transfected Epstein-Barr virus (EBV) gp350 gene was used in membrane immunofluorescence (MIF) and antibody-dependent cellular cytotoxicity (ADCC) assays to analyze the gp350-specific humoral and ADCC responses in groups of EBV-seropositive persons. Results show that there is no correlation between gp350-specific ADCC-mediating antibody titers and MIF or EBV neutralizing antibody titers. For example, sera from patients in the acute phase of infectious mononucleosis, while positive by MIF assay or EBV neutralization test, were not reactive in the ADCC assay. Results also show that nasopharyngeal carcinoma (NPC) patients on MIF present high IgG titers against gp350 compared with healthy persons. Anti-gp350 IgA antibodies were detected in all groups tested; however, titers were highest in the NPC group.
Plaque-reduction neutralization tests were done with eight of nine known representative hantaviruses and immune sera from experimentally infected animals and from patients with hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). Results obtained with animal sera demonstrated each virus to be antigenically unique. Neutralization with the HPS patient sera was highest with Sin Nombre (SN) virus and to a lesser extent with Black Creek Canal (BCC) virus. Sera from Korean HFRS patients reacted best with Hantaan virus, but cross-reactivity with all other viruses except Thottapalayam (TPM) virus was also observed. Sera from Swedish HFRS patients reacted best with Puumala virus but cross-reacted with Prospect Hill, SN, and BCC viruses and to a lesser extent with all of the other viruses except TPM virus.
In this study, 62 water samples were collected from two water treatment plants (WTPs) in Suez Canal cities (Port Said and Ismaillia) and one plant in Cairo (Giza WTP) in addition to the beginning of the two Nile river branches (Rosetta and Damietta). Viruses were concentrated by adsorption-elution ethod sing 142 mm-diameter nitrocellulose membrane of 0.45 microm pore size and eluted with 3% beef extract at pH 9.5. The concentrated samples were inoculated for 3 successive passages in three cell culture types (Vero, BGM and RD). Enterovirus RNAs in CPE-induced samples were extracted by guanidinium thiocyanate/ phenol/chloroform and heat shock methods and detected by RT-PCR and neutralization test. The results showed that eight samples [14.5% (8/62)] contained enteroviruses most of them were polioviruses [87.5% (7/8)] and coxsackievirus type B2 [12.5% (1/8)]. The three cell cultures were of the same sensitivity to detect the isolated viruses. Also, RT-PCR followed by neutralization assay facilitates and accelerate the results. The guanidinium thiocyanate extraction method was more sensitive than heat shock method. The results turned our attention to review our technology of water treatment and disinfection step in addition to the selection of suitable intake for the drinking water treatment plants.
During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites.
Rabbit antisera were raised against plaque-purified influenza virus strains of A/Victoria/75 and A/Texas/77 isolated from Seattle influenza patients. The antigenic specificity of hemagglutinins was compared by hemagglutination inhibition (HI) and kinetic neutralization tests. Anti-A/Victoria/75 had equally high HI titers and neutralization rate constants (kappa values) for A/Victoria/75 and A/Texas/77. In contrast, anti-A/Texas/77 had a high HI titer and kappa value to A/Texas/77 and a low HI titer and kappa value to A/Victoria/75. Similar results were obtained with antisera to recombinants with hemagglutinin specific for A/Victoria/3/75 or A/Texas/1/77 and with irrelevant neuraminidase. Seven wild-type isolates, three each of A/Texas and A/Victoria, and one strain characterized as a bridging strain were tested by HI and kinetic neutralization. Characterization as A/Texas or A/Victoria was confirmed by the results. No significant difference in neuraminidase specific for A/Victoria/75 or A/Texas/77 was hown when recombinants with an irrelevant hemagglutinin were compared by the neuraminidase inhibition test. These results suggest that A/Victoria/75 strains are "senior" to A/Texas/77 strains. The epidemiological implications of this observation are discussed.
A serological survey was conducted in 1985-1987 to determine the presence of infection for Hantavirus in the general population in Argentina, Uruguay, Paraguay and Bolivia, as well as among rodent-exposed laboratory workers in Argentina. Out of 748 individuals tested by immunofluorescence 20 proved positive for Hantaan virus 76/118 strain of whom 16 also reacted against Seoul virus 80/39 strain and 2 against Puumala virus Sotkamo strain. Ten out of 72 Argentine laboratory workers were positive for the first 2 viruses by ELISA, immunofluorescence and/or plaque reduction neutralization test, in 4 of whom recent infection was demonstrated by IgM antibody presence. Inapparent Hantavirus infection was thus demonstrated for the first time in 2.7% of regional inhabitants, together with 13.9% among rodent-exposed laboratory workers. Our data established the existence of human Hantavirus infection nearly 10 years before the recognition of clinical cases of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in Argentina.
Comparative clinical trials of live attenuated and detergent-split subunit influenza virus vaccines were undertaken with 1048 volunteers in Western Australia. Volunteers were divided into three main groups, each of which received either live virus vaccine or a saline control administered intranasally, or subunit vaccine injected subcutaneously. No differences were recorded between the three groups in their post-vaccination symptoms. Serum samples were collected at various times up to 50 weeks after vaccination, and antibody titres were measured by haemagglutination-inhibition (HI) tests and, for 231 volunteers, by virus neutralization tests. The two vaccines were almost equivalent in inducing seroconversion in vaccinees with pre-trial HI titres of 96 or less, but the subunit vaccine stimulated a higher geometric mean HI antibody titre. The longevity of the HI antibody response was greater for the live virus vaccine. The height of the response and the longevity of neutralizing antibody were the same for both vaccines. Both vaccines provided a high degree of protection against epidemic A/England/42/72 influenza, and some protection against A/Port Chalmers/1/73 influenza.
A rapid simple method for the typing of wild HSV isolates based on selective inhibition of HSV-2 replication by the pyrimidine analogue oxauracil (2H-1,3-oxazine-2,6(3H)-dione) has been developed. Chi-square analysis of the results of typing of 30 wild strains by serum neutralization tests, immunofluorescence, and oxauracil-inhibition indicates no significant differences in assignment of types to the isolates. The oxauracil-inhibition test obviates confusion of typing resulting from use of immune sera containing cross-reactive antibodies. We conclude oxauracil typing is a simple, rapid, and precise test which can be performed in any laboratory capable of isolating HSV.