Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV

A novel virus of pigs, swine hepatitis E virus (swine HEV), was recently identified and shown to be antigenically and genetically related to human HEV. In the present study, we attempted to infect specific-pathogen-free (SPF) pigs experimentally with swine HEV or with human strains of HEV. Serum samples collected from naturally infected pigs were used as the source of swine HEV. Pigs inoculated intravenously with serum samples containing swine HEV seroconverted to anti-HEV 4 to 8 weeks postinoculation, and the virus spread to an uninoculated pig. Swine HEV was detected in nasal and rectal swab materials as early as 2 weeks postinoculation and for 4 to 8 weeks thereafter. Viremia appeared 4 to 6 weeks postinoculation and lasted 1 to 3 weeks. The inoculated pigs appeared clinically normal and serum liver enzymes were not significantly elevated. In contrast, pigs were not infected when inoculated intravenously with about 10(5) monkey infectious doses of one of two human strains of HEV (Sar-55 or Mex-14).     

Antibodies against the measles matrix polypeptide after clinical infection and vaccination

Sera from patients exposed to measles virus were investigated for the presence of antibodies against each of the viral antigens. All sera with measurable neutralizing titers contained antibodies against the two surface proteins (the glycoprotein and fusion protein), the nucleocapsid protein, and one of the internal proteins (P2). However, only sera from individuals with clinical symptoms of measles infection (natural measles and atypical measles) contained antibodies against the measles virus matrix protein. Levels of matrix-specific antibodies were highest in patients with atypical measles infection.     

Seroprevalence of orthopox virus specific antibodies in red foxes (Vulpes vulpes) in the Federal State Brandenburg, Germany

The prevalence of orthopox virus (OPV)-specific antibodies in 1,040 red foxes (Vulpes vulpes) was evaluated on a large scale in the German Federal State Brandenburg. Serum samples were selected from 809 communities within the study area from January 1991 to September 1994 by simple random sampling. Screening was carried out by an indirect enzyme-linked immunosorbent assay (ELISA). Orthopox virus-specific antibodies were found in 162 (16%) of the 1,040 fox sera. Furthermore 154 (15%) sera were considered suspect positive. The specificity of the antibodies detected in ELISA-positive and suspect positive sera was confirmed by Western blotting. Presence of OPV-antibodies occurred in 291 communities. No correlation of OPV-antibodies findings to latitude or characteristic topographical and ecological peculiarities of the study area was found. Although the causative agent is still unknown we believe that orthopox viruses probably have a ubiquitous presence among red foxes.     

Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.     

Comparison of cardiac findings at necropsy in octogenarians, nonagenarians, and centenarians

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2196-561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196-561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG+ IgA- IgM-; n = 35), group B (IgG+ IgA+ IgM+; n = 21), group C (IgG+ IgA+ IgM-; n = 5), and group D (IgG+ IgA- IgM+; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2196-561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2196-561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.     

The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle

A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed. The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E. coli. Experimental and field sera from naive, vaccinated and infected cattle were examined. Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection. In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates). A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results. Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection. The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys. The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.     

Seroprevalence of hepatitis E virus in an adult urban population from Burundi

The prevalence of antibodies to the hepatitis E virus (HEV) was measured in a group of 129 adults from Bujumbura, Burundi, using an ELISA. The prevalence of anti-HEV IgG was 14%, much lower than that of hepatitis A virus (HAV) (97.7%). In addition to the lability of antibodies to HEV, this difference might be explained by the extensive availability of good-quality drinking water in the city. The presence of serologic markers of HBV (77.6%), HCV (27.1%), and human immunodeficiency virus (30.2%) was not associated with that of anti-HEV.     

Seroepidemiology of hepatitis E virus in the Egyptian Nile Delta

The seroendemicity of hepatitis E virus (HEV) in an entire village population located in the Egyptain Nile Delta is described. Serum specimens were obtained from 68% of the total population of 1,850 villagers. The lack of serum specimen was greatest in the youngest age group (< 5). Commercially available enzyme immunoassays (EIA) for antibody to hepatitis A virus (anti-HAV), to hepatitis B virus core antigen (anti-HBc), to second-generation hepatitis C virus (anti-HCV) core and nonstructural antigen, and to hepatitis E virus (HEV) were used. Only repeated reactive sera were coded as positive. Stool specimens were examined for Schistosoma mansoni by the Kato method and standard methods for the examination of the liver and spleen by ultrasonography were used. Unadjusted for nonrespone, the seroprevalence of anti-HEV was 17.2% (SE +/- 1.1). Anti-HEV seroprevalence increased by age and was not associated statistically with any of the other viral markers including HCV. Anti-HAV seroprevalence was consistently > 95%, even in the youngest age group (< 5). The overall sero-endemicity of HEV was higher than reported elsewhere and appears not to have been introduced into the village population recently.     

Torsional properties of stainless steel and nickel-titanium endodontic files

American cutaneous leishmaniasis (ACL) presents a spectrum of clinical and immunological manifestations. Since the nature of the cellular response appears to play a fundamental role in determining the characteristics of the immunoglobulin isotype of specific antibody responses, we have compared the relative levels of specific antibodies of the four IgG isotypes against Leishmania in sera from patients with different clinical manifestations of ACL. Using a specific antibody capture assay, significant levels of antibodies of the IgG1, 2 and 3 isotypes were detected in localized cutaneous leishmaniasis (LCL); the average level of IgG4 antibodies was low and they were not detected in 10/20 sera. Sera from muco-cutaneous leishmaniasis (MCL) gave a comparatively strong IgG1 response. Sera from diffuse cutaneous leishmaniasis (DCL), the rare form characterized by antigen-specific anergy of cell-mediated immunity, showed highly significant levels of IgG4 antibodies compared to antibody levels of this isotype in the other groups; IgG1 and IgG2 levels were also elevated. Based on other studies of the relationship between the IgG isotype response and cell-mediated immunity, these results confirm a Th1-like CD4+ T cell response in LCL and MCL and a significant Th2-like response in DCL.     

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive (P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV.     

Use of the protective antigen of Erysipelothrix rhusiopathiae in the enzyme-linked immunosorbent assay and latex agglutination

To establish a safe and convenient serodiagnostic method for swine erysipelas, a purified protective protein antigen of Erysipelothrix rhusiopathiae, which included a large amount of protective protein (64 kDa protein), was used for enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) test. In the ELISA, the antisera to four different serovars (1a, 2, 5 and 20) of E. rhusiopathiae exhibit a positive reaction, while antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) exhibit a negative reaction. In the LA test, the antisera to three different serovars (1a, 2 and 5) of E. rhusiopathiae reacted with P64-sensitized latex beads, while the antiserum to serovar 20 (2553 strain) did not. Moreover, the antisera to other species of bacteria (Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Rhodococcus equi and Corynebacterium pseudotuberculosis) did not in this test. Comparing the results of the growth agglutination (GA), ELISA and LA tests of 284 swine sera, there was a high degree of correlation among the results. The detection of anti-E. rhusiopathiae antibodies in the GA, ELISA and LA tests were compared using sera from pigs immunized with P64, alkaline extract (AE) and live-cell vaccine (LV). In all three tests, anti-E. rhusiopathiae antibodies could be detected 1 week after immunization. The serum antibody titre as determined by the LA test increased moderately, as did that by the GA test, while that determined by ELISA increased rapidly. These results suggested that ELISA could be used to monitor changes in anti-E. rhusiopathiae antibody titre and the LA test could be used in the screening test for swine erysipelas.     

An analysis of the central pathway of vestibulo-sympathetic responses

The sera of 159 patients with monoclonal gammopathies were examined for the presence of anti-thyroglobulin (Tg) activity. An enzyme-linked immunosorbent assay was employed. Thirty-one (19.5%) sera were found to bind Tg. The activity against Tg was further confirmed by using purified immunoglobulins and employing competition assays. The anti-Tg antibodies were found in the sera of patients with IgG, IgM and IgA gammopathies. Anti-Tg antibodies were more frequent among patients with IgG gammopathy. Autoantibodies to Tg are found in patients with Hashimoto's thyroiditis, Graves' disease and occasionally in patients with thyroid carcinoma. Natural autoantibodies directed against human Tg have been detected, as well, in healthy subjects. None of the patients in the present study whose serum was found to contain high titers of anti-Tg human monoclonal antibodies had any clinical or biochemical evidence of thyroid disease. Our results of a high incidence of anti-Tg activity in the sera of patients with monoclonal gammopathies support previous reports of autoantibody properties characteristic of these immunoglobulins.     

Prevalence of specific anti-Cryptosporidium IgG, IgM and IgA antibodies in cat sera using an indirect immunofluorescence antibody test

Sera from 258 healthy and sick domestic and feral cats were screened for specific anti-Cryptosporidium antibodies using an indirect immunofluorescence antibody test (IFA). Sera were positive for IgG, IgM and IgA antibodies in 192 (74%), 84 (32%) and 67 (26%) samples, respectively. Antibody was not detected at dilutions of 1:10 and 1:20 or greater in any of eight specific pathogen-free kittens. IgM and IgA antibody classes were more prevalent in sick than in healthy domestic cats. The presence of IgM and/or IgA antibodies indicated early infection. However, these antibody classes were present in sera from cats either positive or negative for Cryptosporidium infection by faecal examination. Pronounced polar fluorescence was observed in the sporozoites in positive samples under fluorescence microscopy. The higher prevalence of specific anti-Cryptosporidium antibodies and the absence of Cryptosporidium oocysts in faecal samples from some IFA-positive animals suggests that detection of these antibodies in sera from cats could be helpful for the diagnosis of feline cryptosporidiosis.     

Protection of turkeys against haemorrhagic enteritis by monoclonal antibody and hexon immunization

Virus-neutralizing monoclonal antibodies specific for the hexon of haemorrhagic enteritis virus (HEV), a turkey adenovirus, were examined for their ability to confer passive protection against haemorrhagic enteritis (HE) in turkeys. A high dose of antibody prevented clinical disease and reduced virus replication in experimentally infected birds. This suggests that virus neutralization might be an important mechanism for protection against HE. Subsequently, the use of the hexon protein as a subunit vaccine was investigated by immunizing birds with affinity-purified HEV hexon. The birds were tested for the appearance of hexon-specific antibodies in their sera, for protection from clinical disease, and prevention of virus replication after challenge with virulent HEV (HEV-V). Regardless of whether birds were immunized with native or denatured hexon, high ELISA antibody titres were produced to each immunogen. A virus-neutralizing antibody response was induced by immunization with the native hexon but not by immunization with the denatured protein. All turkeys twice immunized with a dose of at least 1 microgram, and four out of five birds immunized with two doses of 0.3 micrograms of purified native hexon, were protected against virus-induced disease and virus replication. In contrast, birds inoculated with denatured hexon were not protected. These results demonstrate the importance of the native (trimeric) structure of the hexon protein for eliciting a protective immune response. The impact of these results on the development of a vaccine for HE in turkeys produced by recombinant DNA technology is discussed.     

Quality of life for critical care patients: a concept analysis

In order to investigate the infection rate of Hantaan virus in Taiwan, a total of 6,536 human serum samples were collected from residents, selected by stratified random sampling, from 19 townships covering four different ethnic groups: Aborigines, Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese. Serum samples were screened for Hantaan virus antibodies by indirect immunofluorescence. The prototype Hantaan virus (76/118)-infected Vero E6 cells were used as the viral antigen for the antibody detection. Among 6,536 human serum samples, 403 (6.2%) samples had Hantaan virus antibodies. The seropositive rates for males and females were 6.1% and 6.2%, respectively. A higher seropositive rate was found among Aborigines on the Orchid Islets (11.5%) and Fukien Taiwanese on the Penghu Islets (11.6%), while the lowest rate was observed among Hakka Taiwanese in the south of Taiwan (2.5%). In comparing with different ethnic groups, the highest prevalence was found among Fukien Taiwanese (8.1%) and the lowest among Mainland Chinese (4.9%). Among the different geographical areas, the highest positive rate was found in western Taiwan (7.1%) and the lowest in southern Taiwan (5.4%). Hantaan virus antibodies were also detected in 22 of 548 (4.0%) rat serum samples. The highest seropositive rate was found in rat sera collected from the Orchid Islets (21.4%). None of the rat sera collected from Hsinchu, Miaoli, Changhua, Nantu, Yunlin, Chiayi, Tainan, and Penghu Counties were positive. Hantaan virus antibodies were found in rats: Rattus rattus (20%), Bandicota indica (9.0%), Rattus norvegicus (8.3%), Bandicota nemorivaga (6.3%), Rattus losea (4.2%), and Apodemus agrarius (1.6%). Hantaan virus antibodies were not detected in rat sera collected from species of Rattus coxinga, Rattus culturatus, Mus musculus, Mus caroli, Suncus murinus, and Apodemus semotus. The results show that the Hantaan or Hantaan-related virus exists and is distributed widely in both human and rats in Taiwan.     

Prevalence of herpes simplex virus type 1- and 2- specific antibodies among the acute, recurrent, and provoked types of female genital herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P < 0.005), and 55% for the recurrent type (P < 0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies

An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV and PPRV belonging to different lineages, experimental sera from cattle vaccinated for a RPV of Asian lineage, and field sera from cattle and sheep/goat populations known to be positive (West Africa) and negative (Korea) for RPV and PPRV were used for the evaluation. Morbillivirus cELISA results on the panel of experimental RPV and PPRV antisera showed high correlation (r=0.97) between the whole virus and the rRPV N antigens, suggesting that the rRPV N contains a ruminant morbillivirus-specific antigenic determinant recognized by the P-13A9 and it may be suitable as an ELISA antigen in place of the whole virus. Morbillivirus cELISA detected anti-RPV and anti-PPRV antibodies in all reference RPV and PPRV antisera containing VN titers >/=1:8, suggesting that the assay can simultaneously detect antibodies against RPV and PPRV. Anti-RPV antibody was detected by morbillivirus cELISA in vaccinated cattle as early as the VNT and continued to be detectable by both the cELISA and the VNT until termination of the study. When applied to field samples from Africa, morbillivirus cELISA showed good agreement with a RP cELISA kit (kappa value of 0.86) in bovine sera and with a peste des petits ruminant cELISA kit (kappa value of 0.81) in caprine/ovine sera. Usefulness of morbillivirus cELISA using the rRPV N protein was discussed.     

Prevalence of Fasciola infection among school children in Sharkia Governorate, Egypt

This study was performed on 1350 school children from 9 different villages in Sharkia Governorate to investigate the real situation of endemicity of fascioliasis in the area. Stool examination using modified Kato thick smear method was performed to detect Fasciola infection and other parasites. Those with negative stool samples were examined serologically by ELISA test to detect anti-Fasciola IgG. All cases with positive anti-Fasciola IgG were further examined by circum-oval precipitin test (COPT) against viable S. mansoni eggs to exclude the crossly reacted Schistosoma infections. Sixty nine cases were found to pass Fasciola eggs in their stool samples (5.1%). Anti-Fasciola IgG was detected in the sera of 231 children (17.1%) using ELISA test. Eighty four out of the 231 children were found positive by COPT and were considered as schistosomal cases. The remaining 147 who gave negative COPT were considered as Fasciola infections. All of the 69 Fasciola positive stool cases were found positive by ELISA test and negative by COPT test. The sensitivity of stool analysis was 47% versus 100% sensitivity of ELISA, whereas the specificity of ELISA was 63%. The total number of Fasciola positive cases by ELISA and stool analysis were 147 cases among 1350 children indicating a prevalence of 10.9% among school children in Sharkia Governorate. This results highlighting the importance of health education and snail control in decreasing the high prevalence.