不同动物制备的抗血清对病毒抗原免疫反应的差异

血清学技术是病毒诊断、鉴定、分类及亲缘关系分析的重要手段。一般常用以制备抗病毒血清的动物是家兔,但也有采用其它动物的,如蛙、羊、豚鼠、鸡及小鼠等。本文比较了Balb/c小鼠、昆明种小鼠和新西兰大白兔对长叶车前花叶病毒上海分离株(RMVsh)和烟草花叶病毒普通株(TMVc)的免疫反应特征。     

Study of the immunological activity of blood in various rickettsial and viral infections]

The content of various types of antibodies were studied in the blood serum of laboratory animals in experimental tick-borne rickettsiosis of Northern Asia. This was done by a simultaneous staging of indirect hemagglutination, indirect hemolysis, compliment fixation under cold conditions, and agglutination with Proteus antigen OX19 tests. Immune horse sera to the influenza virus were also studied with the aid of several serological tests. The data obtained pointed to the significant differences in the properties of individual types of antibodies. Immunological activity of the sera under study depended on the correlation in them of various types of antibodies at various periods of the infectious process in combination with the influence upon the immunogenesis of the individual and species immunological reactivity.     

Formation of group and intergroup antibodies in humans with leptospirosis infections

The authors studied regularities attending the biosynthesis of group (homologous) and intergroup (heterologous) antibodies in man during the whole cycle of leptospirosis infection, and for three months after it. Leptospira of the serological groups Pomona, Grippotyphosa, Icterohaemorrhagiae, Hebdomadis, the most prominent in the morbidity structure at present, served as etiological causative agents of the disease. Biosynthesis of both homologous and heterologous antibodies had its specific features for each serological group of the disease, and was characterized by the quadripol dynamics of 19S-macro and 7S-microglobulin antibodies production in leptospiroses of serological groups Pomona and Gryppotyphosa, whereas in leptospiroses of serological groups Icterohaemorrhagiae and Hebdomadis a graphically dipolic synthesis dynamics of 19S-macroglobulin antibodies alone was noted.     

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus)

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.     

The use of protein A-sandwich ELISA as a means for quantifying serological relationships between members of the tobamovirus group

Indirect protein A sandwich ELISA (PAS-ELISA) was used to determine the serological relationship between eight tobamoviruses with antisera to 26 viruses and virus strains within the group. Very distant relationships were determined by trapping virus with heterologous antiserum and detecting it with homologous antiserum, while near and close relationships were differentiated by using heterologous antiserum each time. The results were esssentially consistent with previously recorded relationships determined by tube precipitin and other serological tests. Since PAS-ELISA requires much less antiserum than many conventional tests and does not require the purification of IgG or virus, it may offer many advantages in the detection of serological relationships.     

Electro-Blot Immunoassay—A Means for Studying Serological Relationships among Plant Viruses?

The coat proteins of tymo-, tombus-, como-, nepo-, tobamo-, potex-, carla- and potyviruses were subjected to sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis, electro-blotted onto nitrocellular membranes and reacted with homologous and heterologous antìsera to intact plant viruses. Immune complexes were detected after reaction with alkaline phosphatase-labelled goat anti-rabbit antibodies. SDS coat proteins of comoviruses reacted only with antisera which were homologous for their intact particles. In all other groups, however, a rather broad range of serological relationships was detected. With a number of viruses, serological relations to other members of the same group were demonstrated for the first time. SDS coat proteins of maclura mosaic virus, a possible potyvirus, and of beet necrotic yellow vein virus, a possible tobamovirus, did not react with antisera to definitive members of the respective groups. Several unexpected reactivities of SDS coat proteins of viruses from one group with antisera to viruses in other groups were, however, also observed. This indicated that electro-blot immunoassay, like other serological methods, cannot be used reliably to assign a virus to a certain group. The SDS coat proteins of several tombusviruses were able to mimic serological reactions by a direct binding of enzymelabelled antibodies which occurred also without serum treatment.     

Characterization of the Antiviral Activity for Influenza Viruses M1 Zinc Finger Peptides

We sought to investigate the cellular uptake and antiviral activity for the M1 zinc finger peptides derived from influenza A and influenza B viruses in vitro. No cellular uptake was detected by fluorescent microscopy for the synthetic zinc finger peptides. When flanked to a cell permeable peptide Tp10, the zinc finger recombinant proteins were efficiently internalized by MDCK cells. However, no antiviral activity was detected against homologous or heterologous virus infections for the synthetic peptides or the Tp10-flanked recombinant proteins, regardless treated with or without Zn²⁺. Nevertheless, MDCK cell constitutively expressing the M1 zinc finger peptides in cell nuclei potently inhibited replication of homologous, but not heterologous influenza viruses. Adenoviral vector delivered M1 zinc finger peptides also exhibited potent antiviral activity against homologous viruses challenge. Transduction at 100 PFU dose of recombinant adenovirus efficiently protected 99% of the cells from 100 TCID₅₀ of different virus infections for both peptides. These results brought new insight to the antiviral researches against influenza virus infections.     

Neutralizing antibodies to bovine herpesvirus-1 (BHV-1) and bovine parainfluenza-3 (PI-3) viruses in cattle in Tunisia

A serological survey was conducted in an attempt to detect antibodies to bovine respiratory viruses in cattle from several localities around Tunis. Blood was collected from approximately 10% of the animals in each of the 44 farms visited and tested for specific antibodies against bovine herpesvirus-1 (BHV-1) and bovine parainfluenza-3 (PI-3) viruses, by ELISA and serum neutralization (SN). Antibodies to PI-3 and BHV-1 viruses were demonstrated in 55.3% and 25.9% animals, respectively. An overall 21.2% of the 170 animals tested had antibodies to both viruses. The incidence of antibody presence varied at different location. A correlation of the presence of BHV-1 antibody with breed and age of the animals was observed; however, no such relationship for PI-3 antibodies appeared to exist.     

Insertion of primary syncytium-inducing (SI) and non-SI envelope V3 loops in human immunodeficiency virus type 1 (HIV-1) LAI reduces neutralization sensitivity to autologous, but not heterologous, HIV-1 antibodies.

The aim of the study was to investigate the influence of V3 loops from naturally occurring viruses on the neutralization sensitivity of a molecularly cloned virus. A selection of well-defined syncytium-inducing (SI) and non-SI V3 loops of a single human immunodeficiency virus type 1-infected individual (H594) and the V3 regions of two SI laboratory strains were inserted in an infectious molecular clone of human immunodeficiency type 1 LAI. Neutralization was performed with a heterologous serum pool and autologous patient serum, using the virus reduction neutralization assay and peripheral blood lymphocytes as target cells. High sensitivity of the chimeric viruses containing the laboratory strain V3 regions to neutralization by H594 sequential sera as well as the heterologous serum pool was found. A statistically significant correlation between the sensitivities of these viruses was seen. In contrast, insertion of the primary isolate NSI and SI envelope V3 loops significantly reduced the neutralization by autologous serum but not by the heterologous serum pool. No correlation was found between the neutralization of the viruses with laboratory strain-derived V3 regions and the viruses with primary isolate V3 domains. We conclude that heterologous antibodies are able to neutralize infectious molecular clones with V3 loops of both SI and NSI viruses, regardless of whether they originated from laboratory strains or primary isolates. However, serum of patient H594 discriminated between the two types of viruses and showed reduced neutralization of the viruses with the autologous NSI and SI primary isolate V3 loops. These results indicated that the neutralization sensitivity of the viruses depended on the capacity of the V3 region to influence the conformation of the virus envelope. These V3-dependent conformational changes partially explain the neutralization sensitivity of laboratory strains and the relative neutralization resistance of primary isolates.     

Serological Evidence of Influenza A Viruses in Frugivorous Bats from Africa

Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona- and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes – H17N10 and H18N11 – in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5μl of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.     

Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

Sequential immunization with V3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary isolates with a matching narrow-neutralization sequence motif

An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.     

Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8–12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.     

Studies on prophylactic efficacy of N-2-hydroxyethyl palmitamide (Impulsin) in acute respiratory infections. Serologically controlled field trials.

The results of three serologically controlled double blind field trials in army units are presented. The evaluation of results according to morbidity, regardless of aetiology, showed a significant reduction in acute respiratory diseases (ARD) after administration of Impulsin. In the 1973 trial (901 volunteers), 22.7% of ARD cases were found in the Impulsin group contrary to 34.4% in the placebo group (P less than 0.0002). The relevant values in the 1974 trial (610 volunteers) were 19.7% and 40.7% (P less than 0.002) and in the 1975 trial (353 volunteers) 10.6% and 28.8% (P less than 0.004). The study of the immunological background in representative sets of volunteers allowed determination of the aetiology, the proportion of asymptomatic infections and possible deformation of results due to preexisting protective antibodies. Manifestation rate (MR) expressing the proportion of sick persons out of all sensitive subjects with serologically proved infection was found useful. This indicator is relatively independent of randomization and is more sensitive as compared to the incidence rate. In the 1973 trial, influenza A 2 England was prevalent, the MR of infection being 15.4% in the Impulsin group and 44.9% in the placebo group (P less than 0.0002). After elimination of persons with preinfection antibodies greater than or equal to 1:256 the corresponding values of MR were 17.6% and 46.6% (P less than 0.005), reflecting the "relatively clean effect" of Impulsin. In the 1974 trial, where influenza B Hong-Kong was prevalent, MR was 14.3% and 57.1%, respectively (P less than 0.001). Preinfection antibodies were negligible. The preliminary prophylactic index of the drug seemed to be 4.3 for combined adenoviral infections (trials 1973 and 1974 taken together). In the 1975 trial, the results of serological examination were unsatisfactory. Antibodies vs. influenza A Port Chalmers were found in 24.5% of ARD only. The differnce is aetiologically unclarified ARD was statistically significant. Although displaying a significant limitation of clinical infections, the administration of Impulsin did not seem to have any influence on the formation of antibodies.     

Plasmodium knowlesi in the marmoset (Callithrix jacchus).

Common marmosets were shown to be susceptible to Plasmodium knowlesi malaria. The morphology of the parasite was indistinguishable from the observed in the natural host (Macaca fascicularis) and the common laboratory model (Macaca mulatta). A differential susceptibility to P. knowlesi was observed in the 8 marmosets studied. Multiplication rates of parasites were variable over 24 h periods. Five animals died of a fulminating infection within 12--17 days after challenge. Three animals recovered spontaneously from infection and were subsequently resistant to challenge with homologous and heterologous variants and strains of P. knowlesi. This resistance was maintained for intervals up to 100 days between challenge infections.     

Protection Patterns in Duck and Chicken after Homo- or Hetero-Subtypic Reinfections with H5 and H7 Low Pathogenicity Avian Influenza Viruses: A Comparative Study

Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza.     

Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections.

A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56- to 58- and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.     

Oligomeric Recombinant H5 HA1 Vaccine Produced in Bacteria Protects Ferrets from Homologous and Heterologous Wild-Type H5N1 Influenza Challenge and Controls Viral Loads Better than Subunit H5N1 Vaccine by Eliciting High-Affinity Antibodies

Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.     

The Use of a Simple Electron Microscope Serology Procedure to Observe Relationships of Seven Potyviruses

The relationships between bean yellow mosaic (BYMV), bean common mosaic (BCMV), clover yellow vein (CYVV), lettuce mosaic (LMV), potato virus Y (PVY), turnip mosaic (TuMV) and celery mosaic (CeMV) viruses were studied in homologous and heterologous reactions, using simple and relatively rapid electron microscope serology decoration tests. The degree of relationship between these viruses was assessed by the intensity of antibody coating when the viruses were decorated by heterologous antibodies. A close relationship was observed between BYMV and CYVV, and between BYMV and LMV but not between CYVV and LMV. CeMV was quite closely related to BYMV and CYVV. Antibodies to BCMV and BYMV intensely decorated different strains of their own virus, but decoration was negligible in cross reactions.     

Bovine Respiratory Syncytial Virus (BRSV) Pneumonia in Beef Calf Herds Despite Vaccination

The present report describes the clinical, pathological, serological and virological findings in calves from 2 larger Danish beef herds experiencing outbreaks of pneumonia. The calves had been vaccinated with an inactivated bovine respiratory syncytial virus (BRSV) vaccine 2 months prior to the outbreak. The clinical signs comprised nasal discharge, pyrexia, cough and increased respiratory rates. A total of 28 calves died in the 2 herds. The laboratory investigations revealed that BRSV was involved and probably initiated both outbreaks. Furthermore, the serological results suggested that the vaccine induced only sparse levels of antibodies probably due to the presence of maternally derived antibodies at the time of vaccination. Necropsy findings in 5 calves revealed changes typical for infectious pneumonia with involvment of BRSV. In conclusion, vaccination of calves against BRSV in 2 Danish beef herds failed to protect the calves against severe or even fatal BRSV mediated respiratory disease 2 months later.