Protection of chickens vaccinated with combinations of commercial live infectious bronchitis vaccines containing Massachusetts, Dutch and QX-like serotypes against challenge with virulent infectious bronchitis viruses 793B and IS/1494/06 Israel variant 2.

Infectious bronchitis virus (IBV) is a coronavirus which affects chickens of all ages. IBV mainly causes respiratory disease but can also result in reduced weight gain, reduced egg production, increased frequency of abnormal eggs and increased rates of mortality. Vaccination is the most important way to control the disease. Nevertheless, novel strains of infectious bronchitis (IB) continue to emerge in the field. In order to respond promptly, combinations of existing IB vaccines are frequently tested to see whether they can provide cross-protection. The efficacy of a combination of vaccines based on Massachusetts, Dutch and QX-like IB strains against emerging IB Israel variant 2 and IB 793B strains was assessed by means of four challenge studies. At least 80% of the birds vaccinated with IB H120 (Mass type) combined with IB D274 (Dutch type) followed by a QX-like IB vaccine booster or vaccinated with a combination of IB H120, IB D274 and QX-like IB were protected against a challenge with IB 793B. In addition, IB 1263 (Mass type) boosted by QX-like IB showed an 85% protection following challenge with IB 793B. A combination of IB H120 and IB D274 boosted by QX-like IB vaccine conferred 70% protection whilst H120 and IB D274 combination on its own showed 61.1% protection against Israel variant 2 challenge. IB 1263 boosted by a QX-like IB vaccine showed 50% protection against IB Israel variant 2. Therefore, it can be concluded that a combination of the IB H120, IB D274 and QX-like IB confers broad protection against different non-related virulent IB strains.

The use of RT-PCR for determination of separate end-points for the strains IB H120 and IB D274 in titration of the combination vaccine Poulvac IB® primer.

Poulvac IB® Primer is a lyophilized vaccine containing two attenuated infectious bronchitis strains in one vial, IB H120 and IB D274. For quantification of the viral content of the vaccine, dilution series of the final product are inoculated in embryonated chicken eggs. After the incubation period of seven days standard practice is for the embryos to be taken from each egg and examined visually for IB specific lesions; these readings are used to determine an end-point in viral titrations. The result is a titre value to which both strains contribute. However, it is not clear what the live virus titre is for strain IB H120 and for strain IB D274. In order to determine end-points in the titration for each of the two strains, we collected the allantoic fluids from each egg after the incubation period and tested these for the presence of IB H120 and IB D274 by a strain specific reverse phase PCR. Based on the data obtained by PCR we were able to determine an end-point for each of the two strains. For a given commercial batch of Poulvac IB primer we determined titres of 10(6.31) EID50 per vial for IB H120 and 10(6.59) EID50 for IB D274 using PCR for end-point determination. These end-points matched well with the end-point determined for both strains cumulatively after visual examination, i.e. 10(6.67) EID50 per vial. It is concluded that PCR is a suitable means to determine end-points in titrations of live viruses.

Efficacy and safety of an attenuated live QX-like infectious bronchitis virus strain as a vaccine for chickens.

The attenuation of infectious bronchitis (IB) QX-like virus strain L1148 is described. The virus was passaged multiple times in embryonated specific pathogen free (SPF) chicken eggs, and at different passage levels samples were tested for safety for the respiratory tract and kidneys in 1-day-old SPF chickens. There was a clear decrease in pathogenicity for the respiratory tract and kidneys when the virus had undergone a large number of passages. Passage level 80 was investigated for safety for the reproductive tract in 1-day-old and 7-day-old SPF chickens. In 1-day-old chickens, 12.5% of the vaccinated birds had macroscopic lesions. No lesions were observed if the chickens had been vaccinated at 7 days of age. Passage level 80 was investigated for its ability to spread from vaccinated to non-vaccinated chickens and for dissemination in the body. The virus was able to spread from vaccinated chickens to groups of non-vaccinated chickens, and in the vaccinated birds the virus was found frequently in oro-pharyngeal and cloacal swabs. A fragment of the hypervariable region of the S1 protein of passage level 80 was sequenced and revealed nucleotide changes resulting in two amino acid substitutions. Passage level 80 was given additional passages to levels 82 and 85. Both passage levels were tested for efficacy in SPF chickens and passage level 85 was tested for efficacy in commercial chickens with maternally derived antibodies (MDA) against a challenge with QX-like strain IB D388. In both SPF chickens and chickens with MDA, the vaccines based on strain IB L1148 were efficacious against challenge.

Determination of optimal conditions for thawing and diluting cell-bound CVI 988 Marek's disease vaccine and stability of the diluted vaccine.

The cell-associated vaccine strain CVI 988, which is the active component of several commercial Marek's disease vaccines, normally is frozen and stored in liquid nitrogen. In order to ascertain good efficacy of the vaccine, it is crucial that the right procedures are followed for thawing and diluting of the virus. In the study presented here, ampoules containing the frozen product were taken from storage in liquid nitrogen and were thawed in a water bath at 27 C, which is similar to a lukewarm bath, and in a water bath at 37 C, with and without agitation. The effect of the thawing procedure on the live virus titer of the vaccine was investigated. Samples of thawed vaccine were diluted in diluent with different temperatures, and live virus titers were determined directly after dilution and after incubation of the diluted vaccine at different temperatures. The results show that directly after thawing in the water baths at 27 C and 37 C, with or without agitation, the live virus titers for CVI 988 were all in the same range. After incubation of the thawed virus at both temperatures for 15 min, the live virus titers were still in the same range. Decreases in live virus titers were observed after incubation for 4 hr. Live virus titration of the vaccine in diluent in different temperatures revealed that the highest titers were found with diluent at a temperature of 30 C to 37 C and the lowest titers in diluent at a temperature of 5 C. In addition, a combination product containing cell-associated CVI 988 and cell-associated herpesvirus of turkeys (HVT) was tested. For this combination product, the titers for HVT also were highest in diluent with a high temperature (i.e., 37 C), whereas the titers for CVI were highest in diluent at a temperature of 22 C. Both strains had relatively low titers in diluent at 5 C. After incubation of the diluted vaccine at the various temperatures for several hours, again, live virus titrations were done. Live virus titers were most stable with diluent at temperatures of 22 C. Vaccine virus diluted in diluent at 37 C could be stabilized by placing the diluted vaccine at 5 C directly after diluting. After evaluation of these data, the following is recommended. For thawing of the vaccine, a water bath at approximately 27 C, which is similar to a lukewarm bath, is preferred. For diluting the vaccine, diluent should be used at a temperature of 22 C or higher. If diluted in diluent at temperatures higher than 22 C, the diluted vaccine should be stored under cooling in order to avoid titer losses.

Increased virulence of Marek's disease virus type 1 vaccine strain CV1988 after adaptation to qt35 cells.

CVI988/Rispens strain of Marek's disease virus type I (MDV-1) is widely used as efficient vaccine to control Marek's disease (MD) in chicken flocks. Similarly to other live MD vaccine viruses it is propagated in freshly prepared chicken embryo fibroblasts (CEF). In this study, MDV-1 CVI988/Rispens strain was adapted to QT35 cells. The adapted virus, designated QT-CVI, exhibited similar cytopathic effect (CPE) in vitro to that of parental virus propagated in CEE In contrast, QT-CVI induced MD symptoms typical for mild MDV-1 strains after injection to birds. For identification of differentially expressed transcripts that might be involved in increased virulence of QT-CVI, we performed subtractive suppression hybridization (SSH). Subtracted PCR products mapped within MDV-1 BamHI-A and -H fragments and differential gene expression was also confirmed by Northern blot analysis with probes derived from these regions. To examine possible divergence at the virus genome level, PCR analysis was carried out. The BamHI-H fragment- specific 132 bp repeats were present at variable copy number, ranging from 2 to more than 30 copies in both CVI988/ Rispens and QT-CVI DNAs. PCR assays with primers mapping at the US/lRS junction identified CVI988/ Rispens-specific insertion of 116 bp in the region upstream of the ICP4 open reading frame (ORF). PCR analysis was positive also for DNA from non-infected QT35 cells and was consistent with the observation of Yamaguchi et al. (J Virol. 74, 10176-10186, 2000) who have found that QT35 cells carry a latent MDV-1 genome. It is likely, that adaptation of CVI988/Rispens to QT35 cells resulted in reactivation of an endogenous MDV-1 or at least in induction of expression of virulence-related transcripts that have consequently led to QT-CVI pathogenicity for chickens.

Glycoprotein gD of MDV lacks functions typical for alpha-herpesvirus gD homologues.

Glycoprotein D (gD) belongs to family of conserved structural proteins of alpha-herpesviruses. During productive infection of cells by herpes simplex virus 1 (HSV-1) gD has several important functions, is involved in virus penetration to and release from infected cells and is one of main targets of neutralizing antibodies. Similar functions are shared also by other alpha-herpesvirus gD homologues. Surprisingly, in previous studies it was found that MDV gD expression could not be detected during infection in vitro using immunological methods. In this study we have analyzed expression of MDV gD and its biological consequences. In vitro expression using rabbit reticulocyte lysate and/or overexpression in transfected cells showed that the second ATG codon is required for synthesis of mature, glycosylated gD. In addition, it was found that gD overexpression is neither toxic for transfected cells nor is involved in membrane fusion. After MDV infection of a proprietary cell line stably transfected with plasmid overexpressing MDV gD, no viral particles could be found in culture. On the other hand, cells overexpressing the MDV gD were sensitive to MDV infection in similar way as parental, non-transfected cells. From our study and results of other authors we propound the following conclusions: (i) MDV gD expression is blocked during in vitro infection at transcription level; (ii) MDV gD is lacking many important functions characteristic for other alpha-herpesvirus gD homologues; (iii) overexpression of single MDV gD does not result in production of mature infectious MDV particles.

Efficacy of a combination vaccine containing MDV CVI 988 strain and HVT against challenge with very virulent MDV.

With the emergence of very virulent Marek's disease virus (MDV) strains, vaccines based on herpesvirus of turkeys (HVT) appear to be not powerful enough to confer full protection, whereas in chicken flocks vaccinated with MDV CVI 988 strain protective immunity sometimes is generated not early enough for full protection. For this reason combination vaccines containing HVT as well as CVI 988 have been developed. In this paper the beneficial effect of combining both types of virus strains in one vaccine for early protection is shown in a vaccination challenge experiment, in which one-day-old chickens were vaccinated with suboptimal dosages of the monovalent vaccines and the same dosages in a combination vaccine. After 5 days the chickens were challenged with a very virulent MDV strain and subsequently observed for a period of approx. 50 days. It appeared that the combination vaccine provided better early protection than the monovalent vaccines. In addition, the combination vaccine was tested as vaccine administered in ovo. It appeared that after in ovo vaccination the vaccine conferred adequate protection against challenge with a very virulent MDV strain, 5 days after hatch, and that protection after in ovo vaccination was similar to that obtained after subcutaneous vaccination with the same combination vaccine.

Mucosal immunoadjuvant activity of liposomes: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with an influenza subunit vaccine and coadministered liposomes.

This paper reports on a novel immunoadjuvant activity of liposomes. An influenza subunit preparation, containing the isolated viral surface antigens, was incorporated in a liposomal formulation. Administration of this vaccine to mice via the intranasal (i.n.) route resulted in a stimulated serum IgG response relative to the response to i.n. immunization with the antigen alone. In addition, the liposomal vaccine induced a secretory IgA (sIgA) response in the mucosa of the lungs and nasal cavity. Both serum IgG and sIgA responses persisted up to at least 21 weeks postimmunization. Immune stimulation was observed with negatively charged liposomes consisting of phosphatidylcholine (PC), cholesterol and dicetylphosphate (DCP), but not with zwitterionic liposomes, consisting of PC and cholesterol alone. Remarkably, for stimulation of serum IgG responses and induction of an sIgA response, liposomes could be simply mixed with the antigen. Moreover, i.n. administration of empty liposomes up to 48 h prior to i.n. immunization with the subunit antigen also resulted in immune stimulation, indicating that the liposomes did not exert their adjuvant effect by acting as a carrier for the antigen. The liposomal vaccine conferred protection against infection. It is concluded that liposomes, administered i.n., provide a promising adjuvant system for stimulation of antibody responses in general, and mucosal sIgA responses in particular.

Immune inhibition of virus release from human and nonhuman cells by antibody to viral and host cell determinants.

Immune inhibition of release of the DNA viruses, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and surprisingly two herpes viruses, bovine mamillitis and equine abortion, were not inhibited by either anti-viral or anti-host sera. Using the herpes simplex virus model, inhibition of virus release was detected in different cells of human and nonhuman origin with cross-inhibition between cell lines of different origin; thus, this form of immunotherapy may not require antibody to be tissue or organ specific. Evidence of inhibition of virus release from neoplastic and leukemic cell lines suggests possible application of this approach to control of virus-mediated leukoproliferative pathology (e.g. Burkitt's lymphoma or adult T cell leukemia).

Virus neutralizing activity induced by synthetic peptides of glycoprotein D of herpes simplex virus type 1, selected by their reactivity with hyperimmune sera from mice.

Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30. 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9-21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9-21, 10-24, 151-165, 216-232, 282-301 and with peptide 340-354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10-24, 151-165, 216-232, 244-267, 260-274, 270-284, 260-284, 282-301, 300-314, 340-354, 348-362 and 355-369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10-24, which covers the same epitope as peptide 9-21, neutralized HSV-1 in vitro.

Reactivity of human sera with overlapping synthetic peptides of herpes simplex virus type 1 glycoprotein D.

Thirty eight human sera, seropositive for herpes simplex virus (HSV) and 56 human sera, seronegative for HSV by immunofluorescence and by ELISA, were investigated for reactivity with a series of overlapping synthetic peptides of HSV type 1 glycoprotein D (gD-1). Thirty four out of the 38 human sera positive for HSV reacted with peptides located between residues 300 and 369; the HSV-negative sera reacted with six of the gD-1 peptides, but with none of the peptides within residues 300 to 369.

The influence of different adjuvants on the immune response to a synthetic peptide comprising amino acid residues 9-21 of herpes simplex virus type 1 glycoprotein D.

The immuno-modulating properties of different adjuvant systems on the murine humoral and cellular immune response to a synthetic peptide comprising amino acid residues 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were investigated. For immunization, the peptide was conjugated to ovalbumin or bovine serum albumin by glutaraldehyde and the adjuvants used in this study were Freund's complete adjuvant (FCA), aluminium hydroxide, the Ribi adjuvant system (RAS) and two non-ionic block polymer surfactants, viz. L101 and 31R1, in oil in water emulsions. High anti-peptide antibody titers were obtained after immunization with FCA, aluminium hydroxide, RAS and L101. All adjuvants, except RAS, stimulated the induction of delayed type hypersensitivity obtained after immunization with peptide 9-21 coupled to ovalbumin and elicited by injection of purified HSV-1 virions in the footpad. Challenge with a lethal dose of HSV-1 showed that mice immunized with peptide 9-21 coupled to ovalbumin in combination with FCA, RAS and L101, respectively, were significantly protected. Although immunization with peptide 9-21 coupled to ovalbumin combined with aluminium hydroxide stimulated induction of delayed type hypersensitivity, no significant protective immunity against the challenge was generated.

Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro.

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro.

The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme-linked immunosorbent assay.

Rabbits were immunized with synthetic peptides of herpes simplex virus type 1 glycoproteins, coupled to a carrier protein with glutaraldehyde. Antibodies directed against the peptides were determined in an enzyme-linked immunosorbent assay (ELISA). Either free peptides or peptides coupled with glutaraldehyde to another carrier protein than the one used for immunization were used as the coating antigen. When conjugated peptides were used as the coat, it was necessary in some instances to correct the antibody titers for a substantial amount of antibody activity against glutaraldehyde. When free peptides were used, optimal coating conditions with regard to pH and ionic strength had to be determined, since some peptides failed to coat under standard conditions, at pH 9.6. The results show that some peptides needed stringent pH conditions while others could be coated in a broad pH range. The addition of 0.6 M NaCl had a favorable effect on peptide coating.

Immunological properties of an N-terminal fragment of herpes simplex virus type 1 glycoprotein D expressed in Escherichia coli.

The N-terminal fragment, comprising residues -5 to 55 of herpes simplex virus type 1 glycoprotein D was expressed as a beta-galactosidase fusion protein in Escherichia coli. This gD-fusion protein reacts with monoclonal antibody LP 14 directed against glycoprotein D of HSV. Antisera obtained after immunization of rabbits with purified gD-fusion protein react with HSV-1 gD in a Western blot and with N-terminal synthetic peptides of gD. In addition, these antisera are able to neutralize viral infectivity in vitro.

Structural properties and reactivity of N-terminal synthetic peptides of herpes simplex virus type 1 glycoprotein D by using antipeptide antibodies and group VII monoclonal antibodies.

To investigate the contribution of individual amino acids to the antigenicity of the N-terminal region of herpes simplex virus type 1 glycoprotein D, a series of 14 overlapping synthetic peptides within residues 1 to 30 were examined for their reactivity with monoclonal antibody LP14 (a group VII monoclonal antibody; in herpes simplex virus mutants resistant to LP14, arginine 16 is substituted by histidine) and two antipeptide antisera (antipeptide 9-21 and antipeptide 1-23). Maximal binding was achieved with peptides 9-21, 10-30, 9-30, and 8-30 and the chymotryptic fragment 9-17 of peptide 9-21, suggesting that a major antigenic site is located within residues 10 through 17. Lysine 10 was shown to be essential for high reactivity, either by binding directly to the antibody molecule or by stabilizing an ordered structure of the peptide. The importance of ordered structure was demonstrated by a decrease in reactivity after sodium dodecyl sulfate treatment of peptides 9-21 and 8-30.

Competition for sulfate and ethanol among desulfobacter, desulfobulbus, and desulfovibrio species isolated from intertidal sediments.

Competition for sulfate and ethanol among Desulfobacter, Desulfobulbus, and Desulfovibrio species isolated from estuarine sediments was studied in energy-limited chemostats. Desulfovibrio baculatus was the most successful competitor for limiting amounts of sulfate and ethanol, followed by Desulfobulbus propionicus. The success of Desulfovibrio baculatus was dependent on the availability of sufficient iron. Of the three species studied, Desulfobacter postgatei was the least successful competitor for limiting amounts of sulfate. Although stimulating the growth of Desulfobacter postgatei, addition of Ca-saturated illite particles to culture media did not affect the outcome of competition for sulfate. Thus, under sulfate limitation acetate accumulated. This phenomenon was briefly discussed in relation to the flow of electrons during anaerobic mineralization in marine and estuarine sulfate-limited sediments.

Competition for L-lactate betweenDesulfovibrio, Veillonella, andAcetobacterium species isolated from anaerobic intertidal sediments.

Almost equal numbers ofDesulfovibrio, Veillonella, andAcetobacterium species were found in agar shake dilutions of anaerobic intertidal brackish sediments applying L-lactate as the only energy source and sulfate as electron acceptor. Pure cultures of these bacteria were studied in more detail in batch cultures as well as in L-lactate-limited chemostats. The maximal specific growth rates on L-lactate were determined in washout experiments and amounted to 0.16, 0.30, and 0.06 h(-1) forDesulfovibrio baculatus H.L21,Veillonella alcalescens NS.L49, andAcetobacterium NS.L40, respectively. Competition for L-lactate was studied in energy-limited chemostats at a dilution rate of 0.02 h(-1).D. baculatus H.L21 turned out to be the best competitor at low L-lactate concentrations provided that sufficient sulfate and iron were present.V. alcalescens NS.L49 was favored by the absence of sulfate and iron. Coexistence ofD. baculatus H.L21 andV. alcalescens NS.L49 was observed in a L-lactate-limited chemostat with additional sulfate and citrate. Syntrophic growth ofV. alcalescens NS.L49 andAcetobacterium NS.L40 occurred in a L-lactate-limited chemostat in the absence of sulfate. No coexistence betweenD. baculatus H.L21 andAcetobacterium NS.L40 was observed in a L-lactate-limited chemostat without sulfate. Addition of calcium-saturated illite to an energy-limited mixed culture ofV. alcalescens NS.L49 andAcetobacterium NS.L40 induced iron limitation and subsequent washout of theAcetobacterium species. Finally, the ecological niches of the 3 species in relation to the consumption of lactate were discussed.