P.A. van Rijn

Potential new Culicoides vector of bluetongue virus in northern Europe

have swept across the Mediterranean Basin and affected 15 countries, many of which had never experienced the disease previously (Purse and others 2005). In August 2006, following an unprec-edented warm summer, bluetongue took a significant ‘leap’ northwards, appearing unexpectedly in northern Europe and affecting parts of the Netherlands, Belgium, Germany and northern France. To date, the disease has spread across approximately 100,000 km

Vaccination of cattle against bovine viral diarrhoea.

This brief review describes types and quality (efficacy and safety) of bovine viral diarrhoea virus (BVDV) vaccines that are in the market or under development. Both conventional live and killed vaccines are available. The primary aim of vaccination is to prevent congenital infection, but the few vaccines tested are not highly efficacious in this respect, as shown in vaccination-challenge experiments. Vaccination to prevent severe postnatal infections may be indicated when virulent BVDV strains are prevalent. Live BVDV vaccines have given rise to safety problems. A complication for the development of BVDV vaccines is the wide antigenic diversity among wild-type BVDV. There is ample room for improvement of both the efficacy and safety of BVDV vaccines, and it may be expected that better vaccines, among which marker vaccines, will be launched in the future.

Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge

Envelope glycoprotein E2, formerly called E1 or gp51-54, of classical swine fever virus (CSFV) expressed in insect cells protects swine from classical swine fever. Monoclonal antibodies directed against epitopes of domains B and C and subdomain A1 are neutralizing. The domains are located on two structural antigenic units in a proposed model of the antigenic structure of E2. One unit consists of nonconserved antigenic domains B and C and the other contains highly conserved antigenic domain A. We produced several mutant E2 proteins by use of the baculovirus expression system. Two selected mutants were E2 proteins in which one of the two structural antigenic units, unit B/C or unit A, was deleted. The protective capacity of the mutant E2 proteins was investigated in an immunization experiment in pigs. Titres of the neutralizing responses in pigs immunized with mutant E2 proteins were all comparable with that of intact E2. These vaccinated pigs were protected against an intranasal lethal CSFV challenge, indicating that the immune response induced by one structural antigenic unit of E2 can protect pigs against classical swine fever.

A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health

Real-time, reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods in the field of molecular diagnostics and research. The potential of this format to provide sensitive, specific and swift detection and quantification of viral RNAs has made it an indispensable tool for state-of-the-art diagnostics of important human and animal viral pathogens. Integration of these assays into automated liquid handling platforms for nucleic acid extraction increases the rate and standardisation of sample throughput and decreases the potential for cross-contamination. The reliability of these assays can be further enhanced by using internal controls to validate test results. Based on these advantageous characteristics, numerous robust rRT-PCRs systems have been developed and validated for important epizootic diseases of livestock. Here, we review the rRT-PCR assays that have been developed for the detection of five RNA viruses that cause diseases that are notifiable to the World Organisation for Animal Health (OIE), namely: foot-and-mouth disease, classical swine fever, bluetongue disease, avian influenza and Newcastle disease. The performance of these tests for viral diagnostics and disease control and prospects for improved strategies in the future are discussed.

Chimeric classical swine fever viruses containing envelope protein ERNS or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response.

Three chimeric classical swine fever virus (CSFV)/bovine viral diarrhoea virus (BVDV) full-length DNA copies were constructed, based on the infectious DNA copy of the CSFV vaccine strain C. The antigenic region of E2 and/or the complete E(RNS) gene were replaced by the analogous sequence of BVDV II strain 5250. Viable chimeric virus Flc11, in which E(RNS) was replaced, was directly recovered from supernatant of SK6.T7 cells transfected with full-length DNA. Viable chimeric virus Flc9, in which E2 was replaced, resulted in recovery of virus only when SK6.T7 transfected cells were maintained for several passages. However, no virus could be recovered after replacement of both E(RNS) and E2, even after 10 cell passages. Both Flc9 and Flc11 grow in swine kidney cells (SK6), stably maintain their heterologous BVDV sequences and, as assessed by monoclonal antibody typing and radio-immunoprecipitation assays, express their heterologous proteins. Flc9 showed a slower growth rate on SK6 cells than Flc11 and wild-type Flc2 virus. Replacement of E(RNS) or E2 of C-strain-based chimeric viruses did not alter cell tropism compared to wild-type C-strain virus for SK6 and FBE cells. Both Flc9 and Flc11 induced E2 or E(RNS) antibodies, which could be discriminated from those induced after wild-type virus infection, even after repeated vaccination. Furthermore, pigs were completely protected against a lethal CSFV challenge. These results indicate the feasibility of introduction of marker antigens in a live-attenuated marker C-strain vaccine for CSFV.

Culicoides chiopterus as a potential vector of bluetongue virus in Europe.

SIR, — In August 2006 bluetongue appeared for the first time in northern Europe and by December had affected over 2000 sheep and cattle holdings in the five eu member states of Belgium, France, Luxembourg, Germany and the Netherlands. Following a mild winter, bluetongue recrudesced explosively in

Classical Swine Fever Virus Erns Deletion Mutants: trans-Complementation and Potential Use as Nontransmissible, Modified, Live-Attenuated Marker Vaccines

ABSTRACT An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein Erns of classical swine fever virus (CSFV) was used to rescue CSFV Erns deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of Erns from this cell line were indistinguishable from those of CSFV Erns. Two Erns deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of Erns (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of Erns antibodies, due to a deletion of 66 amino acids in Erns. The Erns deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by Erns-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that Erns from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) Erns in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.

Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of Erns or E2 of CSFV

Three mutants with deletions in the E2 gene of the infectious DNA copy of the classical swine fever virus (CSFV) strain-C were constructed: one missing the B/C domain of CSFV-E2 between amino acids (aa) 693 and 746, one missing the A domain between aa 800 and 864, and one missing the complete E2 between aa 689 and 1062. All three CSFV-E2 deletion mutants were unable to generate viable virus, indicating that each of the antigenic domains of E2 is essential for viability of CSFV. To rescue the CSFV-E2 deletion mutants SK6 cell lines constitutively expressing glycoprotein E2 of CSFV were generated. The rescued viruses infected and replicated in SK6 cells as demonstrated by expression of viral proteins, but this primary infection did not result in reproduction of infectious virus. Thus, these E2 complemented viruses are considered non-transmissible. In previous experiments, we showed that simultaneous injection of E(rns) complemented virus (Flc23) via intradermal (ID), intramuscular (IM) or intranasal (IN) routes conferred protection to pigs against a lethal challenge with CSFV [J. Virol. 74 (2000) 2973]. Here, we evaluate different routes of application (ID, IM or IN) with E(rns) complemented virus Flc23 in order to find the best route for complemented CSFVs. Intradermal injection with Flc23 protected pigs against a lethal CSFV challenge, whereas intramuscular injection induced partial protection, and intranasal injection did not mediate a protective immune response in pigs at all. We used the intradermal route of vaccination to test the E2 complemented viruses. Vaccination of pigs via the intradermal route with the E2 complemented CSFVs also resulted in the induction of antibodies and in (partial) protection against CSFV challenge. Pigs vaccinated with E2 complemented virus Flc4 (deletion B/C domain) survived a lethal CSFV challenge, whereas partial protection was induced in pigs vaccinated with Flc47 (deletion E2) or Flc48 (deletion A domain) E2 complemented viruses. Serological data demonstrate that these E2 complemented mutant viruses are, in combination with well known diagnostic tests based on E2, potential marker vaccines for CSF.

Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia

Four antigenic domains (A, B, C and D) on envelope glycoprotein E1 (gp51-54) of hog cholera virus strain Brescia have been specified by using 13 monoclonal antibodies (MAbs) that recognize non-conserved and conserved epitopes. It was shown that the non-conserved epitopes map to the N-terminal half of E1 by analysis of chimeric E1 proteins of strains Brescia and C. Conserved epitopes, however, could not be mapped using this approach. Here we describe mapping of both conserved and non-conserved epitopes on E1 by the use of an extensive set of single and double deletion mutants of E1 of strain Brescia. Deletion mutants were transiently expressed in COS1 cells and analysed by immunostaining with the 13 MAbs directed against strain Brescia and four MAbs directed against strain C. All MAbs bound to the N-terminal half of E1, i.e. amino acids 690 to 866 encoded by the sequence of strain Brescia. Domain B and one epitope in domain C are located between residues 690 and 773. Other epitopes in domain C are located on an extended region, i.e. between residues 690 and 800. Conserved epitopes of domain A are mapped between residues 766 and 866, whereas the only non-conserved epitope in this domain is located between residues 766 and 813. Domain D, represented by one MAb, is located in the same region as this non-conserved epitope of domain A, i.e. between residues 766 and 800. The results suggest the presence of two distinct antigenic units on E1, one consisting of domains B and C and the other consisting of domain A.

An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV)

Envelope glycoprotein E2 is the most immunogenic protein of classical swine fever virus (CSFV). In a proposed model of the antigenic structure of E2, the N-terminal half of E2 forms two independent structural antigenic units, A and BC. E2 without transmembrane region (E2-TMR) is expressed and secreted into the medium of insect cells by use of the baculovirus expression system. The immune response induced by E2 protects pigs against CSFV. Recently, we showed that the protective immune response to a homologous CSFV challenge can be induced by a single unit, A or BC, of E2. An indirect blocking ELISA, or complex trapping blocking assay (CTB) based on both units is routinely used worldwide for serological diagnosis of CSFV infections. Here we show that E2-TMR is secreted into the medium as a homodimer. This E2 homodimer was used to develop a CTB detecting antibodies directed against one immunogenic unit of E2. Thus, the protective immune response induced by E2 containing one unit was not detected with a modified CTB based on the other unit, whereas immune responses induced by a variety of low virulent CSFV strains were detected with such a modified CTB. These results indicate that a deletion E2 protein in combination with a modified CTB are feasible as CSF marker vaccine and accompanying differentiating diagnostic test.