Malte Dauber

Porcine Teschoviruses Comprise at Least Eleven Distinct Serotypes: Molecular and Evolutionary Aspects

ABSTRACT Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.

Sequencing of Porcine Enterovirus Groups II and III Reveals Unique Features of Both Virus Groups

ABSTRACT The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5′-cloverleaf structure.

Porcine reproductive and respiratory syndrome virus Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

Detection and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus by Multiplex Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV) are classified into the two distinct genotypes “North American (NA, type 2)” and “European (EU, type 1)”. In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV), characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

Vaccination with Newcastle Disease Virus Vectored Vaccine Protects Chickens Against Highly Pathogenic H7 Avian Influenza Virus

Abstract A recombinant Newcastle disease virus (NDV) was engineered to express the hemagglutinin (HA) gene of avian influenza virus (AIV) subtype H7. The HA gene was inserted between the genes encoding NDV fusion and hemagglutinin–neuraminidase proteins. Within the H7 open reading frame, an NDV gene end-like sequence was eliminated by silent mutation. The expression of H7 protein was detected by western blot analysis and indirect immunofluorescence. The existence of H7 protein in the envelope of recombinant Newcastle disease virions was shown by immunoelectron microscopy. The protective efficacy of recombinant NDVH7m against virulent NDV, as well as against highly pathogenic avian influenza virus (HPAIV), was evaluated in specific-pathogen-free chickens. After a single immunization, all chickens developed NDV-specific, as well as AIV H7-specific, antibodies and were completely protected from clinical disease after infection with a lethal dose of virulent NDV or the homologous H7N1 HPAIV, while all control animals died within four days. Shedding of AIV challenge virus was strongly reduced compared to nonvaccinated control birds. Furthermore, the immunized birds developed antibodies against the AIV nucleoprotein after challenge infection. Thus, NDVH7m could be used as a marker vaccine against subtype H7 avian influenza.

Characterization of a Novel Picornavirus Isolate from a Diseased European Eel (Anguilla anguilla)

ABSTRACT A novel picornavirus was isolated from specimens of a diseased European eel (Anguilla anguilla). This virus induced a cytopathic effect in eel embryonic kidney cells and high mortality in a controlled transmission study using elvers. Eel picornavirus has a genome of 7,496 nucleotides that encodes a polyprotein of 2,259 amino acids. It has a typical picornavirus genome layout, but its low similarity to known viral proteins suggests a novel species in the family Picornaviridae.

Koi herpes virus: do acipenserid restitution programs pose a threat to carp farms in the disease-free zones?

In 2003 and subsequent years, a substantial number of Polish fish farms experienced mass mortality of carp (Cyprinus carpio)—their major culture species. Clinical signs have led to the assumption that these heavy losses may have been caused by infection with the koi herpes virus (KHV), which was first isolated and diagnosed in carps from the Rybacka Stacja Doświadczalna (the Fisheries Research Station) of the Agricultural University in Szczecin (Sadowski and Kempter 2004, Bergmann et al. 2006). In the wake of this finding, many research centres in Poland have initiated projects focusing on KHV, in its different aspects, and attempted to develop diagnostic tests for carp cultures (Kempter et al. 2008a). According to the State Veterinary Research Institute in Pulawy (Panstwowy Instytut Weterynaryjny – Panstwowy Instytut Badawczy), this problem is not only related to the carp market and the stocking material turnover. The research projects carried out at the Pulawy institute focuses mainly on the other species, suspected of being a vector for KHV. This assumption is based on the fact that the clinical signs have hitherto not been observed in any other species except Cyprinus carpio (common carp and koi). Many research facilities, such as the the ACTA ICHTHYOLOGICA ET PISCATORIA (2009) 39 (2): 119–126 DOI: 10.3750/AIP2009.39.2.06

Identification of structural proteins of koi herpesvirus

Abstract As a prerequisite for development of improved vaccines and diagnostic tools for control of the fish pathogen koi herpesvirus, or cyprinid herpesvirus 3 (CyHV-3), we have started to identify putative viral envelope and capsid proteins. The complete or partial CyHV-3 open reading frames ORF25, ORF65, ORF92, ORF99, ORF136, ORF138, ORF146, ORF148, and ORF149 were expressed as bacterial fusion proteins, which were then used for preparation of monospecific rabbit antisera. All of the sera that were obtained detected their target proteins in cells transfected with the corresponding eukaryotic expression plasmids. However, only the type I membrane proteins pORF25, pORF65, pORF99, pORF136 and pORF149 and the major capsid protein pORF92 were sufficiently abundant and immunogenic to permit unambiguous detection in CyHV-3-infected cells. In indirect immunofluorescence tests (IIFT), sera from naturally or experimentally CyHV-3-infected carp and koi predominantly reacted with cells transfected with expression plasmids encoding pORF25, pORF65, pORF148, and pORF149, which represent a family of related CyHV-3 membrane proteins. Moreover, several neutralizing monoclonal antibodies raised against CyHV-3 virions proved to be specific for pORF149 in IIFT of transfected cells and in immunoelectron microscopic analysis of CyHV-3 particles. Since pORF149 appears to be an immunorelevant envelope protein of CyHV-3, a recombinant baculovirus was generated for its expression in insect cells, and pORF149 was shown to be incorporated into pseudotyped baculovirus particles, which might be suitable as diagnostic tools or subunit vaccines.

Isolation and molecular characterization of a second serotype of the encephalomyocarditis virus

Encephalomyocarditis virus (EMCV) and Theilovirus are the two species of the Cardiovirus genus. Whereas theiloviruses comprise several sero-/genotypes, all known EMCV isolates are serologically very similar and are thought to belong to one serotype, named EMCV-1. Here, a novel EMCV type is described. Strain RD 1338 (D28/05) was isolated from a wood mouse (Apodemus sylvaticus) in Germany and can be distinguished from EMCV-1 by serological and molecular means. Failure of EMCV-1 specific hyperimmune sera to neutralize RD 1338 suggests a distinct serotype. The viral genome was de novo sequenced using next-generation Illumina/Solexa technologies. Considerable differences of the BC-loop/loop I/loop II sequences of VP1, the VP2 puff and the VP3 knob provide a structural basis for deviant serological properties. Sequence alignments reveal amino acid identities of 75 percent for the P1 region and 84 percent for the P2 and P3 regions when comparing RD 1338 to EMCV-1 strains and some 60 percent and less than 50 percent amino acid identities, respectively, for comparisons with theilovirus strains. Phylogenetic analyses of the P1, 2C and 3CD gene regions support the establishment of an EMCV-2 serotype. In contrast to the theilovirus sero-/genotypes that show a narrow host range, EMCV-1 infects a wide variety of hosts. The host range of EMCV-2 remains to be determined.

Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture

A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established. One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF). This mAb is very well suited for diagnosis of PEV infections. However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM). Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM. Both mAbs presumably recognize conformation-dependent epitopes of the virus.