Molecular detection of hepatitis E virus in German domestic pigs.

In industrial countries, Hepatitis E virus (HEV) transmission to humans is predominantly assumed to be a zoonotic infection. Recently, it has been demonstrated that about 50% of domestic pigs in Germany carry HEV-specific antibodies. However, further investigations concerning the distribution of HEV in different age groups of German domestic pigs, phylogenetic analyses and the viral load in the porcine liver are still pending. Liver samples of all age groups from herds in a pig-dense region in north-western Germany were investigated for the presence and quantity of HEV RNA and subsequently genotyped. Out of 251 liver samples, 34 contained ORF2-specific RNA, whereas 19 samples were positive using ORF1-specific primers, resulting in an overall detection rate of 13.5% and 7.6%, respectively. Especially nursery pigs and growers were tested positive for viral RNA. Furthermore, determination of the HEV copy numbers revealed high replication levels. Up to 10(9) genome copies per g of liver tissue could be detected suggesting a likely high degree of viral spread to the environment. In the HEV-positive liver samples we found no hints for pathohistological changes reflecting the HEV status.The HEV sequences showed marked diversity but could be assigned to HEV genotype 3 without exception. However, by comparing two different genomic fragments, we found indications for infections with two different HEV variants in domestic pigs. Apart from this, the current study confirms the outcome of our recent serological HEV survey and for the first time gives direct proof of HEV infections in the German domestic pig population.

Molecular evidence of very virulent infectious bursal disease viruses in chickens in Ethiopia.

Infectious bursal disease virus (IBDV) is an important immunosuppressive pathogen of chickens worldwide. The introduction and evolution of IBDV in most African countries, especially in Ethiopia, remains unclear. We have investigated IBDV isolates obtained from commercial broilers, indigenous chickens, and pullets. The hypervariable region of the virus protein (VP) 2 and the 5' two-thirds of VP1 of 11 IBDV isolates were characterized by RT-PCR and further sequencing. All isolates were identified as very virulent (vv) IBDV based on the predicted amino acid (aa) sequences of the VP2 protein. Interestingly, the sequence analysis of the 5' two-thirds of VP1 indicated that the Ethiopian IBDV strains have aa residues typical for vvIBDV and for attenuated IBDV strains. Among all IBDV strains included in this study for phylogenetic comparison of VP2 nucleotide sequences, Ethiopian strains form a cluster within the vvIBDV lineage. We have also shown that Ethiopian IBDV strains have mutations in the VP1 region. Their roles in IBDV virulence may require further in vivo studies. As depicted in this study, the nucleotide and aa sequence analysis of VP1 in addition to VP2 is necessary to obtain a clear picture of the molecular evolution of IBDV.

New insights into the antigenic structure of the glycoprotein E(rns) of classical swine fever virus by epitope mapping.

The E(rns) glycoprotein of classical swine fever virus (CSFV) has been studied in detail concerning biochemical and functional properties, whereas less is known about its antigenic structure. In order to define epitopes recognized by CSFV-specific antibodies, the binding sites of seven E(rns)-specific monoclonal antibodies were investigated. Mapping experiments using chimeric E(rns) proteins, site-directed mutagenesis and an overlapping peptide library identified one antigenic region located between amino acids (aa) 55 to 110 on the E(rns) protein of CSFV Alfort/187. The domain comprises three linear motifs *(64)TNYTCCKLQ(72), (73)RHEWNKHGW(81), and (88)DPWIQLMNR(96), respectively, and two aa at position 102 and 107 that are crucial for the interaction with antibodies. Additionally, the presentation of the epitope in a correct conformation is mandatory for an efficient antibody binding. These findings allow a better understanding of the organization and the structure of the E(rns) and provide valuable information with regard to the development of E(rns)-based diagnostic tests.

Description of the first acute bovine diarrhea virus-2 outbreak in Israel.

This is the first report of an acute and fatal outbreak of bovine diarrhea virus (BVDV)-2 infection in Israel. The clinical presentation varied with the age of the affected animals with a bovine-respiratory-complex-like syndrome in young stock, and diarrhea and dysentery only in the lactating stock. Enteritis first appeared in one shed of post-parturient cows; it spread for 6 weeks, until at least 30% of the lactating stock contracted enteritis or dysentery. At the same time, dairy calves aged 10-90 days exhibited severe respiratory disease. Of 79 animals that died, 13/350 (3.7%) were adult lactating cows, and 66/1100 (6%) were young feedlot calves. Phylogenetic analysis of the isolated virus revealed a 95% identity with the corresponding genome parts of various BVDV type 2 sequences. The route of introduction of BVDV-2 into Israel could not be elucidated.

Fatal epizootic equine herpesvirus 1 infections in new and unnatural hosts.

In a zoological collection, four black bears (Ursus americanus) died from neurological disease within six months. Independently in a geographically different zoo, two Thomson's gazelles (Eudorcas thomsoni) and 18 guinea pigs (Cavia porcellus f. dom.) suffered from neurological disorders. In addition, guinea pigs showed abortions and stillbirths. All affected animals displayed a non suppurative meningoencephalitis with intranuclear inclusion bodies. Immunohistology demonstrated equine herpes virus antigen and ultrastructurally herpes viral particles were detected. Virus isolation and molecular analysis identified neurotropic equine herpesvirus (EHV) 1 strains in both epizootics. There is serological evidence of a possible virus transmission from other equids to the affected animals. Cross-species transmission of EHV-1 should be considered in the management of captive wild equids and ungulates, particularly with respect to fatal disease in irreplaceable species.

[Hepatitis E--a new zoonotic disease in Germany?]. / Hepatitis E--eine neue Zoonose in Deutschland?

Hepatitis E virus (HEV) is the most frequent pathogen of a fecal-oral transmitted hepatitis in humans in many developing countries. It is endemic in the Middle East, in India, in Southeast Asia, central Asia as well as in Central and South America. It can be predominantly found in young adults. The mortality rate comes up to 2% whereas in pregnant women death rate can reach about 25%. Until a few years ago, acute hepatitis E was thought to be rare in industrialized countries: most cases were found in persons with a corresponding travel history. However, after improvement of the diagnostic possibilities, an increasing number of autochthonous cases with acute hepatitis E have become evident. Besides, a relatively high seroprevalence of 1 to 5% in the population in industrialized countries is noticeable which is converse to the only sporadic occurrence of acute hepatitis E. As a reason for this an animal reservoir for HEV is being discussed. The first HEV recovering from an animal succeeded in 1997 from a pig in the USA. Genetic typing showed close relationship between the porcine HEVand human HEV types. This confirmed the suspicion on a zoonotic potential of HEV. Transmission experiments revealed that non-human primates were sensitive to the porcine HEV and pigs could be infected with human HEV. Besides, occupational groups with close contact to pigs evidently have an increased risk for HEV infections. In addition, reports of acute hepatitis E after the consumption of undercooked wild boar meat supported the zoonotic potential of HEV. In the domestic pig and wild boar population in several European countries the virus is ubiquitous. In Germany a high percentage of the wild boars was proven to be infected with HEV. In contrast, studies of HEV circulation in domestic pigs are still pending.

Prevalence of Hepatitis E virus-specific antibodies in sera of German domestic pigs estimated by using different assays.

Hepatitis E virus is the causative agent of an acute hepatitis in humans. In industrialized countries, autochthonous hepatitis E cases in the past were mainly of undetermined origin, whereupon nowadays some cases may be linked to zoonotic transmission of HEV from pigs and wild boars. In contrast to several European countries the HEV status of German domestic pigs and a possible risk of transmission are unknown so far. Here, a novel peptide-based ELISA was used to detect HEV-specific antibodies in 1072 sera from German domestic pigs resulting in an average seroprevalence of 49.8% indicating widespread HEV infections in these animals. A comparative testing of 321 randomly selected sera revealed a seroprevalence of 64.8% when using a commercially available ELISA and 43.9% for the novel peptide-based ELISA but concordant results were obtained in both tests only for 56.1% of the sera. Additional re-testing of 23 randomly selected sera with a modified commercially available immunoblot revealed discordant results also. The use of different antigens and the measurement of different immunoglobulin classes are considered to be responsible for the observed variations of the results. Though the present study revealed a high seroprevalence of HEV in the German domestic pig population and a potential risk of transmission to humans, the differing results of the tests highlight the necessity of a standardization of serological assays for comparative seroprevalence and longitudinal studies.

Formation of bovine viral diarrhea virus E1-E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains.

The envelope of bovine viral diarrhea virus (BVDV) contains the glycoproteins Erns, E1 and E2. Complementation of a recombinant vesicular stomatitis virus (VSV) with BVDV glycoproteins resulted in infectious pseudotyped viruses. To elucidate the specific role of each of the single envelope glycoproteins during viral entry, pseudotypes were generated bearing the BVDV envelope proteins in different combinations. Pseudoviruses that contained E1 and E2 but not Erns were infectious, indicating that Erns is dispensable for virus entry. VSV/BVDV pseudotypes with chimeric proteins (the ectodomain of the BVDV glycoprotein and the transmembrane domain of the VSV-G protein) were not infectious. The fact that E1-E2 heterodimers were not detected if one of the proteins was chimeric indicated that the heterodimers are crucial for BVDV entry. It was shown by site-directed mutagenesis that the charged amino acids in the transmembrane domains of BVDV E1 (lysine and arginine) and the charged amino acid in the transmembrane domain of E2 (arginine) play a key role in heterodimer formation. Pseudoviruses bearing the mutation E2-R/A, where the charged amino acid was substituted by alanine, were not infectious, supporting the hypothesis that E1-E2 heterodimers are essential for BVDV entry.

[Feasibility of serological bulk milk testing as a method for BVD surveillance]. / Untersuchungen zur Eignung der serologischen Tankmilchuntersuchung als Einstieg in die BVD-Uberwachung.

A commercial ELISA detecting antibodies against bovine viral diarrhoea Virus (BVDV) was analysed for its applicability for bulk-milk screening. Detection limits were analysed using native and concentrated milk samples (milk treated with rennet and ammonium sulfate precipitated) from 10 cows whose sera showed different reactivity levels in the ELISA and from two cows which gave birth to persistently infected calves during the last year. Further this and a second commercial ELISA were used to screen 591 randomly selected bulk-milk samples. To clarify discrepancies thirty-nine herds were included in a follow-up study. A second bulk-milk sample and serum samples from 10 young cattle of 6 to 28 month of age per herd were analysed for antibodies against BVDV. The results of this second testing and the detection of viremic animals in 4 herds confirmed the results from initial bulk-milk testing with both tests. The analysed test is suitable for bulk-milk testing although its application is limited by vaccination.

Bovine viral diarrhoea eradication and control programmes in Europe.

The economic impact of BVDV infections has led a number of countries in Europe to start eradication or control programmes. While in both cases the primary step is identification and elimination of persistently infected (PI) animals, the strategy applied thereafter is dependent on the density and seroprevalence of the regional cattle population. One of the first countries to design and implement an eradication programme was Sweden in 1993, a country with a relatively low cattle density and no vaccination. For screening, an indirect antibody ELISA for serum, milk and bulk milk samples is being used. The basics of the Swedish model are no vaccination, voluntary participation, and financing of the entire scheme by the subscribing farmers. BVDV-free herds are certified and permanently checked. While in 1993 only about 35% of the herds were seronegative, about 87% were BVDV-free in 2001. The aim of control programmes in high density areas with high seroprevalence is to minimize economic losses by reducing the incidence of PI animals and thereby virus circulation (German model). Participation is voluntary, and parts of the costs are carried by the public animal insurance (Tierseuchenkasse). Screening is performed using an antigen capture ELISA with blood or serum. In Lower Saxony, for example, a herd is declared BVDV unsuspicious if all animals up to 36 months are BVDV antigen negative and the female offspring older than six months is vaccinated twice (an inactivated vaccine is used for basic immunization, and an attenuated live virus vaccine for boosting).