Mutational Analysis of the Bovine Hepacivirus Internal Ribosome Entry Site.

In recent years, hepatitis C virus (HCV)-related viruses were identified in several species, including dogs, horses, bats, and rodents. In addition, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV internal ribosome entry site (IRES) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf, and IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' nontranslated region. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with large amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, the absence of pronounced effects in a bovine hepatocyte cell line expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.IMPORTANCE Several members of the family Flaviviridae, including HCV, have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated viral internal ribosome entry sites (IRES), to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. We also studied the possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.

An Outbreak of Porcine Reproductive and Respiratory Syndrome Virus in Switzerland Following Import of Boar Semen.

An outbreak of porcine reproductive and respiratory syndrome virus (PRRSV) occurred in November 2012 in Switzerland (CH), traditionally PRRSV-free. It was detected after a German boar stud informed a semen importer about the detection of PRRSV during routine monitoring. Tracing of semen deliveries revealed 26 Swiss sow herds that had used semen from this stud after its last negative routine monitoring and 62 further contact herds. All herds were put under movement restrictions and examined serologically and virologically. As a first measure, 59 sows from five herds that had previously been inseminated with suspicious semen were slaughtered and tested immediately. Investigations in the stud resulted in 8 positive boars with recent semen deliveries to CH (Seven with antibodies and virus, one with antibodies only). In one boar out of six tested, virus was detected in semen. Of the 59 slaughtered sows, five from three herds were virus-positive. In one herd, the virus had spread, and all pigs were slaughtered or non-marketable animals euthanized. In the remaining herds, no further infections were detected. After confirmatory testings in all herds 3 weeks after the first examination gave negative results, restrictions were lifted in January 2013, and Switzerland regained its PRRSV-free status. The events demonstrate that import of semen from non-PRRS-free countries--even from negative studs--poses a risk, because monitoring protocols in boar studs are often insufficient to timely detect an infection, and infections of sows/herds occur even with low numbers of semen doses. The outbreak was eradicated successfully mainly due to the high disease awareness of the importer and because immediate actions were taken before clinical or laboratory diagnosis of a single case in the country was made. To minimize the risk of an introduction of PRRSV in the future, stricter import guidelines for boar semen have been implemented.

Expression of a truncated hepatitis E virus capsid protein in the protozoan organism Leishmania tarentolae and its application in a serological assay.

Zoonotic infections with hepatitis E virus (HEV) genotype 3 are presumably transmitted via contaminated pig meat products, which raises the necessity for enhanced serological surveillance of pig herds. The aim of the study was to set up a novel protein expression system to overcome the well-known problems in (HEV-) protein expression using the standard Escherichia coli tools such as inclusion body formation and loss of protein conformation. A recombinant strain of the protozoan organism Leishmania tarentolae (L. tarentolae) was therefore established. A fragment of HEV ORF2 coding for a truncated capsid protein of a porcine HEV strain was cloned and parts of the plasmid DNA were introduced into the Leishmania genome, resulting in stably transformed cells. Via a C-terminal His-tag the recombinant HEVΔORF2 protein could be purified and concentrated directly from the medium, resulting in a total protein amount of approximately 1.4 mg/l Leishmania culture. The recombinant protein was coated on ELISA plates and was proven to be highly reactive and well-suited to be applied in a serological assay. By investigating 144 porcine sera, the in-house assay detected specific antibodies in 43.1% of the samples and demonstrated a higher sensitivity than a commercially available antibody test. Taken together, it was shown that L. tarentolae exhibits a remarkable alternative expression strategy for viral antigens with considerable advantages of a eukaryotic protein expression host.