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

Pea-derived vaccines demonstrate high immunogenicity and protection in rabbits against rabbit haemorrhagic disease virus

Vaccines against rabbit haemorrhagic disease virus (RHDV) are commercially produced in experimentally infected rabbits. A genetically engineered and manufactured version of the major structural protein of RHDV (VP60) is considered to be an alternative approach for vaccine production. Plants have the potential to become an excellent recombinant production system, but the low expression level and insufficient immunogenic potency of plant-derived VP60 still hamper its practical use. In this study, we analysed the expression of a novel multimeric VP60-based antigen in four different plant species, including Nicotiana tabacum L., Solanum tuberosum L., Brassica napus L. and Pisum sativum L. Significant differences were detected in the expression patterns of the novel fusion antigen cholera toxin B subunit (CTB)::VP60 (ctbvp60(SEKDEL)) at the mRNA and protein levels. Pentameric CTB::VP60 molecules were only detected in N. tabacum and P. sativum, and displayed equal levels of CTB, at approximately 0.01% of total soluble protein (TSP), and traces of detectable VP60. However, strong enhancement of the CTB protein content via self-fertilization was only observed in P. sativum, where it reached up to 0.7% of TSP. In rabbits, a strong decrease in the protective vaccine dose required from 48-400 microg potato-derived VP60 [Castanon, S., Marin, M.S., Martin-Alonso, J.M., Boga, J.A., Casais, R., Humara, J.M., Ordas, R.J. and Parra, F. (1999) Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J. Virol. 73, 4452-4455; Castanon, S., Martin-Alonso, J.M., Marin, M.S., Boga, J.A., Alonso, P., Parra, F. and Ordas, R.J. (2002) The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants. Plant Sci. 162, 87-95] to 0.56-0.28 microg antigenic VP60 (measured with VP60 enzyme-linked immunosorbent assay) of crude CTB::VP60 pea extracts was demonstrated. Rabbits immunized with pea-derived CTB::VP60 showed anti-VP60-specific antibodies, similar to RikaVacc((R))-immunized rabbits, and survived RHDV challenge

Comparison of Porcine Epidemic Diarrhea Viruses from Germany and the United States, 2014

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States

Natural infection of pregnant cows with Schmallenberg virus--a follow-up study.

Schmallenberg virus (SBV), an orthobunyavirus discovered in European livestock in late 2011 for the first time, causes premature or stillbirth and severe fetal malformation when cows and ewes are infected during pregnancy. Therefore, cattle of two holdings in the initially most affected area in Germany were closely monitored to describe the consequence for fetuses and newborn calves. Seventy-one calves whose mothers were naturally infected during the first five months of pregnancy were clinically, virologically, and serologically examined. One calve showed typical malformation, another one, born without visible abnormalities, was dead. Two cows aborted during the studied period; spleen and brain samples or meconium swabs were tested by real-time PCR, in none of the fetuses SBV-specific RNA was detectable and the tested fetal sera were negative in a commercially available antibody ELISA. In contrast, in nine clinically healthy calves high SBV-antibody titers were measurable before colostrum intake, and in meconium swabs of six of these animals viral RNA was present as well. The mothers of all nine seropositive calves were presumably infected between days 47 and 162 of gestation, which is within the critical timeframe for fetal infection suggested for SBV and related viruses

Six Years (2011–2016) of Mandatory Nationwide Bovine Viral Diarrhea Control in Germany—A Success Story

Bovine viral diarrhea (BVD) is one of the most important infectious diseases in cattle, causing major economic losses worldwide. Therefore, control programs have been implemented in several countries. In Germany, an obligatory nationwide eradication program has been in force since 2011. Its centerpiece is the detection of animals persistently infected (PI) with BVD virus, primarily based on the testing of ear tissue samples of all newborn calves for viral genome or antigen, and their removal from the cattle population. More than 48,000 PI animals have so far been detected and removed. Between the onset of the program and the end of 2016, the prevalence of these animals among all newborn calves decreased considerably, from 0.5% to less than 0.03%. The number of cattle holdings with PI animals likewise decreased from 3.44% in 2011 to only 0.16% in 2016. Since a large number of naïve, fully susceptible animals are now confronted with BVD virus, which is still present in the German cattle population, the challenge of the coming years will be the identification of remaining PI animals as quickly and efficiently as possible, and the efficient protection of BVD-free farms from reinfection

Detection of antibodies against Schmallenberg virus in wild boars, Belgium, 2010-2012

peer reviewedIn the summer/fall of 2011, a nonspecific febrile syndrome characterized by hyperthermia and drop in milk production with occasional reports of watery diarrhea and abortion was reported among dairy cows on farms in northwestern Europe. Further, in November 2011, an enzootic outbreak of malformed neonates emerged in several European countries, with stillbirth and birth at term of lambs, kids and calves with neurological signs or malformations of the head, spine, or limbs. Both syndromes were associated with the presence in the blood (adults) or in the central nervous system (newborns) of a new Shamonda/Sathuperi-like orthobunyavirus, provisionally named Schmallenberg virus (SBV) after the town in Germany where the first positive clinical samples were identified. Defining as precisely as possible the host range of the newcomer is a key point to predict the outcome of the emergence of SBV disease in Europe. In this respect, it must be pointed out that orthobunyaviruses infect more animal species than those in which the foetus is damaged. Recently, serological evidence for SBV infection in wild ruminant species (Cervus elaphus and Capreolus capreolus) was reported (Linden et al., 2012). In the present study, the objective was to seek after serological evidence of SBV infection among wild boars living in a geographical area where exposure to infected insect vectors was high in 2011, as judged from the very high seroprevalence reported among cattle in that region. About 700 animals were sampled during the 2010-2012 hunting seasons. All serum samples collected during the fall of 2010 were seronegative. On the contrary, apparent seroprevalence among wild boars in 2011 was ~27% and started to decline in 2012 (~11%). Acquired immunity against the new virus was thus already very high in the wild boar populations sampled in the fall 2011, suggesting that the new virus had quickly spread throughout the region since its emergence about 250 km northeast in the late summer 2011. The drop in seroprevalence recorded in 2012 suggests that the virus was no more circulating in the region

Summary of calves borne in both farms in North Rhine-Westphalia, Germany.

<p>The first number indicates the total number of calves in the respective group, inside the parentheses the number of calves is given separately for both holdings.</p

BVD-2 outbreak leads to high losses in cattle farms in Western Germany

In November 2012, a dairy farmer in the district Kleve first observed a reduction in milk yield, respiratory symptoms, nasal discharge, fever, sporadic diarrhoea and sudden deaths in dairy cows and calves. In the following months, further farms were found infected with cattle showing similar clinical signs. An epidemiological investigation was carried out to identify the source of infection, the date of introduction, potential transmission pathways and to analyse the extent of the epidemic. Furthermore, laboratory analyses were conducted to characterise the causative agent. BVDV had been diagnosed in the index herd in December 2012, but due to the atypical clinical picture, the virus was not immediately recognised as the causative agent. Further laboratory analysis showed that this outbreak and subsequent infections in the area were caused by a BVD type 2c virus with a characteristic genome insertion, which seems to be associated with the occurrence of severe clinical symptoms in infected cattle. Epidemiological investigations showed that the probable date of introduction was in mid-October 2012. The high risk period was estimated as three months. A total of 21 affected farms with 5325 cattle were identified in two German Federal States. The virus was mainly transmitted by person contacts, but also by cattle trade and vehicles. The case-fatality rate was up to 60% and mortality in outbreak farms varied between 2.3 and 29.5%. The competent veterinary authorities imposed trade restrictions on affected farms. All persons who had been in contact with affected animals were advised to increase biosecurity measures (e.g. using farm-owned or disposable protective clothing). In some farms, affected animals were vaccinated against BVD to reduce clinical signs as an “emergency measure”. These measures stopped the further spread of the disease

Detailed analyses of newborn calves in 2 farms in North Rhine-Westphalia.

<p>Meconium samples of calves whose ELISA results are marked in red tested positive by real-time RT-PCR; blue ELISA results correspond to negative PCR results. The day of gestation at the estimated time of SBV-infection of the respective cow is symbolized by a light blue area.</p