Establishment of a multiplex real-time PCR assay for the detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of bovine herpesvirus type 1

Bovine herpesvirus type 1 (BoHV-1), an alphaherpesvirus, is a major pathogen of cattle causing different syndromes such as infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV) and infectious balanoposthitis (IBP). BoHV-1 control programs have been initiated in several European countries including Germany. One of the major components is the vaccination with inactivated or attenuated glycoprotein E (gE)-deleted live marker vaccines. The aim of this study was the development of a triplex real-time polymerase chain reaction (qPCR) assay for the sensitive, specific and reliable BoHV-1 detection. A BoHV-1-specific glycoprotein D (gD) assay was combined with a gE-specific qPCR system for differentiation between wild-type strains and gE-negative vaccine virus strains. Finally, an internal control based on amplification of the bovine beta-actin gene was introduced to verify efficient DNA extraction and PCR amplification. The analytical sensitivity of the triplex BoHV-1 qPCR enables the detection of 10 genome copies per reaction. Furthermore, the sensitivity of the newly developed qPCR assay was compared to an OIE-validated qPCR and the “gold standard” method of virus isolation in cell culture using 10-fold dilution series of BoHV-1 in extended semen as well as in cell culture medium. For all preparations, the tested qPCR assays showed comparable results and the sensitivity of the triplex qPCR was equal or even greater than that of virus isolation. A broad spectrum of reference strains and field isolates was detected reliably. The specificity of the test was confirmed using nasal swabs, semen and different organ materials of BoHV-1-negative cattle. Bovine herpesviruses type 2, 4 and 5 and further ruminant herpesviruses, namely bubaline herpesvirus (BuHV-1), caprine herpesvirus type 1 and cervine herpesvirus type 1 and 2 (CvHV-1, -2) were tested as well. The close genetic and serological relationship between these viruses combined with their ability to infect bovines may interfere with diagnostics resulting in false-positive results. All non-BoHV-1 herpesviruses were negative in the gD-specific assay, while BuHV-1, CvHV-1 and -2 were tested positive by the gE-specific qPCR. Consequently, the triplex qPCR offers for the first time the possibility to detect some related herpesviruses and distinguish them from BoHV-1 in addition to the simultaneous differentiation of BoHV-1 wild-type and gE-deleted vaccine strains

infectious serum or culture-grown virus?

Schmallenberg virus (SBV), discovered in Europe in 2011, causes mild transient disease in adult ruminants, but fetal infection can lead to severe malformation in cattle, sheep and goats.To elucidate the pathogenesis of this novel orthobunyavirus, considerable efforts are required. A reliable and standardized infection model is essential for in vivo studies. In the present study, two groups of four cattle were inoculated with either serum passaged in cattle only or cell culture-grown virus. The replication of culture-grown SBV in cattle was reduced compared to virus inoculated via infectious serum. In a second experiment, the infectious serum was titrated in calves; the tested batch contained 102.83 infectious doses per mL. Hence, serum-borne virus that was only passaged in the natural host is a suitable option for a standardized SBV infection model

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

Assembly of Replication-Incompetent African Horse Sickness Virus Particles: Rational Design of Vaccines for All Serotypes.

UNLABELLED: African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis. IMPORTANCE: African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated. These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. Importantly, these candidate vaccine strains showed strong protective efficacy against AHSV infection in an IFNAR(-/-) mouse model

Sequence analysis of Schmallenberg virus genomes detected in Hungary

Since its emergence near the German–Dutch border in 2011, Schmallenberg virus (SBV) has been identified in many European countries. In this study, we determined the complete coding sequence of seven Hungarian SBV genomes to expand our knowledge about the genetic diversity of circulating field strains. The samples originated from the first case, an aborted cattle fetus without malformation collected in 2012, and from the blood samples of six adult cattle in 2014. The Hungarian SBV sequences shared ≥99.3% nucleotide (nt) and ≥97.8% amino acid (aa) identity with each other, and ≥98.9 nt and ≥96.7% aa identity with reference strains. Although phylogenetic analyses showed low resolution in general, the M sequences of cattle and sheep origin SBV strains seemed to cluster on different branches. Both common and unique mutation sites were observed in different groups of sequences that might help understanding the evolution of emerging SBV strains

Serological and Molecular Investigation of SARS-CoV-2 in Horses and Cattle in Switzerland from 2020 to 2022

Horses and cattle have shown low susceptibility to SARS-CoV-2, and there is no evidence of experimental intraspecies transmission. Nonetheless, seropositive horses in the US and seropositive cattle in Germany and Italy have been reported. The current study investigated the prevalence of antibodies against SARS-CoV-2 in horses and cattle in Switzerland. In total, 1940 serum and plasma samples from 1110 horses and 830 cattle were screened with a species-specific ELISA based on the SARS-CoV-2 receptor-binding domain (RBD) and, in the case of suspect positive results, a surrogate virus neutralization test (sVNT) was used to demonstrate the neutralizing activity of the antibodies. Further confirmation of suspect positive samples was performed using either a pseudotype-based virus neutralization assay (PVNA; horses) or an indirect immunofluorescence test (IFA; cattle). The animals were sampled between February 2020 and December 2022. Additionally, in total, 486 bronchoalveolar lavage (BAL), oropharyngeal, nasal and rectal swab samples from horses and cattle were analyzed for the presence of SARS-CoV-2 RNA via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Six horses (0.5%; 95% CI: 0.2–1.2%) were suspect positive via RBD-ELISA, and neutralizing antibodies were detected in two of them via confirmatory sVNT and PVNA tests. In the PVNA, the highest titers were measured against the Alpha and Delta SARS-CoV-2 variants. Fifteen cattle (1.8%; 95% CI: 1.0–3.0%) were suspect positive in RBD-ELISA; 3 of them had SARS-CoV-2-specific neutralizing antibodies in sVNT and 4 of the 15 were confirmed to be positive via IFA. All tested samples were RT-qPCR-negative. The results support the hypotheses that the prevalence of SARS-CoV-2 infections in horses and cattle in Switzerland was low up to the end of 2022

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines