Kore Schlottau

Presence of two different bovine hepacivirus clusters in Germany

During the last years, genetic information of hepaciviruses (family Flaviviridae), whose type species is the human hepatitis C virus, was detected in a wide range of primates and non-primate vertebrates. Here, samples collected from 263 German cattle kept in 22 different holdings were analysed for the presence of hepacivirus N (syn. bovine hepacivirus; BovHepV). One hundred eighty-six cattle that suffered from unspecific clinical signs such as fever and a reduced milk yield as well as 77 apparently healthy animals were included. A total of 39 cattle (14.8%) tested positive for BovHepV by real-time RT-PCR, but a correlation between clinical signs and virus infection could not be found. From 31 of the virus-positive samples, sequences of the NS3 coding region were generated and from two samples, viral sequences of the complete coding region were produced and compared to further European and African BovHepV sequences. Based on the NS3 genomic region, two distinct German BovHepV clusters were identified which differed between each other up to 20% at the nucleotide level, the diversity within the individual clusters reached up to 10%. Based on the full-length sequences, the newly detected virus variants group together with further German and African viruses in a sister relationship to other hepaciviruses from primates and further mammalians, but form distinct clusters within the BovHepV branch. In conclusion, highly diverse hepaciviruses were detected in German cattle further expanding the known phylogenetic diversity of the genus Hepacivirus.

Development of molecular confirmation tools for swift and easy rabies diagnostics

BackgroundAs rabies still represents a major public threat with tens of thousands of deaths per year, particularly in developing countries, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. Rabies diagnosis relies on highly sensitive and specific laboratory tests for detection of viral antigens. Among those tests, at present the immunofluorescence antibody test is the “gold standard test” for rabies diagnosis, followed by virus isolation in either mice or cell culture. Because of the advantages of molecular assays in terms of sensitivity and applicability their approval as confirmatory diagnostic test by international organizations (OIE, WHO) is envisaged. Therefore, the objective was to develop and validate novel molecular assays and RNA extraction methods for rabies that reduce the turnaround time but remain highly sensitive and specific.MethodsHere, novel assays, i.e. HighSpeed RT-qPCR and isothermal recombinase polymerase amplification (RPA) were designed and tested. Furthermore, three magnetic bead-based rapid extraction methods for manual or automated extraction were validated and combined with the new downstream assays.ResultsWhile the conventional column based RNA extraction method showed the highest intra-run variations, all magnetic bead-based rapid extraction methods delivered nearly comparable sensitivity and efficiency of RNA recovery. All newly developed molecular tests were able to detect different rabies virus strains in a markedly reduced timeframe in comparison to the standard diagnostic assays. The observed detection limit for the HighSpeed RT-qPCR was 10 genome copies per reaction, and 1000 genome copies per reaction for the RPA assay.ConclusionMagnetic bead-based rapid RNA extraction methods are highly sensitive and show a high level of reproducibility and therefore, are particularly suitable for molecular diagnostic assays including rabies. In addition, with a detection limit of 10 genome copies per reaction, the HighSpeed RT-qPCR is suitable for rapid ante mortem rabies diagnosis in humans as well as confirmatory test in integrated bite management and subsequent post-exposure prophylaxis.

Multiple detection of zoonotic variegated squirrel bornavirus 1 RNA in different squirrel species suggests a possible unknown origin for the virus

The recently discovered variegated squirrel bornavirus 1 (VSBV-1) caused the death of three squirrel breeders in Germany. Subsequent first screening of squirrels with in vivo collected swab samples and a VSBV-1-specific RT-qPCR revealed not only variegated squirrel infections (Sciurus variegatoides), but also Prevost’s squirrels (Callosciurus prevostii) as positive for VSBV-1 genome. In this study, 328 squirrels were tested using the established RT-qPCR assays. In 16 individual animals VSBV-1 RNA could be detected; 15 individuals were from small breedings and zoological gardens in Germany, with the remaining individual being from a zoological garden in Croatia. Positive animals belonged to the species C. prevostii, C. finlaysonii, and Tamiops swinhoei within the subfamily Callosciurinae and Sciurus granatensis within the subfamily Sciurinae. Repeated non-invasive oral swab sampling in one holding indicated positive animals months after a first negative result. Besides the oral swabs, VSBV-1 was also detected in fecal (pool) samples allowing the future monitoring of squirrel holdings based on RT-qPCR investigation of such samples. The detection in zoological gardens emphasizes the need for further investigations into the transmission route to humans in order to develop rational public health measures for prevention of transmission. Finally, the detection of several closely related VSBV-1 sequences in squirrels from different subfamilies raises questions as to the origin of the virus.

Mutational modifications of hepatitis A virus proteins 2B and 2C described for cell culture-adapted and attenuated virus are present in wild-type virus populations

Studies have identified certain mutations in the 2B and 2C proteins of hepatitis A virus (HAV) as being essential for efficient growth in cultured cells, and it is assumed that these mutations contribute to the attenuated phenotype. We found that mutations supporting cell culture growth already exist in wild-type HAV populations. This suggests that these variants are not entirely generated de novo but are selected from the wild-type population. In a prolonged case of hepatitis A, we found that sequences associated with cell culture adaptation predominated later in infection. This might suggest selection of an attenuated virus population during a prolonged clinical infection.

Suitability of group-level oral fluid sampling in ruminant populations for lumpy skin disease virus detection

The geographic expansion of Lumpy skin disease (LSD) from the near East into the European Union highlighted again the need for appropriate disease detection tools applicable to animal host populations where access to individual animals is difficult. This is of particular importance considering that the clinical manifestation of LSD is often mild making early disease detection challenging under the above-mentioned conditions. Building on positive experiences of group-level oral fluid sampling for pathogen detection as it is known to work for swine herds and wild boar, the concept was transferred to ruminants. Two groups of six cattle were infected experimentally with Lumpy skin disease virus (LSDV) under controlled conditions. Blood as well as oropharyngeal and nasal swab samples were collected at regular intervals. Group samples were obtained by placing cotton gauze around a salt lick block provided commonly as dietary supplement. Pieces of the gauze with visible signs of manipulation were tested in parallel to samples obtained from individual animals. Genome load analysis by qPCR technology revealed LSDV detection window starting from day 2 post infection until day 28 post infection, the end of the animal trial. At the individual level, detection periods varied between animals and type of sample and included intermitted detection. The accumulative character of the alternative sampling method makes it suitable to detect LSDV DNA at group-level even at times of the infection where a selective sampling of individuals from a group - as normally done in LSD surveillance - would have most likely failed in the detection.

A Novel Squirrel Respirovirus with Putative Zoonotic Potential

In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen.

Fatal Encephalitic Borna Disease Virus 1 in Solid-Organ Transplant Recipients.

Fatal Zoonotic Viral Infection after Transplantation Evidence of Borna disease virus 1 infection in humans is limited; in this report, donor-derived Borna virus encephalitis is shown to occur in three solid-organ transplant recipients.

Molecular identification and characterization of nonprimate hepaciviruses in equines

Hepatitis C virus (HCV) is a positive-sense RNA virus belonging to the genus Hepacivirus, family Flaviviridae. Its genome has a length of 9.6 kb and encodes a single polyprotein flanked by two untranslated regions. HCV can cause liver cirrhosis and hepatocellular carcinoma, and approximately 2% of the world’s population is chronically infected. The investigation of pathogenesis is complicated due to the lack of an animal model. The origin of this virus remains unclear, but in the last few years, relatives of HCV were initially identified in dogs and later in horses, rodents, bats and Old World monkeys. Non-primate hepacivirus (NPHV), which infects dogs and horses, is the closest relative to HCV. We established a pan-reactive “panHepaci”-RT-qPCR assay, which is able to detect human HCV as well as equine NPHV, and additionally, an equine-specific “equHepaci”-RT-qPCR for confirmation of positive results. Serum samples from 1158 clinically inconspicuous horses from Germany and several samples from other mammalian species were screened. We found 2.4% of the horses positive for hepacivirus RNA, and furthermore, the “panHepaci”-RT-qPCR assay also detected a hepacivirus in a donkey from Egypt. This virus had only 78% sequence identity in the E2 gene when compared to other known NPHVs. The established method could be useful for screening purposes, since it is likely that related hepaciviruses also occur in other species.