Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera—i.e., Pedacovirus, Nyctacovirus, and Myotacovirus—that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates—including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus—present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

Bat populations harbour a multitude of viruses; some of these are pathogenic or potentially pathogenic in other animals or humans. Therefore, it is important to monitor the populations and characterize these viruses. In this study, the presence of coronaviruses (CoVs) in different species of Danish bats was investigated using active surveillance at different geographical locations in Denmark. Faecal samples were screened for the presence of CoVs using pan-CoV real-time RT-PCR assays. The amplicons, obtained from five different species of bats, were sequenced. Phylogenetic analysis revealed a species-specific clustering with the samples from Myotis daubentonii, showing a close resemblance to coronavirus sequences obtained from the same species of bat in Germany and the United Kingdom. Our results show, for the first time, that multiple, distinct alphacoronaviruses are present in the Danish bat populations.

Insect production offers a sustainable source of nutrients for livestock. This comes with a risk for transmission of pathogens from the insects into the livestock sector, including viruses causing serious diseases, such as African swine fever virus (ASFV), classical swine fever virus and foot-and-mouth disease virus. ASFV is known to survive for a long time within animal meat and byproducts. Therefore, we conducted experimental exposure studies of insects to ASFV using larvae of two key insect species produced for food and feed, the mealworm; Tenebrio molitor, and the black soldier fly, Hermetia illucens. The larvae were exposed to ASFV POL/2015/Podlaskie, via oral uptake of serum or spleen material from ASFV-infected pigs. Using qPCR, the amounts of viral DNA present immediately after exposure varied from ~10^4.7 to 10^7.2 genome copies per insect. ASFV DNA was detectable in the larvae of H. illucens for up to 3 days post exposure and in T. molitor larvae for up to 9 days post exposure. To assess the presence of infectious virus within the larvae and with this, the risk of virus transmission via oral consumption, pigs were fed cakes containing larvae exposed to ASFV. Pigs that consumed 50 T. molitor or 50 H. illucens virus-exposed larvae did not become infected with ASFV. Thus, it appears, that in our experimental setting, the risk of ASFV transmission via consumption of unprocessed insect larvae, used as feed, is low.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

Introduction of African swine fever virus (ASFV) into pig herds can occur via virus-contaminated feed or other objects. Knowledge about ASFV survival in different matrices and under different conditions is required to understand indirect virus transmission. Maintenance of ASFV infectivity can occur for extended periods outside pigs. Current assays detecting ASFV have inherent disadvantages. Cell culture-based assays are labor-intensive and sensitive to contaminants while methods using qPCR detect ASFV DNA with high sensitivity and specificity, but this may not correspond to infectious virus. Here, we have combined the use of these assays to assess the replication of ASFV within cells and determined the effect of pig feces, straw, wood shavings, and mixed feed on ASFV infectivity. In porcine serum, infectious ASFV survived for at least 60 days at 4 °C, 22 °C, and 37 °C; for two days at 50 °C; one day at 60 °C; and ≤5 min at 70 °C. In the presence of feed, straw, or wood shavings, the survival of the virus wasmarkedly shortened. Samples remained positive in the qPCR assay despite the loss of virus infectivity. Thus, it was possible to distinguish between the presence of ASFV DNA and the survival of the infectious virus.

African swine fever virus (ASFV) is known to be very stable within a protein-rich environment and indirect virus transmission can be mediated via oral uptake of different materials. However, experimental studies in pigs have shown that infection by ASFV via the oral route can be difficult to establish. Currently, there is a lack of studies using strict oral inoculations of pigs with different doses of ASFV. Therefore, we aimed to determine the dose of a European genotype II ASFV that is required to establish infection of pigs by the oral route. In this study, 24 pigs were divided into four groups of six. Three of the groups were fed with a low, medium or high dose of the ASFV POL/2015/Podlaskie virus. The pigs in the fourth group served as positive controls and were inoculated intranasally, just once, using the low dose of the virus. All the pigs inoculated intranasally with ASFV succumbed to the infection, while only three of the six pigs that were fed the high dose of the virus became infected. None of the 12 pigs that were fed with either the medium or low dose of the virus became infected, despite receiving up to thirteen doses each. In two of the pigs infected by intranasal inoculation, the presence of a variant form of the ASFV genome was detected. The results obtained in this study underline that ASFV infection is more difficult to establish via the oral route when compared to the intranasal route. The high dose needed in order to establish oral infection could have implications for future strategies using baited vaccines containing infectious live-attenuated ASFV.