Characterization of Pipistrellus pygmaeus Bat Virome from Sweden.

Increasing amounts of data indicate that bats harbor a higher viral diversity relative to other mammalian orders, and they have been recognized as potential reservoirs for pathogenic viruses, such as the Hendra, Nipah, Marburg, and SARS-CoV viruses. Here, we present the first viral metagenomic analysis of Pipistrellus pygmaeus from Uppsala, Sweden. Total RNA was extracted from the saliva and feces of individual bats and analyzed using Illumina sequencing. The results identified sequences related to 51 different viral families, including vertebrate, invertebrate, and plant viruses. These viral families include Coronaviridae, Picornaviridae, Dicistroviridae, Astroviridae, Hepeviridae, Reoviridae, Botourmiaviridae, Lispviridae, Totiviridae, Botoumiaviridae, Parvoviridae, Retroviridae, Adenoviridae, and Partitiviridae, as well as different unclassified viruses. We further characterized three near full-length genome sequences of bat coronaviruses. A phylogenetic analysis showed that these belonged to alphacoronaviruses with the closest similarity (78-99% at the protein level) to Danish and Finnish bat coronaviruses detected in Pipistrellus and Myotis bats. In addition, the full-length and the near full-length genomes of picornavirus were characterized. These showed the closest similarity (88-94% at the protein level) to bat picornaviruses identified in Chinese bats. Altogether, the results of this study show that Swedish Pipistrellus bats harbor a great diversity of viruses, some of which are closely related to mammalian viruses. This study expands our knowledge on the bat population virome and improves our understanding of the evolution and transmission of viruses among bats and to other species.

Transcriptome Analysis of an Aedes albopictus Cell Line Single- and Dual-Infected with Lammi Virus and WNV.

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes' ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes.

Investigation of the Virome and Characterization of Issyk-Kul Virus from Swedish Myotis brandtii Bats.

Bats are reservoirs for many different viruses, including some that can be transmitted to and cause disease in humans and/or animals. However, less is known about the bat-borne viruses circulating in Northern European countries such as in Sweden. In this study, saliva from Myotis brandtii bats, collected from south-central Sweden, was analyzed for viruses. The metagenomic analysis identified viral sequences belonging to different viral families, including, e.g., Nairoviridae, Retroviridae, Poxviridae, Herpesviridae and Siphoviridae. Interestingly, through the data analysis, the near-complete genome of Issyk-Kul virus (ISKV), a zoonotic virus within the Nairoviridae family, was obtained, showing 95-99% protein sequence identity to previously described ISKVs. This virus is believed to infect humans via an intermediate tick host or through contact with bat excrete. ISKV has previously been found in bats in Europe, but not previously in the Nordic region. In addition, near full-length genomes of two novel viruses belonging to Picornavirales order and Tymoviridae family were characterized. Taken together, our study has not only identified novel viruses, but also the presence of a zoonotic virus not previously known to circulate in this region. Thus, the results from these types of studies can help us to better understand the diversity of viruses circulating in bat populations, as well as identify viruses with zoonotic potential that could possibly be transmitted to humans.

Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line.

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs' effects on their host.

Taxonomic updates for the genus Gyrovirus (family Anelloviridae): recognition of several new members and establishment of species demarcation criteria.

The genus Gyrovirus was assigned to the family Anelloviridae in 2017 with only one recognized species, Chicken anemia virus. Over the last decade, many diverse viruses related to chicken anemia virus have been identified but not classified. Here, we provide a framework for the classification of new species in the genus Gyrovirus and communicate the establishment of nine new species. We adopted the 'Genus + freeform epithet' binomial system for the naming of these species.

Taxonomic update for mammalian anelloviruses (family Anelloviridae).

Anelloviruses are small negative-sense single-stranded DNA viruses with genomes ranging in size from 1.6 to 3.9 kb. The family Anelloviridae comprised 14 genera before the present changes. However, in the last five years, a large number of diverse anelloviruses have been identified in various organisms. Here, we undertake a global analysis of mammalian anelloviruses whose full genome sequences have been determined and have an intact open reading frame 1 (ORF1). We established new criteria for the classification of anelloviruses, and, based on our analyses, we establish new genera and species to accommodate the unclassified anelloviruses. We also note that based on the updated species demarcation criteria, some previously assigned species (n = 10) merge with other species. Given the rate at which virus sequence data are accumulating, and with the identification of diverse anelloviruses, we acknowledge that the taxonomy will have to be dynamic and continuously evolve to accommodate new members.

Sindbis virus neutralising antibodies detected in Swedish horses.

A number of viruses transmitted by mosquitoes are well known to cause disease in both humans and horses, ranging from mild fevers to mortal neurological disease. A recently discovered connection between the alphavirus Sindbis virus (SINV) and neurological disease in horses in South Africa initiated this serological study in northern Europe, where the same genotype of SINV (SINV-I) is also highly endemic. We tested 171 serum samples, originally obtained from horses for other reasons from April to October 2019, for presence of SINV neutralising antibodies using a plaque reduction neutralisation test (PRNT). The serum from six horses reduced the plaque count more than 80%, and two out of these reduced the plaque count more than 90%. These horses were sampled in six different regions of Sweden, and included individuals sampled from April to August. This study shows that horses in Sweden have become infected with SINV and developed neutralising antibodies. Potential connections between infection and development of disease are important questions for future studies.

Complete Coding Sequence of a Pasivirus Found in Swedish Pigs.

Here, we report the complete coding sequence of a pasivirus found in the tonsil of a conventionally reared pig from a herd with respiratory disease in Sweden. The genome displays 75% to 87% and 81% to 94% nucleotide and amino acid sequence identity, respectively, to genomes of pasiviruses from other parts of the world.

Viromics Reveal a Number of Novel RNA Viruses in Swedish Mosquitoes.

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.

Insect-specific virus evolution and potential effects on vector competence.

The advancement in high-throughput sequencing technology and bioinformatics tools has spurred a new age of viral discovery. Arthropods is the largest group of animals and has shown to be a major reservoir of different viruses, including a group known as insect-specific viruses (ISVs). The majority of known ISVs have been isolated from mosquitoes and shown to belong to viral families associated with animal arbovirus pathogens, such as Flaviviridae, Togaviridae and Phenuiviridae. These insect-specific viruses have a strict tropism and are unable to replicate in vertebrate cells, these properties are interesting for many reasons. One is that these viruses could potentially be utilised as biocontrol agents using a similar strategy as for Wolbachia. Mosquitoes infected with the viral agent could have inferior vectorial capacity of arboviruses resulting in a decrease of circulating arboviruses of public health importance. Moreover, insect-specific viruses are thought to be ancestral to arboviruses and could be used to study the evolution of the switch from single-host to dual-host. In this review, we discuss new discoveries and hypothesis in the field of arboviruses and insect-specific viruses.

Novel Viruses Found in Antricola Ticks Collected in Bat Caves in the Western Amazonia of Brazil.

In this study, we describe the viral composition of adult Antricoladelacruzi ticks collected in a hot bat cave in the state of Rondônia, Western Amazonia, Brazil. A.delacruzi ticks, are special, compared to many other ticks, in that they feed on both bats (larval blood feeding) and bat guano (nymphal and adult feeding) instead of feeding exclusively on vertebrate hosts (blood feeding). Considering this unique life-cycle it is potentially possible that these ticks can pick up/be infected by viruses not only present in the blood of viremic bats but also by virus shed through the bat guano. The viral metagenomic investigation of adult ticks showed that single-stranded negative-sense RNA viruses were the dominant group of viruses identified in the investigated ticks. Out of these, members of the Nairoviridae family were in clear majority constituting 88% of all viral reads in the data set. Genetic and phylogenetic analyses indicate the presence of several different orthonairoviruses in the investigated ticks with only distant relationship to previously described ones. In addition, identification of viral sequences belonging to Orthomyxoviridae, Iflaviridae, Dicistroviridae, Polycipiviridae, Reoviridae and different unclassified RNA viruses showed the presence of viruses with low sequence similarity to previously described viruses.

Characterisation of the Virome of Tonsils from Conventional Pigs and from Specific Pathogen-Free Pigs.

Porcine respiratory disease is a multifactorial disease that can be influenced by a number of different microorganisms, as well as by non-infectious factors such as the management and environment of the animals. It is generally believed that the interaction between different infectious agents plays an important role in regard to respiratory diseases. Therefore, we used high-throughput sequencing combined with viral metagenomics to characterise the viral community of tonsil samples from pigs coming from a conventional herd with lesions in the respiratory tract at slaughter. In parallel, samples from specific pathogen-free pigs were also analysed. This study showed a variable co-infection rate in the different pigs. The differences were not seen at the group level but in individual pigs. Some viruses such as adenoviruses and certain picornaviruses could be found in most pigs, while others such as different parvoviruses and anelloviruses were only identified in a few pigs. In addition, the complete coding region of porcine parvovirus 7 was obtained, as were the complete genomes of two teschoviruses. The results from this study will aid in elucidating which viruses are circulating in both healthy pigs and in pigs associated with respiratory illness. This knowledge is needed for future investigations into the role of viral-viral interactions in relation to disease development.

Viral metagenomics reveals the presence of highly divergent quaranjavirus in Rhipicephalus ticks from Mozambique.

Background: Ticks are primary vectors for many well-known disease-causing agents that affect human and animal populations globally such as tick-borne encephalitis, Crimean-Congo hemorrhagic fever and African swine fever. In this study, viral metagenomics was used to identify what viruses are present in Rhipicephalus spp. ticks collected in the Zambezi Valley of Mozambique. Methods: The RNA was amplified with sequence-independent single primer amplification (SISPA) and high-throughput sequencing was performed on the Ion Torrent platform. The generated sequences were subjected to quality check and classfied by BLAST. CodonCode aligner and SeqMan were used to assemble the sequences. Results: The majority of viral sequences showed closest sequence identity to the Orthomyxoviridae family, although viruses similar to the Parvoviridae and Coronaviridae were also identified. Nearly complete sequences of five orthomyxoviral segments (HA, NP, PB1, PB2, and PA) were obtained and these showed an amino acid identity of 32-52% to known quaranjaviruses. The sequences were most closely related to the Wellfleet Bay virus, detected and isolated from common eider during a mortality event in the USA. Conclusions: In summary, this study has identified a highly divergent virus with in the Orthomyxoviridae family associated with Rhipicephalus ticks from Mozambique. Further genetic and biological studies are needed in order to investigate potential pathogenesis of the identified orthomyxovirus.

Genetic characterization of a novel picorna-like virus in Culex spp. mosquitoes from Mozambique.

BACKGROUND: Mosquitoes are the potential vectors for a variety of viruses that can cause diseases in the human and animal populations. Viruses in the order Picornavirales infect a broad range of hosts, including mosquitoes. In this study, we aimed to characterize a novel picorna-like virus from the Culex spp. of mosquitoes from the Zambezi Valley of Mozambique. METHODS: The extracted RNA from mosquito pools was pre-amplified with the sequence independent single primer amplification (SISPA) method and subjected to high-throughput sequencing using the Ion Torrent platform. Reads that are classified as Iflaviridae, Picornaviridae and Dicistroviridae were assembled by CodonCode Aligner and SPAdes. Gaps between the viral contigs were sequenced by PCR. The genomic ends were analyzed by 5' and 3' RACE PCRs. The ORF was predicted with the NCBI ORF finder. The conserved domains were identified with ClustalW multiple sequence alignment, and a phylogenetic tree was built with MEGA. The presence of the virus in individual mosquito pools was detected by RT-PCR assay. RESULTS: A near full-length viral genome (9740 nt) was obtained in Culex mosquitoes that encoded a complete ORF (3112 aa), named Culex picorna-like virus (CuPV-1). The predicted ORF had 38% similarity to the Hubei picorna-like virus 35. The sequence of the conserved domains, Helicase-Protease-RNA-dependent RNA polymerase, were identified by multiple sequence alignment and found to be at the 3' end, similar to iflaviruses. Phylogenetic analysis of the putative RdRP amino acid sequences indicated that the virus clustered with members of the Iflaviridae family. CuPV-1 was detected in both Culex and Mansonia individual pools with low infection rates. CONCLUSIONS: The study reported a highly divergent, near full-length picorna-like virus genome from Culex spp. mosquitoes from Mozambique. The discovery and characterization of novel viruses in mosquitoes is an initial step, which will provide insights into mosquito-virus interaction mechanisms, genetic diversity and evolution.

Detection and genetic characterisation of porcine circovirus 3 from pigs in Sweden.

Porcine circovirus 3 (PCV3) is a newly detected circovirus belonging to the family Circoviridae with a circular ssDNA genome of 2000 bp that encodes two proteins-the replicase protein and the capsid protein. PCV3 was discovered for the first time in the US in 2016. After this initial discovery, PCV3 was detected in other parts of the world such as in China, South Korea, Italy and Poland. In this study, 49 tissue samples from Swedish pig herds were screened for PCV3 using PCR and 10 samples were positive and one was uncertain. The entire PCV3 genome and a mini PCV-like virus (MPCLV) were obtained from one of these samples. These two viruses showed a high sequence identity to PCV3 viruses from other countries as well as to MPCLV from the US. However, the sequence identity to PCV1 and 2 was only 31-48% on amino acid level. This is the first detection and complete genetic characterisation of PCV3 in Swedish pigs. It is also interesting to note that one of the positive samples was collected in 1993, showing that PCV3 has been present for a long time.

Single primer isothermal amplification (SPIA) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (SISPA).

Coronaviruses are of major importance for both animal and human health. With the emergence of novel coronaviruses such as SARS and MERS, the need for fast genome characterisation is ever so important. Further, in order to understand the influence of quasispecies of these viruses in relation to biology, techniques for deep-sequence and full-length viral genome analysis are needed. In the present study, we compared the efficiency of two sequence-independent approaches [sequence-independent single primer amplification (SISPA) and single primer isothermal amplification (SPIA, represented by the Ovation kit)] coupled with high-throughput sequencing to generate the full-length genome of bovine coronavirus (BCoV) from a nasal swab. Both methods achieved high genome coverage (100% for SPIA and 99% for SISPA), however, there was a clear difference in the percentage of reads that mapped to BCoV. While approximately 45% of the Ovation reads mapped to BCoV (sequence depth of 169-284 944), only 0.07% of the SISPA reads (sequence depth of 0-249) mapped to the reference genome. Although BCoV was the focus of the study we also identified a bovine rhinitis B virus (BRBV) in the data sets. The trend for this virus was similar to that observed for BCoV regarding Ovation vs. SISPA, but with fewer sequences mapping to BRBV due to a lower amount of this virus. In summary, the SPIA approach used in this study produced coverage of the entire BCoV (high copy number) and BRBV (low copy number) and a high sequence/genome depth compared to SISPA. Although this is a limited study, the results indicate that the Ovation method could be a preferred approach for full genome sequencing if a low copy number of viral RNA is expected and if high sequence depth is desired.

Seroprevalence and risk factors for peste des petits ruminants and selected differential diagnosis in sheep and goats in Tanzania.

Introduction: Livestock husbandry is critical for food security and poverty reduction in a low-income country like Tanzania. Infectious disease is one of the major constraints reducing the productivity in this sector. Peste des petits ruminants (PPR) is one of the most important diseases affecting small ruminants, but other infectious diseases may also be present. Objective: The objective of this study was to determine the seroprevalence and risk factors for exposure to PPR, contagious caprine pleuropneumonia (CCPP), foot-and-mouth disease (FMD), bluetongue (BT), and bovine viral diarrhoea (BVD) in sheep and goats in Tanzania. Methods: Serum samples were collected in 2014 and 2015, and analysed using enzyme-linked immunosorbent assays to detect antibodies to the five pathogens. Results and discussion: This is the first description of seroprevalence of FMD and BT among small ruminants in Tanzania. Risk factor analysis identified sex (female) (OR for 2014: PPR: 2.49, CCPP: 3.11, FMD: 2.98, BT: 12.4, OR for 2015: PPR: 14.1, CCPP: 1.10, FMD: 2.67, BT: 1.90, BVD: 4.73) and increasing age (>2 years) (OR for 2014: PPR: 14.9, CCPP: 2.34, FMD: 7.52, BT: 126, OR for 2015: PPR: 8.13, CCPP: 1.11, FMD: 2.98, BT: 7.83, BVD: 4.74) as risk factors for exposure to these diseases.

Genome Sequence of a Bovine Rhinitis B Virus Identified in Cattle in Sweden.

A bovine rhinitis B virus, identified in a calf from Sweden, was genetically characterized. The complete polyprotein was recovered, and phylogenetic analysis showed that this virus has the highest similarity to a bovine rhinitis B virus previously identified in Mexico.

The Antiviral RNAi Response in Vector and Non-vector Cells against Orthobunyaviruses.

BACKGROUND: Vector arthropods control arbovirus replication and spread through antiviral innate immune responses including RNA interference (RNAi) pathways. Arbovirus infections have been shown to induce the exogenous small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) pathways, but direct antiviral activity by these host responses in mosquito cells has only been demonstrated against a limited number of positive-strand RNA arboviruses. For bunyaviruses in general, the relative contribution of small RNA pathways in antiviral defences is unknown. METHODOLOGY/PRINCIPAL FINDINGS: The genus Orthobunyavirus in the Bunyaviridae family harbours a diverse range of mosquito-, midge- and tick-borne arboviruses. We hypothesized that differences in the antiviral RNAi response in vector versus non-vector cells may exist and that could influence viral host range. Using Aedes aegypti-derived mosquito cells, mosquito-borne orthobunyaviruses and midge-borne orthobunyaviruses we showed that bunyavirus infection commonly induced the production of small RNAs and the effects of the small RNA pathways on individual viruses differ in specific vector-arbovirus interactions. CONCLUSIONS/SIGNIFICANCE: These findings have important implications for our understanding of antiviral RNAi pathways and orthobunyavirus-vector interactions and tropism.

Viral Metagenomic Analysis Displays the Co-Infection Situation in Healthy and PMWS Affected Pigs.

The development of high-throughput sequencing technologies have allowed the possibility to investigate and characterise the entire microbiome of individuals, providing better insight to the complex interaction between different microorganisms. This will help to understand how the microbiome influence the susceptibility of secondary agents and development of disease. We have applied viral metagenomics to investigate the virome of lymph nodes from Swedish pigs suffering from the multifactorial disease postweaning multisystemic wasting syndrome (PMWS) as well as from healthy pigs. The aim is to increase knowledge of potential viruses, apart from porcine circovirus type 2 (PCV2), involved in PMWS development as well as to increase knowledge on the virome of healthy individuals. In healthy individuals, a diverse viral flora was seen with several different viruses present simultaneously. The majority of the identified viruses were small linear and circular DNA viruses, such as different circoviruses, anelloviruses and bocaviruses. In the pigs suffering from PMWS, PCV2 sequences were, as expected, detected to a high extent but other viruses were also identified in the background of PCV2. Apart from DNA viruses also RNA viruses were identified, among them were a porcine pestivirus showing high similarity to a recently (in 2015) discovered atypical porcine pestivirus in the US. Majority of the viruses identified in the background of PCV2 in PMWS pigs could also be identified in the healthy pigs. PCV2 sequences were also identified in the healthy pigs but to a much lower extent than in PMWS affected pigs. Although the method used here is not quantitative the very clear difference in amount of PCV2 sequences in PMWS affected pigs and healthy pigs most likely reflect the very strong replication of PCV2 known to be a hallmark of PMWS. Taken together, these findings illustrate that pigs appear to have a considerable viral flora consisting to a large extent of small single-stranded and circular DNA viruses. Future research on these types of viruses will help to better understand the role that these ubiquitous viruses may have on health and disease of pigs. We also demonstrate for the first time, in Europe, the presence of a novel porcine pestivirus.