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Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health. The RdRp sequence is similar but not identical to previously detected sequences in the feces of two different species of bats (T. brasiliensis and Molossus molossus) in Brazil. In common with the experience of others doing similar work, attempts to isolate the virus in cell cultures were unsuccessful. We surmise that this and highly related alphacoronavirus are carried by Brazilian free-tailed bats living in a wide eco-spatial region. As various coronaviruses (CoVs) that affect humans emerged from bats, our study raises the question whether CoVs such as the one detected in our work are yet-to-be-detected pathogens of humans and animals other than bats.

Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host.

In the last decade, many porcine epidemic diarrhoea (PED) outbreaks have been reported by several countries in Asia whereas only a few Member States of the European Union (EU) have reported PED clinical cases and/or PED virus (PEDV)-seropositive animals. This alphacoronavirus was first reported in the USA in May 2013, followed by rapid spread throughout the country and outbreaks reported by several countries in the Americas. The recent PEDV-EU isolates have high level of sequence identity to PEDV-Am isolates. Based on nucleotide sequencing, multiple variants of PEDV are circulating in Europe, the Americas and Asia but any difference in virulence and antigenicity is currently unknown. Serological cross-reactivity has been reported between PEDV isolated in Europe and in the Americas; however no data regarding cross-protection are available. The impact of different PEDV strains is difficult to compare between one country and another, since impact is dependent not only on pathogenicity but also on factors such as biosecurity, farm management, sanitary status or herd immune status. However, the clinical signs of PEDV infections in naive pigs are similar in different countries with mortalities up to 100% in naive newborn piglets. The impact of recently reported PED outbreaks in Asia and the USA seems to be more severe than what has been described in Europe. Infected animals, faeces, feed and objects contaminated with faeces are matrices that have been reported to transmit PEDV between farms. Infectious PEDV has been detected in spray-dried porcine plasma (SDPP) in one study but the origin of the infectious PEDV in SDPP is not clear. Detection of porcine deltacoronavirus (PDCoV) has been reported in a few countries but only limited testing has been done. Based on the currently available information, it seems that PDCoV would have a lower impact than PEDV.

Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV., IMPORTANCE The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.

To the Editor: Bats play a critical role in the transmission and origin of zoonotic diseases, primarily viral zoonoses associated with high case-fatality rates, including those caused by Nipah virus (NiV) and severe acute respiratory syndrome (SARS)–like coronavirus (CoV) infections (1). Recently, the World Health Organization (WHO) reported 44 confirmed cases of human infection with Middle East respiratory syndrome CoV, resulting in 22 deaths. Full-genome and phylogenetic analyses of these Middle East respiratory syndrome CoVs have been published elsewhere (2). The identified viruses from 2 patients (previously referred to as England/Qatar/2012 and EMC/2012) are genetically related and belong to group C betacoronavirus, which is most related to CoVs from Nycteris bats in Ghana and Pipistrellus bats in Europe (2,3). In addition, bat CoVs HKU4 and HKU5 originated from Tylonycteris pachypus and Pipistrellus abramus bats, respectively, in the People’s Republic of China (4). Bats are also known to harbor and transmit nonviral zoonotic pathogens, including the fungal pathogen Histoplasma capsulatum, which causes histoplasmosis in humans (5). Bat guano is sold for use as a fertilizer in several countries, including Thailand, Indonesia, Mexico, Cuba, and Jamaica. The practice of collecting and harvesting bat guano may pose a considerable health risk because guano miners have a high level of contact and potential exposure to bat-borne pathogens. To assess pathogens in bat guano, we examined bat guano from a cave in the Khao Chong Phran Non-hunting Area (KCP-NHA) in Ratchaburi Province, Thailand, where bat guano was sold as agricultural fertilizer, for the presence of NiV, CoV, and H. capsulatum fungi. Bats from 14 species in 7 families have been found roosting within this area. Tadarida plicata bats are the most abundant species (2,500,000 bats), and 3 other species of bats found at the site each had thousands of members: Taphozous melanopogon, Taphozous theobaldi, and Hipposideros larvatus. A random sample of dry bat guano, ≈100 g, was collected in a sterile plastic bag weekly from the main cave at KCP-NHA from September 2006 through August 2007. The specimens were sent for analysis by express mail (at room temperature within 2–3 days) to the WHO Collaborating Centre for Research and Training in the Viral Zoonoses Laboratory at Chulalongkorn University. Samples were frozen immediately at –80°C until nucleic acids were extracted and PCR assays were run. A total of 52 collected bat guano specimens were examined in this study. Two aliquots of feces from each weekly specimen (104 samples total) were screened for CoV, NiV, and H. capsulatum by PCR. RNA was extracted from 10 mg of fecal pellet by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). CoV RNA was detected by using nested reverse transcription PCR with the degenerated primers to amplify the RNA-dependent RNA polymerase (RdRp) gene (6). NiV RNA was detected by duplex nested reverse transcription PCR (7). To detect H. capsulatum and other fungi, we extracted genomic DNA directly from bat guano by using the silica-guanidine thiocyanate protocol, NucliSense Isolation Reagent (bioMerieux, Boxtel, the Netherlands), according to the manufacturer’s protocol. We tested for fungal ribosomal DNA (rDNA) in extracted total nucleic acid specimens by using the PCR protocol designed to amplify all rDNA from 4 major fungus phyla at the internal transcribed spacer 1 and 2 regions (8). Four (3.8%) of 104 samples were positive for CoV. They were collected on September 2, 2006 (KCP9), October 26, 2006 (KCP12), November 14, 2006 (KCP15), and March 4, 2007 (KCP31). Three of the 4 positive CoV sequences (KCP9, KCP12, and KCP15) were identical at 152 nt of the RdRp region (ATCGTGCTATGCCTAATATGTGTAGGATTTTTGCATCTCTCATATTAGCTCGTAAACACAATACTTGTTGTAGTGTTTCAGACCGCTtTtATAGACTTGCaAACGAGTGTGCGCAAGTCTTGAGTGAGTATGTGCTATGTGGTGGTGGCTAT) and phylogenetically clustered with the group C betacoronavirus (Figure), with 76%, 80%, and 77% nt identity to bat CoV HKU4, bat CoV HKU5, and human CoV EMC and England1_CoV, respectively. The other CoV sequence (KCP31: ATCGTGCACTTCCCAATATGATACGCATGATTTCCGCCATGATTTTGGGATCAAAGCATGTTACTTGCTGTGACACATCTGATAAGTATTACCGTCTTTGTAATGAGCTtGCACAAGTTTTGACAGAGGTTGTTTATTCTAATGGTGGTTTC) showed 82% nt identity with bat CoV HKU8, an alphacoronavirus. Although we recognize that longer sequences or full genomes may alter the topology of the phylogeny slightly and give stronger branch support, we expect that the overall topology and placement of these CoVs would remain consistent. Samples from particular bat species could not be identified because bats of different species roost in this cave, and samples were pooled during collection for bat guano fertilizer. The detection of CoVs in bat guano from the KCP-NHA cave in Ratchaburi was consistent with the previous finding of alphacoronavirus from Hipposideros armiger bats from the same province in 2007, but those researchers tested fresh bat feces (9). Figure Phylogenetic tree of 4 coronaviruses (CoVs) isolated from bat guano collected in this study (KCP9, KCP12, KCP15, and KCP31); 35 additional human and animal CoVs from the National Center for Biotechnology Information database are included. Construction ... All bat guano samples screened by PCR were negative for NiV and Histoplasma spp. but were positive for group C betacoronavirus. The natural reservoir and complete geographic distribution of this CoV are currently unknown. Although we did not isolate live virus from these samples, the detection of nucleic acid and previous isolation of viruses from bat feces and urine (10) warrants some concern that guano miners might be exposed to bat pathogens in fresh excreta as well as in soil substances. We suggest that guano miners use preventive measures of personal hygiene and improved barrier protection to reduce the possibility of exposure to zoonotic pathogens.

Bats host a broad diversity of coronaviruses (CoVs), including close relatives of human pathogens. There is only limited data on neotropical bat CoVs. We analysed faecal, blood and intestine specimens from 1562 bats sampled in Costa Rica, Panama, Ecuador and Brazil for CoVs by broad-range PCR. CoV RNA was detected in 50 bats representing nine different species, both frugivorous and insectivorous. These bat CoVs were unrelated to known human or animal pathogens, indicating an absence of recent zoonotic spill-over events. Based on RNA-dependent RNA polymerase (RdRp)-based grouping units (RGUs) as a surrogate for CoV species identification, the 50 viruses represented five different alphacoronavirus RGUs and two betacoronavirus RGUs. Closely related alphacoronaviruses were detected in Carollia perspicillata and C. brevicauda across a geographical distance exceeding 5600 km. Our study expands the knowledge on CoV diversity in neotropical bats and emphasizes the association of distinct CoVs and bat host genera.

Coronavirus spike (S) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. Here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of S assembly into mouse hepatitis virus (MHV). Recombinant MHVs exhibited a range of abilities to accommodate the homologous S endodomains from the betacoronaviruses bovine coronavirus and human SARS-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (TGEV), and the gammacoronavirus avian infectious bronchitis virus respectively. Interestingly, in TGEV endodomain chimeras the reverting mutations resulted in stronger S incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. Additionally, MHV S assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. These results indicate an important role for negatively charged endodomain residues in the incorporation of MHV S protein into assembled virions.

The recent emergence of a number of new viral diseases as well as the re-emergence of tuberculosis (TB), indicate an urgent need for new drugs against viral and bacterial infections. Coronavirus nsp1 has been shown to induce suppression of host gene expression and interfere with host immune response. However, the mechanism behind this is currently unknown. Here we present the first nsp1 structure from an alphacoronavirus, Transmissible gastroenteritis virus (TGEV) nsp1. Contrary to previous speculation, the TGEV nsp1 structure clearly shows that alpha- and betacoronavirus nsp1s have a common evolutionary origin. However, differences in conservation, shape and surface electrostatics indicate that the mechanism for nsp1-induced suppression of host mRNA translation is likely to be different in the alpha- and betacoronavirus genera. The Modoc virus is a neuroinvasive rodent virus with similar pathology as flavivirus encephalitis in humans. The flaviviral methyltransferase catalyses the two methylations required to complete 5´ mRNA capping, essential for mRNA stability and translation. The structure of the Modoc NS5 methyltransferase domain was determined in complex with its cofactor S-adenosyl-L-methionine. The observed methyltransferase conservation between Modoc and other flaviviral branches, indicates that it may be possible to identify drugs that target a range of flaviviruses and supports the use of Modoc virus as a model for general flaviviral studies. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is part of the methylerythritol phosphate (MEP) pathway that produces essential precursors for isoprenoid biosynthesis. This pathway is used by a number of pathogens, including Mycobacterium tuberculosis and Plasmodium falciparum, but it is not present in humans. Using a structure-based approach, we designed a number of MtDXR inhibitors, including a novel fosmidomycin-analogue that exhibited improved activity against P.falciparum in an in vitro blood cell growth assay. The approach also allowed the first design of an inhibitor that bridge both DXR substrate and co-factor binding sites, providing a stepping-stone for further optimization.

A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.

Although coronaviruses are known to infect various animals by adapting to new hosts, interspecies transmission events are still poorly understood. During a surveillance study from 2005 to 2010, a novel alphacoronavirus, BatCoV HKU10, was detected in two very different bat species, Ro-BatCoV HKU10 in Leschenault's rousettes ( Rousettus leschenaulti ) (fruit bats in the suborder Megachiroptera) in Guangdong and Hi-BatCoV HKU10 in Pomona leaf-nosed bats ( Hipposideros pomona ) (insectivorous bats in the suborder Microchiroptera) in Hong Kong. Although infected bats appeared to be healthy, Pomona leaf-nosed bats carrying Hi-BatCoV HKU10 had lower body weights than uninfected bats. To investigate possible interspecies transmission between the two bat species, the complete genomes of two Ro-BatCoV HKU10 and six Hi-BatCoV HKU10 strains were sequenced. Genome and phylogenetic analyses showed that Ro-BatCoV HKU10 and Hi-BatCoV HKU10 represented a novel alphacoronavirus species, sharing highly similar genomes except in the genes encoding spike proteins, which had only 60.5% amino acid identities. Evolution of the spike protein was also rapid in Hi-BatCoV HKU10 strains from 2005 to 2006 but stabilized thereafter. Molecular-clock analysis dated the most recent common ancestor of all BatCoV HKU10 strains to 1959 (highest posterior density regions at 95% [HPDs], 1886 to 2002) and that of Hi-BatCoV HKU10 to 1986 (HPDs, 1956 to 2004). The data suggested recent interspecies transmission from Leschenault's rousettes to Pomona leaf-nosed bats in southern China. Notably, the rapid adaptive genetic change in BatCoV HKU10 spike protein by ∼40% amino acid divergence after recent interspecies transmission was even greater than the ∼20% amino acid divergence between spike proteins of severe acute respiratory syndrome-related Rhinolophus bat coronavirus (SARSr-CoV) in bats and civets. This study provided the first evidence for interspecies transmission of coronavirus between bats of different suborders.

Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination., link_to_OA_fulltext

BACKGROUND: Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL(pro)), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: Here, we profiled the substrate specificities of 3CL(pro) from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19 × 8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL(pro) prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL(pro) from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL(pro) prefers P4-Pro and SARS-CoV 3CL(pro) prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences 'VARLQ↓SGF' that can be cleaved efficiently by all 3CL(pro) with relative activity of 1.7 to 3.2, and 'VPRLQ↓SGF' that can be cleaved specifically by IBV 3CL(pro) with relative activity of 4.3. CONCLUSIONS/SIGNIFICANCE: The comprehensive substrate specificities of 3CL(pro) from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.

To the Editor: Porcine coronavirus (PorCoV) HKU15 is a single-stranded, positive-sense, enveloped RNA virus belonging to the genus Deltacoronavirus (family Coronaviridae). PorCoV HKU15 was first identified in 2012 in a surveillance study from China ( 1 ). Until February 2014, however, the role of this virus strain in clinical diseases of pigs had not been reported. We recently reported the detection of PorCoV strain HKU15-OH1987 in feces samples from sows and intestine samples from piglets in Ohio, United States; the infected animals were from swine farms where outbreaks of diarrheal disease had occurred in late January and early February 2014 ( 2 ). Genetic analysis showed that HKU15-OH1987 is closely related to 2 deltacoronavirus strains that were detected in Hong Kong, China, in 2012: HKU15-155 and HKU15-44 ( 2 ). We also demonstrated the presence of histopathologic lesions in the small intestines of PorCoV HKU15–infected piglets with diarrhea (L. Wang, unpub. data). In April 2014, a novel swine enteric coronavirus disease caused by PorCoV HKU15 or porcine epidemic diarrhea virus was reported to the World Animal Health Organization by the US Department of Agriculture (http://www.oie.int/wahis_2/public/wahid.php/Reviewreport/Review?page_refer=MapFullEventReport&reportid=15133). PorCoV HKU15 is now recognized as a key pathogenic cause of diarrheal diseases in pigs in the United States. However, the geographic distribution and genotype diversity of PorCoV HKU15 in this country are still not clear. To further our knowledge of the virus, we analyzed swine samples that had been submitted for diagnosis of diarrheal disease from farms in 10 US states. We report the detection and phylogenetic analyses of PorCoV HKU15 strains from these samples. Between February 7, 2014, when PorCoV HKU15-OH1987 was first identified in Ohio ( 2 ), and April 9, 2014, the Animal Disease Diagnostic Laboratory of the Ohio Department of Agriculture received >2,000 swine samples from farms in 10 US states for diagnosis of diarrheal disease. The states from which samples had been submitted were Minnesota, South Dakota, Nebraska, Illinois, Indiana, Michigan, Kentucky, Pennsylvania, Maryland, and Ohio. Among those samples, 435 were selected to be tested for the presence of PorCoV HKU15. A real-time reverse transcription PCR assay targeting the membrane protein gene was used to identify PorCoV HKU15. Samples with a cycle threshold value of 99.8%). Furthermore, all of the isolates share high nucleotide similarity (98.9%–99.2%) with the 2 PorCoV HKU15 strains in GenBank, HKU15-155 and HKU15-44. A phylogenetic tree constructed by using the entire sequence showed that all PorCoV HKU15 strains from the United States clustered together in 1 clade of the genus Deltacoronavirus with HKU15-155 and HKU15-44 (Technical Appendix Figure, panel A). This finding indicates that 1 genotype of PorCoV HKU15 is currently circulating in multiple US states. This result was further supported by phylogenetic trees constructed by using the full-length amino acids of spike and nucleocapsid proteins (Technical Appendix Figure, panels B, C). Because of limitation of the samples received, only a partial genome sequence was determined for strain MN3092 from Minnesota. However, on the basis of the spike and nucleocapsid protein sequence analyses, it is highly likely that the entire genome of the Minnesota strain is genetically identical to that of the other 8 strains (Technical Appendix Figure, panels B, C). In addition to the 9 states reported in this study, Iowa has also had a recent detection of PorCoV HKU15 ( 3 ). Thus, PorCoV HKU15 has been detected in 10 of the 50 US states, and those 10 states mainly cluster in the midwestern United States (Figure). As with data collected for porcine epidemic diarrhea outbreaks by the US National Animal Health Laboratory Network (http://www.nahln.org/default/), data collected on the geographic location and numbers of PorCoV HKU15 cases is also required to be reported weekly. Earlier reports support avian coronaviruses as the gene source for Deltacoronavirus spp. ( 1 , 4 ). To confirm this, surveillance for PorCoV should be carried out among birds. Moreover, effective control strategies, including vaccine development, should be in place for prevention and control of infections caused by PorCoV HKU15. Technical Appendix Phylogenetic tree of 4 genera of coronaviruses (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus spp.), including 9 US strains of porcine coronavirus HKU15.

Bats may host emerging viruses, including coronaviruses (CoV). We conducted an evaluation of CoV in rhinolophid and vespertilionid bat species common in Europe. Rhinolophids carried severe acute respiratory syndrome (SARS)-related CoV at high frequencies and concentrations (26% of animals are positive; up to 2.4 × 10 8 copies per gram of feces), as well as two Alphacoronavirus clades, one novel and one related to the HKU2 clade. All three clades present in Miniopterus bats in China (HKU7, HKU8, and 1A related) were also present in European Miniopterus bats. An additional novel Alphacoronavirus clade (bat CoV [BtCoV]/BNM98-30) was detected in Nyctalus leisleri . A CoV grouping criterion was developed by comparing amino acid identities across an 816-bp fragment of the RNA-dependent RNA polymerases (RdRp) of all accepted mammalian CoV species (RdRp-based grouping units [RGU]). Criteria for defining separate RGU in mammalian CoV were a >4.8% amino acid distance for alphacoronaviruses and a >6.3% distance for betacoronaviruses. All the above-mentioned novel clades represented independent RGU. Strict associations between CoV RGU and host bat genera were confirmed for six independent RGU represented simultaneously in China and Europe. A SARS-related virus (BtCoV/BM48-31/Bulgaria/2008) from a Rhinolophus blasii (Rhi bla) bat was fully sequenced. It is predicted that proteins 3b and 6 were highly divergent from those proteins in all known SARS-related CoV. Open reading frame 8 (ORF8) was surprisingly absent. Surface expression of spike and staining with sera of SARS survivors suggested low antigenic overlap with SARS CoV. However, the receptor binding domain of SARS CoV showed higher similarity with that of BtCoV/BM48-31/Bulgaria/2008 than with that of any Chinese bat-borne CoV. Critical spike domains 472 and 487 were identical and similar, respectively. This study underlines the importance of assessments of the zoonotic potential of widely distributed bat-borne CoV.

The SARS epidemic has boosted interest in research on coronavirus biodiversity and genomics. Before 2003, there were only 10 coronaviruses with complete genomes available. After the SARS epidemic, up to December 2008, there was an addition of 16 coronaviruses with complete genomes sequenced. These include two human coronaviruses (human coronavirus NL63 and human coronavirus HKU1), 10 other mammalian coronaviruses [bat SARS coronavirus, bat coronavirus (bat-CoV) HKU2, bat-CoV HKU4, bat-CoV HKU5, bat-CoV HKU8, bat-CoV HKU9, bat-CoV 512/2005, bat-CoV 1A, equine coronavirus, and beluga whale coronavirus] and four avian coronaviruses (turkey coronavirus, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13). Two novel subgroups in group 2 coronavirus (groups 2c and 2d) and two novel subgroups in group 3 coronavirus (groups 3b and 3c) have been proposed. The diversity of coronaviruses is a result of the infidelity of RNA-dependent RNA polymerase, high frequency of homologous RNA recombination, and the large genomes of coronaviruses. Among all hosts, the diversity of coronaviruses is most evidenced in bats and birds, which may be a result of their species diversity, ability to fly, environmental pressures, and habits of roosting and flocking. The present evidence supports that bat coronaviruses are the gene pools of group 1 and 2 coronaviruses, whereas bird coronaviruses are the gene pools of group 3 coronaviruses. With the increasing number of coronaviruses, more and more closely related coronaviruses from distantly related animals have been observed, which were results of recent interspecies jumping and may be the cause of disastrous outbreaks of zoonotic diseases.

Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans.

Although many novel members of the Coronaviridae have recently been recognized in different species, the ecology of coronaviruses has not been established. Our study indicates that bats harbor a much wider diversity of coronaviruses than any other animal species. Dating of different coronavirus lineages suggests that bat coronaviruses are older than those recognized in other animals and that the human severe acute respiratory syndrome (SARS) coronavirus was directly derived from viruses from wild animals in wet markets of southern China. Furthermore, the most closely related bat and SARS coronaviruses diverged in 1986, an estimated divergence time of 17 years prior to the outbreak, suggesting that there may have been transmission via an unknown intermediate host. Analysis of lineage-specific selection pressure also indicated that only SARS coronaviruses in civets and humans were under significant positive selection, also demonstrating a recent interspecies transmission. Analysis of population dynamics revealed that coronavirus populations in bats have constant population growth, while viruses from all other hosts show epidemic-like increases in population. These results indicate that diverse coronaviruses are endemic in different bat species, with repeated introductions to other animals and occasional establishment in other species. Our findings suggest that bats are likely the natural hosts for all presently known coronavirus lineages and that all coronaviruses recognized in other species were derived from viruses residing in bats. Further surveillance of bat and other animal populations is needed to fully describe the ecology and evolution of this virus family.

Coronaviruses can infect a variety of animals including poultry, livestock, and humans and are currently classified into three groups. The interspecies transmissions of coronaviruses between different hosts form a complex ecosystem of which little is known. The outbreak of severe acute respiratory syndrome (SARS) and the recent identification of new coronaviruses have highlighted the necessity for further investigation of coronavirus ecology, in particular the role of bats and other wild animals. In this study, we sampled bat populations in 15 provinces of China and reveal that approximately 6.5% of the bats, from diverse species distributed throughout the region, harbor coronaviruses. Full genomes of four coronavirues from bats were sequenced and analyzed. Phylogenetic analyses of the spike, envelope, membrane, and nucleoprotein structural proteins and the two conserved replicase domains, putative RNA-dependent RNA polymerase and RNA helicase, revealed that bat coronaviruses cluster in three different groups: group 1, another group that includes all SARS and SARS-like coronaviruses (putative group 4), and an independent bat coronavirus group (putative group 5). Further genetic analyses showed that different species of bats maintain coronaviruses from different groups and that a single bat species from different geographic locations supports similar coronaviruses. Thus, the findings of this study suggest that bats may play an integral role in the ecology and evolution of coronaviruses.

The existence of coronaviruses in bats is unknown until the recent discovery of bat-SARS-CoV in Chinese horseshoe bats and a novel group 1 coronavirus in other bat species. Among 309 bats of 13 species captured from 20 different locations in rural areas of Hong Kong over a 16-month period, coronaviruses were amplified from anal swabs of 37 (12%) bats by RT-PCR. Phylogenetic analysis of RNA-dependent-RNA-polymerase (pol) and helicase genes revealed six novel coronaviruses from six different bat species, in addition to the two previously described coronaviruses. Among the six novel coronaviruses, four were group 1 coronaviruses (bat-CoV HKU2 from Chinese horseshoe bat, bat-CoV HKU6 from rickett's big-footed bat, bat-CoV HKU7 from greater bent-winged bat and bat-CoV HKU8 from lesser bent-winged bat) and two were group 2 coronaviruses (bat-CoV HKU4 from lesser bamboo bats and bat-CoV HKU5 from Japanese pipistrelles). An astonishing diversity of coronaviruses was observed in bats.

Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horse-shoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals. © 2005 by The National Academy of Sciences of the USA., link_to_OA_fulltext

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS). SCoV-like viruses were isolated from Himalayan palm civets found in a live-animal market in Guangdong, China. Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market. All the animal isolates retain a 29-nucleotide sequence that is not found in most human isolates. The detection of SCoV-like viruses in small, live wild mammals in a retail market indicates a route of interspecies transmission, although the natural reservoir is not known. Civet cats, a raccoon dog, and a ferret badger in an animal market in Gunagdong, China, were infected with a coronavirus identical to the one that causes SARS in humans save for an extra 29-nucleotide sequence. Civet cats, a raccoon dog, and a ferret badger in an animal market in Gunagdong, China, were infected with a coronavirus identical to the one that causes SARS in humans save for an extra 29-nucleotide sequence.

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

Summary Coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on 4 swine breeding farms. Diarrhea was reproduced in experimental pigs with one of the isolates, designated CV777, which was found to be distinct from the 2 known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus.

Coronaviruses, such as severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, pose significant public health threats. Bats have been suggested to act as natural reservoirs for both these viruses, and periodic monitoring of coronaviruses in bats may thus provide important clues about emergent infectious viruses. The Eastern bent-wing bat Miniopterus fuliginosus is distributed extensively throughout China. We therefore analyzed the genetic diversity of coronaviruses in samples of M. fuliginosus collected from nine Chinese provinces during 2011–2013. The only coronavirus genus found was Alphacoronavirus. We established six complete and five partial genomic sequences of alphacoronaviruses, which revealed that they could be divided into two distinct lineages, with close relationships to coronaviruses in Miniopterus magnater and Miniopterus pusillus. Recombination was confirmed by detecting putative breakpoints of Lineage 1 coronaviruses in M. fuliginosus and M. pusillus (Wu et al., 2015), which supported the results of topological and phylogenetic analyses. The established alphacoronavirus genome sequences showed high similarity to other alphacoronaviruses found in other Miniopterus species, suggesting that their transmission in different Miniopterus species may provide opportunities for recombination with different alphacoronaviruses. The genetic information for these novel alphacoronaviruses will improve our understanding of the evolution and genetic diversity of coronaviruses, with potentially important implications for the transmission of human diseases.

Background Porcine epidemic diarrhea (PED) is a syndrome that is characterized by rapidly spreading watery diarrhea affecting pigs of all ages, but with major effects on suckling piglets. The disease, as well as the causative Alphacoronavirus, the Porcine epidemic diarrhea virus (PEDV), was first described in Europe in the 1970s and since then has spread over many Asian and American countries, where it recently led to devastating effects on swine health and pork industry. While the disease was seldom reported in Europe within the last few decades, a few recent reports re-emergence of PED in German pig farms. The hitherto isolated German strain seems to be closely related to a low pathogenic PEDV variant from the USA. This case report describes the first detection of PEDV in Austria. Case presentation Reduced feed uptake and occasional diarrhea were observed in December 2014 in a group of fattening pigs, kept on an Austrian swine farm. The concerned pigs had been recently purchased from Germany. Within a few weeks, diarrhea became apparent also in pigs of Austrian origin, which were kept in a different stable on the same farm. Gastrointestinal symptoms among fattening pigs were generally mild, quickly resolving and did not lead to death. PEDV RNA was identified by RT-qPCR in pooled feces and serum and PEDV antibodies were detectable in serum in both groups of pigs. Phylogenetic analysis of the nearly complete PEDV spike gene shows that the Austrian PEDV strain is highly similar to other strains involved in recent outbreaks in Western and Central Europe. Conclusion This is the first report demonstrating the presence of PEDV in Austria. The virus was probably introduced by purchasing piglets from a German source, which underlines the significance of trans-boundary animal trade for the distribution of highly contagious diseases, such as PED. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0624-1) contains supplementary material, which is available to authorized users.

Bats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil.Three hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus.This study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.