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1.
Proc Natl Acad Sci U S A ; 119(18): e2118126119, 2022 05 03.
Article in English | MEDLINE | ID: covidwho-1815697

ABSTRACT

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.


Subject(s)
Alphacoronavirus , Coronavirus Infections , Swine Diseases , Alphacoronavirus/genetics , Animals , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Coronavirus Infections/epidemiology , Swine
2.
Viruses ; 14(2)2022 02 07.
Article in English | MEDLINE | ID: covidwho-1674829

ABSTRACT

Coronaviruses (CoV) are divided into the genera α-CoVs, ß-CoVs, γ-CoVs and δ-CoVs. Of these, α-CoVs and ß-CoVs are solely capable of causing infections in humans, resulting in mild to severe respiratory symptoms. Bats have been identified as natural reservoir hosts for CoVs belonging to these two genera. Consequently, research on bat populations, CoV prevalence in bats and genetic characterization of bat CoVs is of special interest to investigate the potential transmission risks. We present the genome sequence of a novel α-CoV strain detected in rectal swab samples of Miniopterus fuliginosus bats from a colony in the Wavul Galge cave (Koslanda, Sri Lanka). The novel strain is highly similar to Miniopterus bat coronavirus 1, an α-CoV located in the subgenus of Minunacoviruses. Phylogenetic reconstruction revealed a high identity of the novel strain to other α-CoVs derived from Miniopterus bats, while human-pathogenic α-CoV strains like HCoV-229E and HCoV-NL63 were more distantly related. Comparison with selected bat-related and human-pathogenic strains of the ß-CoV genus showed low identities of ~40%. Analyses of the different genes on nucleotide and amino acid level revealed that the non-structural ORF1a/1b are more conserved among α-CoVs and ß-CoVs, while there are higher variations in the structural proteins known to be important for host specificity. The novel strain was named batCoV/MinFul/2018/SriLanka and had a prevalence of 50% (66/130) in rectal swab samples and 58% (61/104) in feces samples that were collected from Miniopterus bats in Wavul Galge cave. Based on the differences between strain batCoV/MinFul/2018/SriLanka and human-pathogenic α-CoVs and ß-CoVs, we conclude that there is a rather low transmission risk to humans. Further studies in the Wavul Galge cave and at other locations in Sri Lanka will give more detailed information about the prevalence of this virus.


Subject(s)
Alphacoronavirus/genetics , Alphacoronavirus/isolation & purification , Chiroptera/virology , Coronavirus Infections/veterinary , Disease Reservoirs/veterinary , Disease Reservoirs/virology , Genome, Viral , Alphacoronavirus/classification , Animals , Caves/virology , Coronavirus Infections/virology , Evolution, Molecular , Female , Male , Phylogeny , Sequence Analysis, DNA , Sri Lanka
3.
Viruses ; 14(2)2022 01 19.
Article in English | MEDLINE | ID: covidwho-1625724

ABSTRACT

Bats are a reservoir for coronaviruses (CoVs) that periodically spill over to humans, as evidenced by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. A collection of 174 bat samples originating from South Dakota, Minnesota, Iowa, and Nebraska submitted for rabies virus testing due to human exposure were analyzed using a pan-coronavirus PCR. A previously partially characterized CoV, Eptesicus bat CoV, was identified in 12 (6.9%) samples by nested RT-PCR. Six near-complete genomes were determined. Genetic analysis found a high similarity between all CoV-positive samples, Rocky Mountain bat CoV 65 and alphacoronavirus HCQD-2020 recently identified in South Korea. Phylogenetic analysis of genome sequences showed EbCoV is closely related to bat CoV HKU2 and swine acute diarrhea syndrome CoV; however, topological incongruences were noted for the spike gene that was more closely related to porcine epidemic diarrhea virus. Similar to some alphaCoVs, a novel gene, ORF7, was discovered downstream of the nucleocapsid, whose protein lacked similarity to known proteins. The widespread circulation of EbCoV with similarities to bat viruses that have spilled over to swine warrants further surveillance.


Subject(s)
Alphacoronavirus/classification , Alphacoronavirus/genetics , Chiroptera/virology , Disease Reservoirs/veterinary , Disease Reservoirs/virology , Phylogeny , Alphacoronavirus/isolation & purification , Animals , Genome, Viral , Iowa , Midwestern United States , Minnesota , Republic of Korea , Sequence Analysis, DNA , South Dakota , Viral Zoonoses/transmission
4.
Sci Rep ; 11(1): 24145, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1585802

ABSTRACT

Recent studies suggest that coronaviruses circulate widely in Southeast Asian bat species and that the progenitors of the SARS-Cov-2 virus could have originated in rhinolophid bats in the region. Our objective was to assess the diversity and circulation patterns of coronavirus in several bat species in Southeast Asia. We undertook monthly live-capture sessions and sampling in Cambodia over 17 months to cover all phases of the annual reproduction cycle of bats and test specifically the association between their age and CoV infection status. We additionally examined current information on the reproductive phenology of Rhinolophus and other bat species presently known to occur in mainland southeast China, Vietnam, Laos and Cambodia. Results from our longitudinal monitoring (573 bats belonging to 8 species) showed an overall proportion of positive PCR tests for CoV of 4.2% (24/573) in cave-dwelling bats from Kampot and 4.75% (22/463) in flying-foxes from Kandal. Phylogenetic analysis showed that the PCR amplicon sequences of CoVs (n = 46) obtained clustered in Alphacoronavirus and Betacoronavirus. Interestingly, Hipposideros larvatus sensu lato harbored viruses from both genera. Our results suggest an association between positive detections of coronaviruses and juvenile and immature bats in Cambodia (OR = 3.24 [1.46-7.76], p = 0.005). Since the limited data presently available from literature review indicates that reproduction is largely synchronized among rhinolophid and hipposiderid bats in our study region, particularly in its more seasonal portions (above 16° N), this may lead to seasonal patterns in CoV circulation. Overall, our study suggests that surveillance of CoV in insectivorous bat species in Southeast Asia, including SARS-CoV-related coronaviruses in rhinolophid bats, could be targeted from June to October for species exhibiting high proportions of juveniles and immatures during these months. It also highlights the need to develop long-term longitudinal surveys of bats and improve our understanding of their ecology in the region, for both biodiversity conservation and public health reasons.


Subject(s)
Alphacoronavirus/genetics , Betacoronavirus/genetics , COVID-19/transmission , Chiroptera/growth & development , SARS-CoV-2/genetics , Alphacoronavirus/classification , Animals , Asia, Southeastern/epidemiology , Betacoronavirus/classification , COVID-19/epidemiology , COVID-19/virology , Cambodia/epidemiology , Chiroptera/classification , Chiroptera/virology , Epidemics/prevention & control , Evolution, Molecular , Genome, Viral/genetics , Geography , Humans , Longitudinal Studies , Male , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/physiology , Species Specificity
5.
Viruses ; 13(11)2021 10 29.
Article in English | MEDLINE | ID: covidwho-1488763

ABSTRACT

In the last two decades, several coronavirus (CoV) interspecies jumping events have occurred between bats and other animals/humans, leading to major epidemics/pandemics and high fatalities. The SARS epidemic in 2002/2003 had a ~10% fatality. The discovery of SARS-related CoVs in horseshoe bats and civets and genomic studies have confirmed bat-to-civet-to-human transmission. The MERS epidemic that emerged in 2012 had a ~35% mortality, with dromedaries as the reservoir. Although CoVs with the same genome organization (e.g., Tylonycteris BatCoV HKU4 and Pipistrellus BatCoV HKU5) were also detected in bats, there is still a phylogenetic gap between these bat CoVs and MERS-CoV. In 2016, 10 years after the discovery of Rhinolophus BatCoV HKU2 in Chinese horseshoe bats, fatal swine disease outbreaks caused by this virus were reported in southern China. In late 2019, an outbreak of pneumonia emerged in Wuhan, China, and rapidly spread globally, leading to >4,000,000 fatalities so far. Although the genome of SARS-CoV-2 is highly similar to that of SARS-CoV, patient zero and the original source of the pandemic are still unknown. To protect humans from future public health threats, measures should be taken to monitor and reduce the chance of interspecies jumping events, either occurring naturally or through recombineering experiments.


Subject(s)
COVID-19/virology , Chiroptera/virology , Coronavirus Infections/virology , Coronavirus/physiology , Host Adaptation , Severe Acute Respiratory Syndrome/virology , Alphacoronavirus/genetics , Alphacoronavirus/physiology , Animals , COVID-19/transmission , Coronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Coronavirus Infections/veterinary , Host Specificity , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/physiology , SARS Virus/genetics , SARS Virus/physiology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Severe Acute Respiratory Syndrome/epidemiology , Severe Acute Respiratory Syndrome/transmission , Severe Acute Respiratory Syndrome/veterinary
6.
Viruses ; 13(10)2021 10 07.
Article in English | MEDLINE | ID: covidwho-1463837

ABSTRACT

In summer 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was detected on mink farms in Utah. An interagency One Health response was initiated to assess the extent of the outbreak and included sampling animals from on or near affected mink farms and testing them for SARS-CoV-2 and non-SARS coronaviruses. Among the 365 animals sampled, including domestic cats, mink, rodents, raccoons, and skunks, 261 (72%) of the animals harbored at least one coronavirus. Among the samples that could be further characterized, 127 alphacoronaviruses and 88 betacoronaviruses (including 74 detections of SARS-CoV-2 in mink) were identified. Moreover, at least 10% (n = 27) of the coronavirus-positive animals were found to be co-infected with more than one coronavirus. Our findings indicate an unexpectedly high prevalence of coronavirus among the domestic and wild free-roaming animals tested on mink farms. These results raise the possibility that mink farms could be potential hot spots for future trans-species viral spillover and the emergence of new pandemic coronaviruses.


Subject(s)
Alphacoronavirus/isolation & purification , COVID-19/epidemiology , COVID-19/veterinary , SARS-CoV-2/isolation & purification , Alphacoronavirus/classification , Alphacoronavirus/genetics , Animals , Animals, Domestic/virology , Animals, Wild/virology , Cats , Disease Hotspot , Female , Male , Mephitidae/virology , Mice , Mink/virology , Raccoons/virology , SARS-CoV-2/classification , SARS-CoV-2/genetics , Utah/epidemiology
7.
Viruses ; 13(10)2021 10 11.
Article in English | MEDLINE | ID: covidwho-1460086

ABSTRACT

Coronavirus, an important zoonotic disease, raises concerns of future pandemics. The bat is considered a source of noticeable viruses resulting in human and livestock infections, especially the coronavirus. Therefore, surveillance and genetic analysis of coronaviruses in bats are essential in order to prevent the risk of future diseases. In this study, the genome of HCQD-2020, a novel alphacoronavirus detected in a bat (Eptesicus serotinus), was assembled and described using next-generation sequencing and bioinformatics analysis. The comparison of the whole-genome sequence and the conserved amino acid sequence of replicated proteins revealed that the new strain was distantly related with other known species in the Alphacoronavirus genus. Phylogenetic construction indicated that this strain formed a separated branch with other species, suggesting a new species of Alphacoronavirus. Additionally, in silico prediction also revealed the risk of cross-species infection of this strain, especially in the order Artiodactyla. In summary, this study provided the genetic characteristics of a possible new species belonging to Alphacoronavirus.


Subject(s)
Alphacoronavirus/classification , Alphacoronavirus/genetics , Chiroptera/virology , Coronavirus Infections/veterinary , Genome, Viral/genetics , Alphacoronavirus/isolation & purification , Amino Acid Sequence/genetics , Animals , Artiodactyla/virology , Coronavirus Infections/virology , Phylogeny , Republic of Korea , Sequence Alignment , Whole Genome Sequencing
8.
Viruses ; 13(10)2021 10 06.
Article in English | MEDLINE | ID: covidwho-1460085

ABSTRACT

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.


Subject(s)
Alphacoronavirus/isolation & purification , Betacoronavirus/isolation & purification , Chiroptera/virology , Genome, Viral/genetics , Metagenome/genetics , Alphacoronavirus/classification , Alphacoronavirus/genetics , Animals , Betacoronavirus/classification , Betacoronavirus/genetics , Chiroptera/genetics , Computational Biology/methods , Feces/virology , High-Throughput Nucleotide Sequencing , Metagenomics/methods , Moscow , Phycodnaviridae/classification , Phycodnaviridae/genetics , Phycodnaviridae/isolation & purification , Sequence Analysis, DNA
9.
Viruses ; 13(10)2021 10 01.
Article in English | MEDLINE | ID: covidwho-1444333

ABSTRACT

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and ß showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and ß-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10-5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, ß-, and γ-.


Subject(s)
Alphacoronavirus/genetics , Coronavirus Infections/veterinary , Deltacoronavirus/genetics , Gammacoronavirus/genetics , SARS-CoV-2/genetics , Animals , Animals, Wild/virology , Betacoronavirus/genetics , COVID-19/veterinary , Chiroptera/virology , Genome, Viral/genetics , Humans , Livestock/virology , Rodentia/virology
10.
Viruses ; 13(10)2021 09 29.
Article in English | MEDLINE | ID: covidwho-1441884

ABSTRACT

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.


Subject(s)
Alphacoronavirus/genetics , Chiroptera/virology , Alphacoronavirus/pathogenicity , Animals , Base Sequence/genetics , Biological Evolution , China , Chiroptera/genetics , Coronavirus/genetics , Coronavirus/pathogenicity , Coronavirus Infections/virology , Evolution, Molecular , Genetic Variation/genetics , Genome, Viral/genetics , Genotype , Phylogeny , Sequence Analysis, DNA/methods , Viral Proteins/genetics
11.
Emerg Microbes Infect ; 10(1): 1660-1668, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1343597

ABSTRACT

The coronavirus disease (COVID-19) pandemic is a major challenge worldwide. However, the epidemic potential of common human coronaviruses (HCoVs) remains unclear. This study aimed to determine the epidemiological and co-infection characteristics of common HCoVs in individuals with influenza-like illness (ILI) and severe acute respiratory infection (SARI). This retrospective, observational, multicentre study used data collected from patients admitted to nine sentinel hospitals with ILI and SARI from January 2015 through December 2020 in Shanghai, China. We prospectively tested patients for a total of 22 respiratory pathogens using multi-real-time polymerase chain reaction. Of the 4541 patients tested, 40.37% (1833/4541) tested positive for respiratory pathogens and 3.59% (163/4541) tested positive for common HCoVs. HCoV infection was more common in the non-endemic season for respiratory pathogens (odds ratio: 2.33, 95% confidence interval: 1.64-3.31). HCoV-OC43 (41.72%, 68/163) was the most common type of HCoV detected. The co-infection rate was 31.29% (51/163) among 163 HCoV-positive cases, with HCoV-229E (53.13%, 17/32), the HCoV type that was most frequently associated with co-infection. Respiratory pathogens responsible for co-infections with HCoVs included parainfluenza virus, rhinovirus/enterovirus, influenza A virus, and adenovirus. Furthermore, we identified one patient co-infected with HCoV-OC43 and HCoV-NL63/HKU1. The prevalence of common HCoVs remains low in ILI/SARI cases, in Shanghai. However, the seasonal pattern of HCoVs may be opposite to that of other respiratory pathogens. Moreover, HCoVs are likely to co-exist with specific respiratory pathogens. The potential role of co-infections with HCoVs and other pathogenic microorganisms in infection and pathogenesis of ILI and SARI warrants further study.


Subject(s)
Alphacoronavirus , COVID-19/epidemiology , COVID-19/virology , Coinfection/epidemiology , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , SARS-CoV-2 , Adult , Aged , Aged, 80 and over , Alphacoronavirus/classification , Alphacoronavirus/genetics , COVID-19/diagnosis , COVID-19/history , China/epidemiology , Coronavirus Infections/diagnosis , Coronavirus Infections/history , Female , History, 21st Century , Humans , Male , Middle Aged , Prevalence , Public Health Surveillance , Retrospective Studies , SARS-CoV-2/classification , SARS-CoV-2/genetics , Seasons
12.
PLoS One ; 16(6): e0253622, 2021.
Article in English | MEDLINE | ID: covidwho-1286870

ABSTRACT

Porcine epidemic diarrhea virus (PEDV), a leading cause of piglet diarrhea outbreaks, poses a significant danger to the swine industry. The aim of this study was to investigate the epidemic characteristics of PEDV that was circulating in Guangdong province, one of China's major pig producing provinces. Clinical samples were collected from eight pig farms in Guangdong province between 2018 and 2019 and tested for the major porcine enteric pathogens, including PEDV, transmissible gastroenteritis virus (TGEV), Swine enteric coronavirus (SeCoV), Swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (RV). As a result, only PEDV and RV were detected at a rate of 47.0% (16/34) and 18.6% (8/34), respectively. Coinfectoin with PEDV and RV occurred at a rate of PEDV 12.5% (2/16). Subsequently, the full-length S gene sequences of 13 PEDV strains were obtained, and phylogenetic analysis suggested the presence of GII-c group PEDV strains in this region (non-S-INDEL). Two novel common amino acid insertions (55T/IG56 and 551L) and one novel glycosylation site (1199G+) were detected when the CV777 and ZJ08 vaccine strains were compared. Furthermore, intragroup recombination events in the S gene regions 51-548 and 2478-4208 were observed in the PEDV strains studied. In summary, the observations provide current information on the incidence of viral agents causing swine diarrhea in southern China and detailed the genetic characteristics and evolutionary history of the dominant PEDV field strains. Our findings will aid in the development of an updated vaccine for the prevention and control of PEDV variant strains.


Subject(s)
Coronavirus Infections/genetics , Disease Outbreaks , Phylogeny , Porcine epidemic diarrhea virus/genetics , Swine Diseases/genetics , Alphacoronavirus/genetics , Animals , China/epidemiology , Coronavirus Infections/epidemiology , Swine , Swine Diseases/epidemiology , Transmissible gastroenteritis virus/genetics
13.
Viruses ; 13(6)2021 06 04.
Article in English | MEDLINE | ID: covidwho-1282637

ABSTRACT

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.


Subject(s)
Alphacoronavirus/genetics , Astroviridae/genetics , Chiroptera/virology , Genome, Viral , Whole Genome Sequencing , Alphacoronavirus/classification , Alphacoronavirus/isolation & purification , Animals , Astroviridae/classification , Astroviridae/isolation & purification , Denmark , Feces/virology , Genomics/methods , Open Reading Frames , Phylogeny
14.
J Med Virol ; 93(7): 4382-4391, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1263102

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has spread around the globe very rapidly. Previously, the evolution pattern and similarity among the COVID-19 causative organism severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and causative organisms of other similar infections have been determined using a single type of genetic marker in different studies. Herein, the SARS-CoV-2 and related ß coronaviruses Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV,  bat coronavirus (BAT-CoV) were comprehensively analyzed using a custom-built pipeline that employed phylogenetic approaches based on multiple types of genetic markers including the whole genome sequences, mutations in nucleotide sequences, mutations in protein sequences, and microsatellites. The whole-genome sequence-based phylogeny revealed that the strains of SARS-CoV-2 are more similar to the BAT-CoV strains. The mutational analysis showed that on average MERS-CoV and BAT-CoV genomes differed at 134.21 and 136.72 sites, respectively, whereas the SARS-CoV genome differed at 26.64 sites from the reference genome of SARS-CoV-2. Furthermore, the microsatellite analysis highlighted a relatively higher number of average microsatellites for MERS-CoV and SARS-CoV-2 (106.8 and 107, respectively), and a lower number for SARS-CoV and BAT-CoV (95.8 and 98.5, respectively). Collectively, the analysis of multiple genetic markers of selected ß viral genomes revealed that the newly born SARS-COV-2 is closely related to BAT-CoV, whereas, MERS-CoV is more distinct from the SARS-CoV-2 than BAT-CoV and SARS-CoV.


Subject(s)
Alphacoronavirus/genetics , Genome, Viral/genetics , Microsatellite Repeats/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , Base Sequence/genetics , Chiroptera/virology , DNA Mutational Analysis , Genetic Markers/genetics , Genetic Variation/genetics , Humans , Phylogeny , Sequence Alignment , Sequence Homology, Nucleic Acid , Whole Genome Sequencing
16.
J Med Virol ; 93(7): 4382-4391, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1156882

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has spread around the globe very rapidly. Previously, the evolution pattern and similarity among the COVID-19 causative organism severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and causative organisms of other similar infections have been determined using a single type of genetic marker in different studies. Herein, the SARS-CoV-2 and related ß coronaviruses Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV,  bat coronavirus (BAT-CoV) were comprehensively analyzed using a custom-built pipeline that employed phylogenetic approaches based on multiple types of genetic markers including the whole genome sequences, mutations in nucleotide sequences, mutations in protein sequences, and microsatellites. The whole-genome sequence-based phylogeny revealed that the strains of SARS-CoV-2 are more similar to the BAT-CoV strains. The mutational analysis showed that on average MERS-CoV and BAT-CoV genomes differed at 134.21 and 136.72 sites, respectively, whereas the SARS-CoV genome differed at 26.64 sites from the reference genome of SARS-CoV-2. Furthermore, the microsatellite analysis highlighted a relatively higher number of average microsatellites for MERS-CoV and SARS-CoV-2 (106.8 and 107, respectively), and a lower number for SARS-CoV and BAT-CoV (95.8 and 98.5, respectively). Collectively, the analysis of multiple genetic markers of selected ß viral genomes revealed that the newly born SARS-COV-2 is closely related to BAT-CoV, whereas, MERS-CoV is more distinct from the SARS-CoV-2 than BAT-CoV and SARS-CoV.


Subject(s)
Alphacoronavirus/genetics , Genome, Viral/genetics , Microsatellite Repeats/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , Base Sequence/genetics , Chiroptera/virology , DNA Mutational Analysis , Genetic Markers/genetics , Genetic Variation/genetics , Humans , Phylogeny , Sequence Alignment , Sequence Homology, Nucleic Acid , Whole Genome Sequencing
17.
PLoS One ; 16(1): e0244006, 2021.
Article in English | MEDLINE | ID: covidwho-1074162

ABSTRACT

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.


Subject(s)
Alphacoronavirus/genetics , Chiroptera/genetics , Alphacoronavirus/classification , Alphacoronavirus/isolation & purification , Animals , Caves , Chiroptera/classification , Chiroptera/virology , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/metabolism , DNA, Viral/chemistry , DNA, Viral/metabolism , Far East , Feces/virology , Gene Flow , Genetic Variation , High-Throughput Nucleotide Sequencing , Monte Carlo Method , Phylogeny
18.
J Med Virol ; 93(7): 4438-4445, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-986280

ABSTRACT

The hologic panther fusion (PF) platform provides fully automated CE marked diagnostics for respiratory viruses, including the recently discovered severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) by a transcription mediated amplification (TMA) assay, but not for the endemic human coronaviruses (hCoV). Therefore, a laboratory developed test (LDT) comprising a multiplexed reverse transcription polymerase chain reaction (RT-PCR) protocol that detects and differentiates the four hCoV NL63, 229E, HKU1, and OC43 was adapted on the PF. The novel CE marked Aptima SARS-CoV-2 TMA and the LDT for hCoV were validated with 321 diagnostic specimens from the upper and lower respiratory tract in comparison to two SARS-CoV-2 RT-PCRs (PF E-gene RT-PCR and genesig RT-PCR, 157 specimens) or the R-GENE hCoV/hParaFlu RT-PCR (164 specimens), respectively. For the endemic hCoV, results were 96.3% concordant with two specimens discordantly positive in the PF and four specimens discordantly positive in the R-GENE assay. All discordantly positive samples had Ct values between 33 and 39. The PF hCoV LDT identified 23 hCoV positive specimens as NL63, 15 as 229E, 15 as HKU1, and 25 as OC43. The Aptima SARS-CoV-2 TMA gave 99.4% concordant results compared to the consensus results with a single specimen discordantly positive. Moreover, 36 samples from proficiency testing panels were detected and typed correctly by both novel methods. In conclusion, the SARS-CoV-2 TMA and the LDT for hCoV enhanced the diagnostic spectrum of the PF for all coronaviruses circulating globally for a multitude of diagnostic materials from the upper and lower respiratory tract.


Subject(s)
Alphacoronavirus/genetics , COVID-19/diagnosis , Coronavirus 229E, Human/genetics , Coronavirus NL63, Human/genetics , Coronavirus OC43, Human/genetics , SARS-CoV-2/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Respiratory System/virology , Reverse Transcriptase Polymerase Chain Reaction/methods
19.
J Virol ; 94(22)2020 10 27.
Article in English | MEDLINE | ID: covidwho-982189

ABSTRACT

Coronaviruses (CoV) have caused a number of major epidemics in humans and animals, including the current pandemic of coronavirus disease 2019 (COVID-19), which has brought a renewed focus on the evolution and interspecies transmission of coronaviruses. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was recently identified in piglets in southern China, is an alphacoronavirus that originates from the same genus of horseshoe bats as severe acute respiratory syndrome CoV (SARS-CoV) and that was reported to be capable of infecting cells from a broad range of species, suggesting a considerable potential for interspecies transmission. Given the importance of the coronavirus spike (S) glycoprotein in host range determination and viral entry, we report a cryo-electron microscopy (cryo-EM) structure of the SADS-CoV S trimer in the prefusion conformation at a 3.55-Å resolution. Our structure reveals that the SADS-CoV S trimer assumes an intrasubunit quaternary packing mode in which the S1 subunit N-terminal domain (S1-NTD) and the S1 subunit C-terminal domain (S1-CTD) of the same protomer pack together by facing each other in the lying-down state. SADS-CoV S has several distinctive structural features that may facilitate immune escape, such as a relatively compact architecture of the S trimer and epitope masking by glycan shielding. Comparison of SADS-CoV S with the spike proteins of the other coronavirus genera suggested that the structural features of SADS-CoV S are evolutionarily related to those of the spike proteins of the other genera rather than to the spike protein of a typical alphacoronavirus. These data provide new insights into the evolutionary relationship between spike glycoproteins of SADS-CoV and those of other coronaviruses and extend our understanding of their structural and functional diversity.IMPORTANCE In this article, we report the atomic-resolution prefusion structure of the spike protein from swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV is a pathogenic alphacoronavirus that was responsible for a large-scale outbreak of fatal disease in pigs and that was reported to be capable of interspecies transmission. We describe the overall structure of the SADS-CoV spike protein and conducted a detailed analysis of its main structural elements. Our results and analyses are consistent with those of previous phylogenetic studies and suggest that the SADS-CoV spike protein is evolutionarily related to the spike proteins of betacoronaviruses, with a strong similarity in S1-NTDs and a marked divergence in S1-CTDs. Moreover, we discuss the possible immune evasion strategies used by the SADS-CoV spike protein. Our study provides insights into the structure and immune evasion strategies of the SADS-CoV spike protein and broadens the understanding of the evolutionary relationships between coronavirus spike proteins of different genera.


Subject(s)
Alphacoronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/ultrastructure , Alphacoronavirus/genetics , Amino Acid Sequence , Cryoelectron Microscopy , Evolution, Molecular , Immune Evasion , Models, Molecular , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Structural Homology, Protein
20.
Proc Natl Acad Sci U S A ; 117(43): 26915-26925, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-851432

ABSTRACT

Zoonotic coronaviruses represent an ongoing threat, yet the myriads of circulating animal viruses complicate the identification of higher-risk isolates that threaten human health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered, highly pathogenic virus that likely evolved from closely related HKU2 bat coronaviruses, circulating in Rhinolophus spp. bats in China and elsewhere. As coronaviruses cause severe economic losses in the pork industry and swine are key intermediate hosts of human disease outbreaks, we synthetically resurrected a recombinant virus (rSADS-CoV) as well as a derivative encoding tomato red fluorescent protein (tRFP) in place of ORF3. rSADS-CoV replicated efficiently in a variety of continuous animal and primate cell lines, including human liver and rectal carcinoma cell lines. Of concern, rSADS-CoV also replicated efficiently in several different primary human lung cell types, as well as primary human intestinal cells. rSADS-CoV did not use human coronavirus ACE-2, DPP4, or CD13 receptors for docking and entry. Contemporary human donor sera neutralized the group I human coronavirus NL63, but not rSADS-CoV, suggesting limited human group I coronavirus cross protective herd immunity. Importantly, remdesivir, a broad-spectrum nucleoside analog that is effective against other group 1 and 2 coronaviruses, efficiently blocked rSADS-CoV replication in vitro. rSADS-CoV demonstrated little, if any, replicative capacity in either immune-competent or immunodeficient mice, indicating a critical need for improved animal models. Efficient growth in primary human lung and intestinal cells implicate SADS-CoV as a potential higher-risk emerging coronavirus pathogen that could negatively impact the global economy and human health.


Subject(s)
Alphacoronavirus/physiology , Coronavirus Infections/virology , Disease Susceptibility/virology , Virus Replication , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Alphacoronavirus/genetics , Alphacoronavirus/growth & development , Animals , Cells, Cultured , Chlorocebus aethiops , Coronavirus Infections/transmission , Gene Expression , Host Specificity , Humans , Luminescent Proteins/genetics , Mice , Vero Cells , Virus Replication/drug effects
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