Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.

Detection of coronavirus in vampire bats (Desmodus rotundus) in southern Brazil.

The vampire bat (Desmodus rotundus) is a haematophagous animal that feeds exclusively on the blood of domestic mammals. Vampire bat feeding habits enable their contact with mammalian hosts and may enhance zoonotic spillover. Moreover, they may carry several pathogenic organisms, including coronaviruses (CoVs), for which they are important hosts. The human pathogens that cause severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV) and possibly coronavirus disease 2019 (SARS-CoV-2) all originated in bats but required bridge hosts to spread into human populations. To monitor the presence of potential zoonotic viruses in bats, the present work evaluated the presence of CoVs in vampire bats from southern Brazil. A total of 101 vampire bats were captured and euthanized between 2017 and 2019 in Rio Grande do Sul state, southern Brazil. The brain, heart, liver, lungs, kidneys and intestines were collected and macerated individually. The samples were pooled and submitted to high-throughput sequencing (HTS) using the Illumina MiSeq platform and subsequently individually screened using a pancoronavirus RT-PCR protocol. We detected CoV-related sequences in HTS, but only two (2/101; 1.98%) animals had CoV detected in the intestines by RT-PCR. Partial sequences of RdRp and spike genes were obtained in the same sample and the RdRp region in the other sample. The sequences were classified as belonging to Alphacoronavirus. The sequences were closely related to alphacoronaviruses detected in vampire bats from Peru. The continuous monitoring of bat CoVs may help to map and predict putative future zoonotic agents with great impacts on human health.

Animal Reservoirs and Hosts for Emerging Alphacoronaviruses and Betacoronaviruses.

The ongoing global pandemic caused by coronavirus disease has once again demonstrated the role of the family Coronaviridae in causing human disease outbreaks. Because severe acute respiratory syndrome coronavirus 2 was first detected in December 2019, information on its tropism, host range, and clinical manifestations in animals is limited. Given the limited information, data from other coronaviruses might be useful for informing scientific inquiry, risk assessment, and decision-making. We reviewed endemic and emerging infections of alphacoronaviruses and betacoronaviruses in wildlife, livestock, and companion animals and provide information on the receptor use, known hosts, and clinical signs associated with each host for 15 coronaviruses detected in humans and animals. This information can be used to guide implementation of a One Health approach that involves human health, animal health, environmental, and other relevant partners in developing strategies for preparedness, response, and control to current and future coronavirus disease threats.

Data standardization implementation and applications within and among diagnostic laboratories: integrating and monitoring enteric coronaviruses.

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.

PACIFIC: a lightweight deep-learning classifier of SARS-CoV-2 and co-infecting RNA viruses.

Viral co-infections occur in COVID-19 patients, potentially impacting disease progression and severity. However, there is currently no dedicated method to identify viral co-infections in patient RNA-seq data. We developed PACIFIC, a deep-learning algorithm that accurately detects SARS-CoV-2 and other common RNA respiratory viruses from RNA-seq data. Using in silico data, PACIFIC recovers the presence and relative concentrations of viruses with > 99% precision and recall. PACIFIC accurately detects SARS-CoV-2 and other viral infections in 63 independent in vitro cell culture and patient datasets. PACIFIC is an end-to-end tool that enables the systematic monitoring of viral infections in the current global pandemic.

Detection of community-acquired respiratory viruses in allogeneic stem-cell transplant recipients and controls-A prospective cohort study.

BACKGROUND: Community-acquired respiratory viruses (CARV) cause upper and lower respiratory tract infections (URTI/LRTI) and may be life-threatening for recipients of an allogeneic stem cell transplantation (allo-SCT). METHODS: In a prospective study encompassing 4 winter-seasons, we collected throat gargles (TG) at random time points from allo-SCT recipients (patients) and controls and followed them up for at least 3 weeks including repetitive sampling and documentation of symptoms. A Multiplex-PCR system to identify 20 CARV and Mycoplasma pneumoniae was used to detect CARV. RESULTS: One hundred ninety-four patients with 426 TG and 273 controls with 549 TG were included. There were more patients with a positive test result (25% vs 11% in the controls), and the patients had a higher number of positive TG (70 = 16%) compared to controls (32 = 6%) (P < .001). Altogether, 115 viruses were detected. Multiple viruses in one TG (11/48, 34%) and prolonged shedding were only observed in patients (13/48, 27%). Patients had more RSV (18/83, 26%) and adenovirus (15/83, 21%) than controls (both viruses 2/32, 6%). Independent risk factors for the detection of CARV included age >40 years (OR 3.38, 95% CI 1.8-6.4, P < .001) and presence of URTI-symptoms (OR 3.22, 95% CI 1.9-5.5, P < .001). No controls developed a LRTI or died whereas 4/48 (8%) patients developed a LRTI (coronavirus in 2, RSV in 1 and influenza A H1N1 in 1 patient). One patient died of CARV (influenza A H1N1). CONCLUSION: Allo-SCT-recipients have more CARV-infections, exhibit a different epidemiology, have more cases of co-infection or prolonged shedding and have a higher rate of LRTI and mortality.

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses.

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/µL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

Isolation of a Chinook Salmon Bafinivirus (CSBV) in Imported Goldfish Carassius auratus L. in the United Kingdom and Evaluation of Its Virulence in Resident Fish Species.

This is the first record of a fish nidovirus isolated from a consignment of goldfish at the United Kingdom (UK) border. The full-length viral genome was 25,985 nt, sharing a 97.9% nucleotide identity with the Chinook salmon bafinivirus (CSBV) NIDO with two deletions of 537 and 480 nt on the ORF Ia protein. To assess the potential impact on UK fish species, Atlantic salmon, common carp and goldfish were exposed to the virus via an intraperitoneal (IP) injection and bath challenge. Moribundity was recorded in only 8% of IP-injected goldfish. A high viral load, ≈107 of the CSBV PpIa gene, was measured in the kidney of moribund goldfish. Mild histopathological changes were observed in the kidneys of challenged carps. Ultrastructural observations in renal tubule epithelial cells of goldfish showed cylindrical tubes (≈15 nm in diameter) and tubular structures budding spherical virions (≈200 nm in diameter) with external spike-like structures. Negative staining showed both circular and bacilliform virions. Seroconversion was measured in common carp and goldfish but not in Atlantic salmon. This study reinforces the potential risk of novel and emerging pathogens being introduced to recipient countries via the international ornamental fish trade and the importance of regular full health screens at the border inspection posts to reduce this risk.

Human virome in nasopharynx and tracheal secretion samples.

BACKGROUND: In Brazil the implementation of the Sentinel Surveillance System of Influenza began in 2000. Central public health laboratories use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for diagnosis of respiratory viruses, but this protocol identifies only specific targets, resulted in inconclusive diagnosis for many samples. Thus, high-throughput sequencing (HTS) would be complementary method in the identification of pathogens in inconclusive samples for RT-qPCR or other specific detection protocols. OBJECTIVES: This study aimed to detect unidentified viruses using HTS approach in negative samples of nasopharynx/tracheal secretions by the standard RT-qPCR collected in the Federal District, Brazil. METHODS: Nucleic acids were extracted from samples collected in winter period of 2016 and subjected to HTS. The results were confirmed by the multiplex PR21 RT-qPCR, which identifies 21 respiratory pathogens. FINDINGS: The main viruses identified by HTS were of families Herpesviridae, Coronaviridae, Parvoviridae and Picornaviridae, with the emphasis on rhinoviruses. The presence of respiratory viruses in the samples was confirmed by the PR21 multiplex RT-qPCR. Coronavirus, enterovirus, bocavirus and rhinovirus were found by multiplex RT-qPCR as well as by HTS analyses. MAIN CONCLUSIONS: Wide virus diversity was found by different methodologies and high frequency of rhinovirus occurrence was confirmed in population in winter, showing its relevance for public health.

Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea.

Currently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40-65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3'- or 5'-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/µL, 1.92 × 102 copies/µL, 1.74 × 102 copies/µL, and 1.76 × 102 copies/µL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrheal symptoms were collected in Northeastern China from 2017 to 2018 followed by analysis using the assay, and epidemiological investigation results showed that PEDV, TGEV, PoRV, and PDCoV prevalence was 19.05%, 5.21%, 4.32%, and 3.87%, respectively. The assay developed in this study showed higher detection accuracy than conventional RT-PCR method, suggesting a useful tool for the accurate differentiation of the four major viruses causing porcine viral diarrhea in practice.

MOLECULAR IDENTIFICATION OF AVIAN VIRUSES IN NEOTROPIC CORMORANTS ( PHALACROCORAX BRASILIANUS) IN CHILE.

We identified two RNA (paramyxovirus and coronavirus) and two DNA (adenovirus and herpesvirus) viruses in a common aquatic bird, the Neotropic Cormorant ( Phalacrocorax brasilianus), and determined their phylogenetic relationships to other global circulating variants. We analyzed 104 cloacal swabs from individuals collected at locations in Central Chile. Sequences were obtained from amplicons using consensus primers targeting conserved genes of the virus families Paramyxoviridae, Coronaviridae, Adenoviridae, and Herpesviridae. A total of 20.2% of the samples was positive for coronavirus, 8.7% for adenovirus, and 3.8% for herpesvirus. No paramyxoviruses were detected. All coronaviruses were identified as viruses of the Gammacoronavirus genus, closely related to the infectious bronchitis virus clade (bootstrap clade support=75%). All adenovirus samples were identified as Aviadenovirus, related to a gull and falcon adenovirus (Bayesian posterior probability=0.86). The herpesviruses identified were related to the infectious laryngotracheitis virus ( Gallid herpesvirus 1) of the genus Iltovirus (bootstrap clade support=99%). We provide information about the diversity of viruses circulating among apparently healthy Neotropic Cormorants.

Human virome in nasopharynx and tracheal secretion samples

BACKGROUND In Brazil the implementation of the Sentinel Surveillance System of Influenza began in 2000. Central public health laboratories use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for diagnosis of respiratory viruses, but this protocol identifies only specific targets, resulted in inconclusive diagnosis for many samples. Thus, high-throughput sequencing (HTS) would be complementary method in the identification of pathogens in inconclusive samples for RT-qPCR or other specific detection protocols. OBJECTIVES This study aimed to detect unidentified viruses using HTS approach in negative samples of nasopharynx/tracheal secretions by the standard RT-qPCR collected in the Federal District, Brazil. METHODS Nucleic acids were extracted from samples collected in winter period of 2016 and subjected to HTS. The results were confirmed by the multiplex PR21 RT-qPCR, which identifies 21 respiratory pathogens. FINDINGS The main viruses identified by HTS were of families Herpesviridae, Coronaviridae, Parvoviridae and Picornaviridae, with the emphasis on rhinoviruses. The presence of respiratory viruses in the samples was confirmed by the PR21 multiplex RT-qPCR. Coronavirus, enterovirus, bocavirus and rhinovirus were found by multiplex RT-qPCR as well as by HTS analyses. MAIN CONCLUSIONS Wide virus diversity was found by different methodologies and high frequency of rhinovirus occurrence was confirmed in population in winter, showing its relevance for public health.

Description and initial characterization of metatranscriptomic nidovirus-like genomes from the proposed new family Abyssoviridae, and from a sister group to the Coronavirinae, the proposed genus Alphaletovirus.

Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed.

Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States.

BACKGROUND: Emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) in North America, Asia and Europe has caused severe economic loss to the global swine industry. However, the virome of PEDV infected pigs and its effect on disease severity remains unknown. The advancements of sequencing technology have made it possible to characterize the entire microbiome of different body sites for any host. METHODS: The objective of this study was to characterize the RNA virome in PEDV-positive pigs using the hypothesis-free metagenomics approach based on next-generation sequencing. Specifically, 217 PEDV-positive swine fecal swab samples collected from diarrheic piglets over 17 US states during 2015-2016 were analyzed. RESULTS: A Kraken algorithm-based bioinformatics analysis revealed the presence of up to 9 different RNA genera besides PEDV (Alphacoronavirus genus), including Mamastrovirus (52%, 113/217), Enterovirus (39%, 85/217), Sapelovirus (31%, 67/217), Posavirus (30%, 66/217), Kobuvirus (23%, 49/217), Sapovirus (13%, 28/217), Teschovirus (10%, 22/217), Pasivirus (9%, 20/217), and Deltacoronavirus (3%, 6/217). There were 58 out of 217 piglets (27%) have PEDV infection alone whereas the remaining 159 (73%) shed 2 up to 9 different viruses. CONCLUSION: These findings demonstrated that PEDV infected diarrheic pigs had an extensive RNA viral flora consisting of four different families: Astroviridae, Picornaviridae, Caliciviridae, and Coronaviridae.

New viruses associated with canine gastroenteritis.

A number of novel viruses have been associated with canine gastroenteritis in recent years, from viral families as diverse as Caliciviridae and Picornaviridae to Parvoviridae and Circoviridae. The ability of many of these viruses to cause disease is uncertain, but epidemiological studies are continually adding to our knowledge of these potential pathogens. This review presents a summary of the latest research and current understanding of novel viruses associated with canine gastroenteritis.

A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification.

BACKGROUND: Porcine Deltacoronavirus (PDCoV) is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. RESULTS: In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR). This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine kobuvirus (PKoV), porcine astrovirus (PAstV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), and porcine circovirus type 2 (PCV2). By screening a panel of clinical specimens (N = 192), this method presented a similar sensitivity with nested RT-PCR and was 1-2 log more sensitive than conventional RT-PCR in detection of PDCoV. CONCLUSIONS: The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology.

Identification of diverse viruses in upper respiratory samples in dromedary camels from United Arab Emirates.

Camels are known carriers for many viral pathogens, including Middle East respiratory syndrome coronavirus (MERS-CoV). It is likely that there are additional, as yet unidentified viruses in camels with the potential to cause disease in humans. In this study, we performed metagenomic sequencing analysis on nasopharyngeal swab samples from 108 MERS-CoV-positive dromedary camels from a live animal market in Abu Dhabi, United Arab Emirates. We obtained a total of 846.72 million high-quality reads from these nasopharyngeal swab samples, of which 2.88 million (0.34%) were related to viral sequences while 512.63 million (60.5%) and 50.87 million (6%) matched bacterial and eukaryotic sequences, respectively. Among the viral reads, sequences related to mammalian viruses from 13 genera in 10 viral families were identified, including Coronaviridae, Nairoviridae, Paramyxoviridae, Parvoviridae, Polyomaviridae, Papillomaviridae, Astroviridae, Picornaviridae, Poxviridae, and Genomoviridae. Some viral sequences belong to known camel or human viruses and others are from potentially novel camel viruses with only limited sequence similarity to virus sequences in GenBank. A total of five potentially novel virus species or strains were identified. Co-infection of at least two recently identified camel coronaviruses was detected in 92.6% of the camels in the study. This study provides a comprehensive survey of viruses in the virome of upper respiratory samples in camels that have extensive contact with the human population.

Novel coronaviruses, astroviruses, adenoviruses and circoviruses in insectivorous bats from northern China.

Bats are considered as the reservoirs of several emerging infectious disease, and novel viruses are continually found in bats all around the world. Studies conducted in southern China found that bats carried a variety of viruses. However, few studies have been conducted on bats in northern China, which harbours a diversity of endemic insectivorous bats. It is important to understand the prevalence and diversity of viruses circulating in bats in northern China. In this study, a total of 145 insectivorous bats representing six species were collected from northern China and screened with degenerate primers for viruses belonging to six families, including coronaviruses, astroviruses, hantaviruses, paramyxoviruses, adenoviruses and circoviruses. Our study found that four of the viruses screened for were positive and the overall detection rates for astroviruses, coronaviruses, adenoviruses and circoviruses in bats were 21.4%, 15.9%, 20% and 37.2%, respectively. In addition, we found that bats in northern China harboured a diversity of novel viruses. Common Serotine (Eptesicus serotinu), Fringed long-footed Myotis (Myotis fimriatus) and Peking Myotis (Myotis pequinius) were investigated in China for the first time. Our study provided new information on the ecology and phylogeny of bat-borne viruses.

Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR.

Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50-100 copies/mL and 5-100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.

A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

BACKGROUND: Porcine Deltacoronavirus (PDCoV) is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. RESULTS: In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR). This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine kobuvirus (PKoV), porcine astrovirus (PAstV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), and porcine circovirus type 2 (PCV2). By screening a panel of clinical specimens (N = 192), this method presented a similar sensitivity with nested RT-PCR and was 1-2 log more sensitive than conventional RT-PCR in detection of PDCoV. CONCLUSIONS: The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology.