Application of codon pair deoptimization for ORF7-induced attenuation of type I porcine reproductive and respiratory syndrome virus without reduced immune responses.

Codon pair deoptimization (CPD) attenuated type I porcine reproductive and respiratory syndrome virus (PRRSV). Infectious clones covering the full genome of a Korean type I PRRSV (E38) were synthesized, and CPD induced nine synonymous mutants of NSP1 (n = 1) and ORF7 (n = 8). In a trial to rescue live viruses from infectious clones, only four clones with mutations at nt 177 downstream of ORF7 were rescued, which showed a substantial decrease in cellular replication ability. The rescue-failed clones had two common mutation sites with a high minimum free energy and significantly modified RNA secondary structure relative to the original virus. In infected pigs, CPD viruses demonstrated significantly lower replication ability and pathogenicity than the original virus. However, immune response level induced by the attenuated viruses and the original virus was similar. This is the first study to demonstrate that type I PRRSV virulence can be attenuated through CPD application to ORF7.

Unexpected thermal stability of two enveloped megaviruses, Emiliania huxleyi virus and African swine fever virus, as measured by viability PCR (preprint)

Background: The particle structure of Emiliania huxleyi virus (EhV), an algal infecting member of nucleocytoplasmic large DNA viruses (NCLDVs), contains an outer lipid membrane envelope similar to that found in animal viruses such as African swine fever virus (ASFV). Despite both being enveloped NCLDVs, EhV and ASFV are known for their stability in the environment. Method: Here we report for the first time, the application of a viability PCR method to describe the unprecedented and similar virion thermal stability of both EhV and ASFV. This result contradicts the bioassay method that suggests that virus “infectivity” is lost in a matter of seconds (EhV) and minutes (ASFV) at temperature greater than 50 °C. Confocal microscopy and analytical flow cytometry methods was used to validate the viability PCR data for EhV. Results: We observed that both EhV and ASFV particles has unprecedented thermal tolerances. These two NCLDVs are exceptions to the rule that having an enveloped virion anatomy is a predicted weakness, as is often observed in enveloped RNA viruses (i.e., the viruses causing Porcine Reproductive and Respiratory Syndrome (PRRS), COVID-19, Ebola, or seasonal influenza). Using the viability PCR method, we confirm that no PRRSV particles remain after 20 minutes of exposure to temperatures up to 100 °C. Conclusions: This observation has practical implications for industries involved in animal health and food security. Finally, we propose that EhV could be used as a surrogate for ASFV under certain circumstances.

Metagenomic analysis of endemic viruses in oral secretions from Chinese pigs.

BACKGROUND: Pigs are unique reservoirs for virus ecology. Despite the increased use of improved biosecurity measures, pig viruses readily circulate in Chinese swine farms. OBJECTIVES: The main objective of this study was to examine archived swine oral secretion samples with a panel of pan-species viral assays such that we might better describe the viral ecology of swine endemic viruses in Chinese farms. METHODOLOGY: Two hundred (n = 200) swine oral secretion samples, collected during 2015 and 2016 from healthy pigs on six swine farms in two provinces in China, were screened with molecular pan-species assays for coronaviruses (CoVs), adenoviruses (AdVs), enteroviruses (EVs), and paramyxoviruses (PMV). Samples were also screened for porcine circovirus (PCV) 3, porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). RESULTS: Among 200 swine oral secretion samples, 152 (76.0%) were found to have at least one viral detection. Thirty-four samples (17%) were positive for more than one virus, including 24 (70.5%) with dual detection and 10 (29.5%) with triple detection. Seventy-eight (39.0%) samples were positive for porcine AdVs, 22 (11.0%) were positive for porcine CoVs, 21 (10.5%) were positive for IAVs, 13 (6.5%) were positive for PCV, 7 (3.5%) were positive for PMV, six (3.0%) were positive for PRRSV and five (2.5%) were positive for porcine EV. CONCLUSION: Our findings underscore the high prevalence of numerous viruses among production pigs in China and highlight the need for routine, periodic surveillance for novel virus emergence with the goal of protecting pigs.

Characterization and cross-protection of experimental infections with SeCoV and two PEDV variants. (preprint)

Global emergence and re-emergence of Porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus which causes a highly contagious enteric disease, have led to several studies addressing its variability. The aim of this study was to characterize the infection of weaned pigs with Swine enteric coronavirus (SeCoV) -a chimeric virus most likely originated from a recombination event between PEDV and Transmissible gastroenteritis virus, or its mutant Porcine respiratory coronavirus- , and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals; piglets in groups B, C and D were orally inoculated with a PEDV variant (B and D) or SeCoV (C), while piglets in group A were mock inoculated and maintained as controls. At day 20 post-infection all groups were exposed to rPEDV-SeCoV; thus, group D was subjected to a homologous re-challenge, groups B and C to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively) and group A was primary challenged (-/rPEDV-SeCoV). Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposition to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in feces.

Study on the effect of intestinal immunity that the neonatal piglet co-infection with porcine deltacoronavirus and porcine epidemic diarrhoea virus (preprint)

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhoea virus (PEDV) have been often simultaneously detected in coronavirus diarrhea piglets. But the intestinal immune of the interaction between the co-circulating PDCoV and PEDV are unknown. Therefore, the study was conducted to investigate intestinal immunity of neonatal piglets that were exposed with PDCoV first and then PEDV subsequently. The amount and distribution of CD3 + T lymphocytes, B lymphocytes and goblet cells (GCs) in small intestine were analyzed by immunohistochemistry and periodic acid-schiff respectively. The expression of pattern recognition receptors and downstream mediator genes were analyzed by qPCR. The results showed that the number of GCs, CD3 + T lymphocytes and B lymphocytes in the small intestine of the PDCoV + PEDV co-inoculated piglets were increased compared with PEDV single-inoculated piglets. The piglets in the group of PDCoV + PEDV were significantly up-regulated IFN-α and IFN-λ 1 when compared with the PEDV single-inoculated piglets. These results suggest that the PDCoV + PEDV co-infected piglets can superiorly activate intestinal antiviral immunity compared to PEDV single-infected piglets, which provide a new insight into the pathogenesis mechanism of swine enteric coronavirus coinfection that may be used for vaccination in the future.

Detection of respiratory viruses in cases of porcine respiratory disease in nurseries. (preprint)

Respiratory disease in weaned pigs is a common problem in the field, with a complex aetiology of both viruses and bacteria. In the present study, we investigated the presence of eleven viruses in nasal swabs collected from nurseries (fifty-five clinical outbreaks) under the suspicion of swine influenza A virus (swIAV) by cough and fever. The other ten viruses included influenza B (IBV) and influenza D viruses (IDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine respiratory coronavirus (PRCV), Porcine cytomegalovirus (PCMV), porcine circoviruses 2 (PCV2), 3 (PCV3) and 4 (PCV), Porcine parainfluenza 1 virus (PPIV1) and Swine orthopneumovirus (SOV). Twenty-nine swIAV-positive cases and twenty-six cases of swIAV-negative respiratory disease were primarily established. IBV, IBD, PCV4 and PPIV1 were not found in any case, while PRCV, SOV, and PCMV were more likely to be found in swIAV-positive nurseries with respiratory disease ( p <0.05) although, globally, PCV3, PRRSV, and PCMV were the most frequently detected agents on herd level. At an individual level, the prevalence of different viruses was: swIAV 48.6%; PRCV 48.0%; PRRSV 31.6%; SOV 33.8%; PCMV 48.3%, PCV2 36.0%; and PCV3 33.0%. Beyond that, it was common to find animals with low Ct values (< 30) for all agents except for PCV2 and PCV3. When analysed the association between different pathogens, PRCV was the one with the most associations. It positively interacted ( p < 0.05) with swIAV and SOV but was negatively associated ( p < 0.05) with PRRSV and PCVM. Besides these, swIAV and PRRSV were negatively related (p < 0.05). Further analysis of suckling pigs showed that circulation of PRCV, PCMV, SOV, and PCV3 started in the maternities, suggesting a role of the sows in the transmission. Overall, our data may contribute to a better understanding of the complex aetiology and the epidemiology of respiratory disease in weaners. This is the first report of SOV in Spain.

Identification and Characterization of MRNAs, lncRNAs, and MiRNAs Expression Profiles and ceRNA Networks in PEDV Infection (preprint)

Background: Long non-coding RNAs (LncRNAs) are transcripts longer than 200 nucleotides with no protein-coding ability and exert crucial effects on viral infection and host immune responses. Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that seriously affects the swine industry. However, our understanding of the function of lncRNA involved in host-PEDV interaction is limited. Results: : A total of 1197 mRNA transcripts, 539 lncRNA transcripts, and 208 miRNA transcripts were differentially regulated at 24 h and 48 h post-infection. Moreover, gene ontology (GO) and KEGG pathway enrichment analysis showed that DE mRNAs and DE lncRNAs were mainly involved in biosynthesis, innate immunity, and lipid metabolism. Ten differentially expressed genes were randomly selected and validated by reverse-transcription qRT-PCR. In addition, we constructed a miRNA-mRNA-pathway network followed by a lncRNA-miRNA-mRNA ceRNA network. Conclusions: : The present study is the first to reveal the global expression profiles of mRNAs, lncRNAs, and miRNAs during PEDV infection. We comprehensively characterize the ceRNA networks which can provide new insights into the pathogenesis of PEDV.

First isolation and whole genome characterization of Porcine Deltacoronavirus from pigs in Peru (preprint)

Porcine Deltacoronavirus is a newly emergent enteric pathogen affecting swine farms worldwide. It has been detected in several countries in Europe, Asia and North America; yet, it has not been reported in South America. In November 2019, an enteric disease outbreak in a pig farm located in San Martin, Peru; was reported along with submission of three intestinal samples from pigs who succumbed to the disease. Samples were processed for molecular detection by qRT-PCR, viral isolation and further sequencing analysis. A taqman-based RT-PCR was performed to differentiate among the most relevant swine enteric coronaviruses described to date. All samples were positive to Porcine Deltacoronavirus with a cycle threshold (Ct) value between 9-14, revealing a high viral load, while testing negative to Porcine Epidemic diarrhea and Transmissible Gastroenteritis viruses. Following detection, viral isolation was performed using PK-15 and Vero cell lines. After 5 days of inoculation, no cytopathic effect was observed. A second blind passage allowed the observation of cytopathic effect on PK-15 cells, while it remained absent in Vero cells. A fluorescence test using an anti-N monoclonal antibody confirmed viral replication. One sample was processed for whole genome sequencing (NGS). In short, raw reads were imported into CLC genomics and assembled de novo . Out of 479k reads generated from the sample, 436k assembled into a 25501 bp contig which was 99.5% identical to a reference Porcine Deltacoronavirus strain from US within the North American phylogroup. Yet, there are relevant differences at the nucleotide and amino acid levels compared to previously described Porcine Deltacoronavirus strains. Altogether, our findings represent the first report of Porcine Deltacoronavirus in South America, its genomic characterization, which provides information of its evolutionary origin. Thus, this study offers new insights into the molecular epidemiology of Porcine Deltacoronavirus infections in the swine industry.

Antibody Dynamics Simulation -Theory and Application (preprint)

Infectious disease such as COVID-19 poses a considerable threat to public health when a pandemic strain emerges. Constructing a reliable mathematical model helps us quantitatively explain the kinetic characteristics of antibody-virus interactions, which could provide a reasonable prediction toward many sensitive concerns faced by the public, such as how to calculate protection time provided by the specific vaccine. A novel and robust model is developed to integrate antibody dynamics with virus dynamics in the host body. Our model is based on a comprehensive understanding of immunology principles rather than a simple data-fitting attempt by arbitrarily mathematical function selection. The physical-based mechanism would bring this model more reliable and broader prediction performance. This model gives quantitative insights between antibody dynamics and virus loading in the host body. Based on this model, we can estimate the antibody dynamic parameters with high fidelity. We could solve lots of critical problems, such as the calculation of vaccine protection time. We can also explain lots of mysterious phenomena such as antibody inferences, self-reinfection, chronic infection, etc. We suggest the best strategy in prolonging the vaccine protection time is not repeated inoculation but a directed induction of fast binding antibodies. Eventually, it will also inform the future construction of the mathematical model and help us fight against those infectious diseases.

The Horrors of Plague and a Message of Hope (preprint)

In 1347 CE, a Genoese trading ship docked at the Italian town of Messina. Along with trade goods, it brought a mysterious illness. Soon after, Messina descended into chaos as sailors and townspeople began to drop dead. Terrified, people fled the city, leaving behind all they had ever known. This scene would become far too common as the bubonic plague ripped through Europe. By 1351, the plague had killed an estimated twenty-four million people, approximately one-third of Europe’s population. Examination of Europe’s fourteenth-century plague reveals how ubiquitous death can generate fear and anger among the populace; however, it also demonstrates how a pandemic can bring about much-needed social change. The plague resulted in improved medicine, epidemiology, and sanitation as well as enhanced economic and political status for the common people.

Milk Small Extracellular Vesicles-derived miRNAs Restrict Porcine Epidemic Diarrhea Virus Infectivity (preprint)

Background: Virus-caused diseases are a huge challenge to both animals and human beings, especially coronaviruses. Porcine epidemic diarrhea virus (PEDV), a coronavirus, causes acute diarrhea and up to 100% mortality in piglets less than three weeks of age. Maternal immunity provides protection for piglets in resisting PEDV infection. Small extracellular vesicles (sEV) contain bioactive molecules such as miRNAs to exchange genetic and epigenetic information between cells. Our previous study suggested that milk sEV facilitated intestinal tract development and prevented LPS-induced intestine damage. However, the effects of milk sEV on the inhibition of viral infections remain unclear. Results: In this study, through in vivo experiments, we found that porcine milk sEV protected piglets from PEDV-induced diarrhea and death. In vitro, we clarified that this protective effect was partly generated through the inhibition of the PEDV-N protein and HMGB1 by sEV miR-let-7e and miR-27b, respectively. Conclusions: In conclusion, we report that porcine milk sEVs protected piglets from PEDV-induced diarrhea and death by inhibiting virus replication, and this protective effect was partly generated through the inhibition of the PEDV-N and HMGB1 pathways by exosomal miR-let-7e and miR-27b. This study reveals a new antiviral function of milk sEVs, and the results suggest that milk sEVs may act as a mother-offspring transmission pathway for protecting newborns against PEDV infection.

Thermal Fogging with Disinfectant Didecyl Dimethyl Ammonium Bromide Effectively Kills A Coronavirus, An Influenza Virus and Two Indicator Bacteria in Subzero Cold-Chain Environment (preprint)

Background The origin of several local COVID-19 outbreaks in China in 2020 were confirmed to be frozen food or packages contaminated with SARS-CoV-2, revealing the lack of effective disinfection measures in the frozen food chain. Objective To evaluate the disinfection efficacy at −20°C of a recently marketed thermal fogging disinfection product, with disinfectant-antifreeze combination being didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG). Method Carriers with porcine epidemic diarrhea virus (PEDV) YN1, a coronavirus, and a swine influenza virus H1N1 (SIV-H1N1), and three indicator bacteria, E. coli, S. aureus, and B. subtilis endospores, were respectively placed in a −20°C freezer warehouse with or without DDAB-PPG fogging and activities of the microorganisms were tested. Results At −20°C, DDAB-PPG fogging, which fully settles in 3.5−4.5 hours, fully inactivated PEDV of 10 −3.5 TCID 50 /0.1ml and SIV-H1N1 of 2 6 hemagglutination titer within 15-30 min, and inactivated S. aureus and E. coli vegetative cells (10 6 /ml) within 15 or 60 min, respectively, but had little effect on B. subtilis spores. The bactericidal effect lasted at least 3 hours for bacteria on carrier plates and for 6 hours for airborne bacteria. Conclusions A practical subzero temperature disinfection technology was confirmed its efficacy in killing enveloped viruses and vegetative bacteria. It would help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity. Highlights First report of efficacy of disinfectant didecyl dimethyl ammonium bromide (DDAB) at subzero temperatures The customized thermal fogging machine makes fine droplets of DDAB-propylene glycol (PPG) fog which can suspend for ~4 hours at −20°C DDAB-PPG thermal fogging at −20°C effectively inactivated a coronavirus and an influenza virus DDAB-PPG thermal fogging at −20°C for effectively killed S. aureus and E. coli DDAB-PPG thermal fogging at −20°C inactivated airborne microorganism for up to 6 h

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.

In silico screening of JAK-STAT modulators from the antiviral plants of Indian traditional system of medicine with the potential to inhibit 2019 novel coronavirus (preprint)

Aim: The present study was aimed to identify the lead hits from reported anti-viral Indian medicinal plants to modulate the proteins through the JAK-STAT pathway and to identify the proteins that share the domain with coronavirus (COVID19) associated proteins i.e. 3CLpro, PLpro, and spike protein. Methods: . The reported anti-viral plants were screened from the available databases and published literature; their phytoconstituents were retrieved, gene-expression was predicted and the modulated proteins in JAK-STAT pathway were predicted. The interaction between proteins was evaluated using STRING and the network between phytoconstituents and proteins was constructed using Cytoscape. The druglikeness score was predicted using MolSoft and the ADMET profile of phytoconstituents was evaluated using admetSAR2.0. The domain of three proteins i.e. 3CLpro, PLpro, and spike protein of coronavirus was compared using NCBI blastP against the RCSB database. Results: . The majority of the phytoconstituents from the anti-viral plants were predicted to target TRAF5 protein in the JAK-STAT pathway; among them, vitexilactone was predicted to possess the highest druglikeness score. Proteins targeted in the JAK-STAT pathways were also predicted to modulate the immune system. Similarly, the docking study identified sesaminol 2-O-β-D-gentiobioside to possess the highest binding affinity with spike protein. Similarly, phylogeny comparison also identified the common protein domains with other stains of microbes like murine hepatitis virus strain A59, avian infectious bronchitis virus, and porcine epidemic diarrhea virus CV777. Conclusion: Although, the present study is based on computer simulations and database mining, it provides two important aspects in identifying the lead hits against coronavirus. First, targeting the JAK-STAT pathway in the corona-infected host by folk anti-viral agents can regulate the immune system which would inhibit spreading the virus inside the subject. Secondly, the well-known targets of coronavirus i.e. 3CLpro, PLpro, and spike protein share some common domains with other proteins of different microbial strains.

Ultra-High-Resolution CT Follow-Up in Patients with Imported Early-Stage Coronavirus Disease 2019 (COVID-19) Related Pneumonia (preprint)

Background: An ongoing outbreak of mystery pneumonia in Wuhan was caused by coronavirus disease 2019 (COVID-19). The infectious disease has spread globally and become a major threat to public health. Purpose: We aim to investigate the ultra-high-resolution CT (UHR-CT) findings of imported COVID-19 related pneumonia from the initial diagnosis to early-phase follow-up. Methods: This retrospective study included confirmed cases with early-stage COVID-19 related pneumonia imported from the epicenter. Initial and early-phase follow-up UHR-CT scans (within 5 days) were reviewed for characterizing the radiological findings. The normalized total volumes of ground-glass opacities (GGOs) and consolidations were calculated and compared during the radiological follow-up by artificial-intelligence-based methods. Results: Eleven patients (3 males and 8 females, aged 32-74 years) with confirmed COVID-19 were evaluated. Subpleural GGOs with inter/intralobular septal thickening were typical imaging findings. Other diagnostic CT features included distinct margins (8/11, 73%), pleural retraction or thickening (7/11, 64%), intralesional vasodilatation (6/11, 55%). Normalized volumes of pulmonary GGOs (p=0.003) and consolidations (p=0.003) significantly increased during the CT follow-up. Conclusions: The abnormalities of GGOs with peripleural distribution, consolidated areas, septal thickening, pleural involvement and intralesional vasodilatation on UHR-CT indicate the diagnosis of COVID-19. COVID-19 cases could manifest significantly progressed GGOs and consolidations with increased volume during the early-phase CT follow-up.

A human monoclonal 1 antibody blocking SARS-CoV-2 infection (preprint)

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV). This cross-neutralizing antibody targets a communal epitope on these viruses and offers potential for prevention and treatment of COVID-19.