Establishment and application of triple PCR method for CPV, CCoV and CRV

Canine parvovirus (CPV), canine coronavirus (CCoV) and canine rotavirus (CRV) are the three main causative viruses of diarrhea in dogs with similar clinical symptoms;thereby it is necessary to establish a high effective molecular detection method for differentiating the above pathogens. By optimizing the primer concentration and annealing temperature, a triple PCR method was established for simultaneous detection of CPV, CCoV and CRV, and then the specificity, sensitivity and repeatability of the method were tested. The results showed that the target fragments of CPV VP2 gene (253 bp), CCoV ORF-1b gene (379 bp) and CRV VP6 gene (852 bp) could be accurately amplified by the triple PCR method with high specificity, the detection limits of CPV, CCOV and CRV were 6.44x10-1 pg/L, 8.72x10-1 pg/L and 8.35x10-1 pg/L respectively with high sensitivity, and the method had good stability. Using this triple PCR method, 135 canine diarrhea fecal samples collected in Chengdu region from 2019 to 2020 were detected, and compared with those of single PCR method. The detection rates of CPV, CCoV and CRV were 16.30%, 20.74% and 4.44%, respectively, and the total infection rate was 51.11% (65/135) with 20.00% (13/65) co-infection rate. The detection results were consistent with three single PCR methods. In conclusion, CPV/CCoV/CRV triple PCR method successfully established in this paper can be applied as an effective molecular method to detection of related pathogens and to the epidemiological investigation.

Establishment of a duplex fluorescent quantitative PCR assay for detection of Porcine circovirus type 2 and type 3

To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3), specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested. Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies.L-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181), the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181). The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181). The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.

Effect of Drebrin protein on the proliferation of PEDV in Vero cells

Previous studies have revealed that developmental regulated brain protein (Drebrin) is involved in cell- to-cell communication, nerve transmission, tumor metastasis, spermatogenesis and other life activities, but there are few studies on viruses. The aim of the current research was therefore, to study the function of Drebrin and its effect on the proliferation of porcine epidemic diarrhea virus (PEDV). The Drebrin gene was cloned according to the Drebrin gene sequence (XM_008015438.2) of Chlorocebus sabaeus registered by GenBank, and the phylogenetic tree was constructed to analyze its homology. The results showed that the CDS region of Vero cells Drebrin gene was 2088 bp long, encoding 695 amino acids, and was relatively conserved and had high homology with all species. To investigate the effect of Drebrin on the proliferation of PEDV in Vero cells, the eukaryotic expression vector pcDNA3.1-Drebrin-Flag was constructed. After transfection of Vero cells with different concentrations of pcDNA3.1-Drebrin-Flag, cells were infected with PEDV. Our results showed that overexpression of Drebrin in Vero cells could significantly inhibit the intracellular PEDV mRNA level and N protein expression, reduce the extracellular virus titer and inhibit the proliferation of PEDV. Further study on the interaction between Drebrin and PEDV S proteins by laser confocal technique was also performed. The results showed that Drebrin and S protein were co-located in the cytoplasm, suggesting that the two proteins may interact with each other. This study demonstrated for the first time that Drebrin can inhibit PEDV proliferation in Vero cells, laying a foundation for further research in to Drebrin function and provides a valuable information for anti-PEDV research.

Development and application of a multiplex fluorescent quantitative RT-PCR for detection of Porcine epidemic diarrhea virus, porcine coronavirus and porcine acute diarrhea syndrome coronavirus

To develop a multiplex fluorescent quantitative RT-PCR for the detection of porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV), in this study, specific primers/probes were designed based on the conserved regions of M, M and N gene sequences of PEDV, PDCoV and SADS-CoV, respectively. After optimization of the reaction conditions, a multiplex fluorescent quantitative RT-PCR for PEDV, PDCoV and SADS-CoV was established. The results of specificity assay showed that the method was positive for detection of PEDV, PDCoV and SADS-CoV, and negative for detection of porcine transmissible gastroenteritis virus, porcine rotavirus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus, classical swine fever virus and foot-and-mouth disease virus. The results of sensitivity assay showed that the detection limit of this method for PEDV, PDCoV, and SADS-CoV plasmids standard was 1.0x101 copies/L, and had a good linear relationship with their Ct values in the range of 101 copies/L to 106 copies/L. The results of repeatability assay showed that the coefficients of variation (CVs) of intra- and inter-assay reproducibility ranged from 0.33% to 2.53%, indicating good repeatability and stability. To evaluate the effects of the developed method, 100 clinical samples collected from different parts of Henan province were used for detection of these three viruses and compared with those of single RT-PCR and standard methods. The results of multiplex fluorescent quantitative RT-PCR showed that the positive rates of PEDV, PDCoV and SADS-CoV were 38% (38/100), 14% (14/100) and 5% (5/100), respectively. There was no mixed infection. The coincidence rate with the standard detection methods of PEDV and PDCoV was 100%, and the sensitivity was higher than that of single RT-PCR. In this study, a specific, sensitive and rapid multiplex fluorescent quantitative RTPCR method was established for the first time, which could be used for the differential detection of PEDV, PDCoV and SADS-CoV, and laid a foundation for the differential diagnosis and control of porcine diarrheal diseases.

Historical perspective: other human coronavirus infectious diseases, SARS and MERS

Alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1) are common causes of upper respiratory tract infection in humans. SARS-CoV-1 and MERS-CoV emerged in 2002 and 2012, respectively, with the potential of causing severe and lethal disease in humans, termed SARS and MERS, respectively. Bats appear to be the common natural source of SARS-like coronaviruses including SARS-CoV-1, but their role in MERS-CoV is less clear. Civet cats and dromedary camels are the intermediary animal sources for SARS-CoV-1 and MERS-CoV, respectively. Nosocomial outbreaks are hallmarks of SARS and MERS. MERS patients with comorbidities or immunosuppression tend to progress more rapidly to respiratory failure and have a higher case fatality rate than SARS patients. SARS has disappeared since 2004, while there are still sporadic cases of MERS in the Middle East. Continued global surveillance is essential for SARS-like coronaviruses and MERS-CoV to monitor changing epidemiology due to viral variants.Copyright © ERS 2021.

Incidence and sero-surveillance of feline viruses in Korean cats residing in Gyeonggi-do

Incidences of major feline viral diseases provide basic information for preventing viral disease in cats. Despite the growing interest in feline viral diseases, sero-surveillances have been lacking. In this study, we analyzed the diagnoses of feline viral diseases and conducted a sero surveillance of feline panleukopenia virus (FPV), feline calicivirus (FCV), feline herpesvirus-1 (FHV-1), and feline infectious peritonitis virus (FIPV) in Korean cats. Of the 204 confirmed cases since 2015, the numbers of diagnoses for FPV, FIPV, FCV, feline influenza virus, and FHV-1 were 156, 32, 12, 3, and 1 case, respectively. In total, 200 sera, collected between 2019 and 2021, were screened for the presence of antibodies against FPV, 2 FCVs, FHV-1, and FIPV using a hemagglutination inhibition test and a virus-neutralizing assay (VNA). The overall seropositive rates in cats tested for FPV, the 2 FCVs, FHV-1, and FIPV were 92.5%. 42.0%, 37.0%, 52.0%, and 14.0%, respectively. A low correlation (r = 0.466) was detected between the VNA titers of 2 FCV strains. The highest incidence and seropositive rate of FPV reveal that FPV is circulating in Korean cats. The low r-value between 2 FCVs suggests that a new feline vaccine containing the 2 kinds of FCVs is required.

Coinfection in the cases of feline panleukopenia: a literature review

Coinfection caused by bacteria, parasites, or viruses complicates almost all feline panleukopenia virus (FPV) infections. Pathogens that colonize the gastrointestinal tract, Clostridium perfingens, Clostridium piliforme, Cryptosporidium spp, Giardia spp, Tritrichomonas fetus, canine parvovirus type 2,Salmonella sp., feline coronavirus, feline bocavirus, and feline astrovirus were isolated in the presence of FPV infection. Complex mechanisms between viruses, bacteria, protozoa, and hosts contribute to the pathogenesis and severity of coinfection. Prompt and accurate diagnosis, vaccination precautions, and appropriate treatment play important roles in reducing morbidity and mortality. This article outlines the etiology, pathogenesis, diagnosis, prevention, and treatment that can help veterinarians and pet owners improve their knowledge of managing the diseases.

Restrospective study of hemogram profile in effusive feline infectious peritonitis

Feline Infectious Peritonitis (FIP) is highly mortality disease in cats. The reliable and fast diagnosis is crucial to the best prognosis. The aim of this study to evaluate the hemogram profile in cats infected with effusive FIP. Twenty cats had been diagnosed effusive FIP at Animal Clinic Department of Internal Medicine, Faculty Veterinary Medicine, Universitas Gadjah Mada were used in the study. The diagnosis were based on clinical examination, ultrasound, x-ray, rivalta test, and rapid test. The hemogram profile were analyzed include routine hematology and serum biochemistry. Hemogram profile in effusive FIP showed the decreased hematocrit, hyperproteinemia, and leukocytosis with an average 22.9+or-7.4%;9.0+or-2.2 g/dL;22425+or-4116 cells/mm3 respectively. Erythrocyte, hemoglobin and fibrinogen levels were still in the normal range. The results of differential leukocytes revealed that 90% cats had neutrophilia and 75% lymphopenia with an average 20066+or-3337 cells/mm3 and 1861+or-1818 cells/mm3 respectively. The blood chemistry profile showed 60% of cats experienced increase in SGPT and SGOT with an average 138.4+or-72.3 IU/L and 101+or-60.5 IU/L respectively. Hyperglobulinemia was found in 90% samples with an average 6.7+or-0.8 g/dL. All cats have a low albumin:globulin ratio with an average 0.3+or-0.1. The hemogram profile of effusive FIP were: leukocytosis, neutrophilia, lymphopenia, hyperglobulinemia, and decreased albumin-globulin ratio..

Interaction between target peptide and complementary peptide by bimolecular fluorescence complementary technology

[Objective] Using the bimolecular fluorescence complementation (BiFC) technology, the present experiment aimed to study the interaction relationship and localization of the target peptide and the complementary peptide based on the porcine epidemic diarrhea virus (PEDV) S protein receptor binding site peptide in living cells, so as to provide the foundation and theoretical support for the further use of the peptide in the detection of porcine epidemic diarrhea virus. [Method] The target peptide was designed according to the physical and chemical characteristics of the target protein, such as the amino acid composition, the type of charge, the ability to form intennolecular hydrogen bonds, the strength of polarity, and hydrophobicity;According to the amino acid composition of the target protein, a complementary peptide that interacted with it in theory was designed, and the target peptide and complementary peptide were predicted and analyzed by using bioinfonnatics tools;The target peptide and complementary peptide were inserted into the pBiFC-VC155 and pBiFC-VN173 vector, which was double digested by the EcoRI/XhoI and NotI/SalI, respectively, verified by enzyme digestion and sequencing, and then transfected into Vero cells to study the interaction between the target peptide and the complementary peptide, and the precise localization of BiFC complex in cells. [Result] Bioinfonnatics analysis showed that the target peptide and complementary peptide had hydrophilic and hydrophobic domains, respectively, and the hydrophilic domains were both positively and negatively charged, which could generate electrostatic attraction. The results of enzyme digestion and sequencing showed that the pBiFC-VC155-target peptide and pBiFC-VNI73-complementary peptide plasmids were successfully constructed;Cell transfection experiments showed that the target peptide and complementary peptide could form BiFC complexes in Vcro cells after co-transfection of recombinant plasmids, indicating that they could interact with each other;Indirect immuttolluorescence assay confirmed that the BiFC complex was mainly distributed in the nucleus. [Conclusion] It was confirmed that the peptide designed based on the PEW/ S protein receptor binding site can interact with each other in living cells, demonstrating the feasibility of the peptide for detection.

Improved virus inhibition by microencapsulated monoclonal antibody against porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has been affecting the swine industry, especially in suckling pigs in with a high mortality rate. Among all the strategies to overcome PEDV, boosting mucosal immunity in pig intestine via oral administration appears to be more efficient than other routes. However, there are biological obstacles such as acidic environment that could damage biologics, a product from organisms often used for PEDV treatment. The plant-derived 2C10 monoclonal antibody (mAb) from Nicotiana benthamiana produced by transient expression was revealed as one of the potential candidates against PEDV through oral delivery. Herein, we demonstrated the calcium-alginate microencapsulation system to protect the 2C10 mAb from the harsh condition in the stomach and to be released the 2C10 mAb when arriving in the intestine. The pH-responsive encapsulated 2C10 mAb microbeads were constructed from the calcium-alginate system. The microbeads were well-tolerated under the acidic environment of simulated gastric fluid (SGF) and were digested under the alkaline condition of simulated intestinal fluid (SIF). The encapsulated 2C10 mAb in the SPF-treated microbeads exhibited high virus neutralization efficiency in Vero cells when compared to the unencapsulated 2C10 mAb treated by SPF that cannot neutralize the virus. For these reasons, calcium-alginate microencapsulation system is an attractive platform to be considered as a candidate for the next generation of oral vaccine development.

Duplex Taq man qPCR for detecting porcine epidemic diarrhea and transmissible gastroenteritis viruses and epidemic study in Fujian

Objective: A Taq Man probe-based duplex real-time PCR for rapid detection of porcine epidemic diarrhea virus(PEDV) and transmissible gastroenteritis virus(TGEV) was developed. A study was conducted using the methodology to analyze the related 2019-2021 epidemic occurred in Fujian. Method: Specific primers and probes labeled with FAM and VIC were designed to amplify the N gene of PEDV and the S gene of TGEV, respectively. A reaction system for the assay was established, optimized, and tested for sensitivity, specificity, and repeatability. The assay was used for the viral detection on297 suspected clinic specimens collected from 2019 to 2021 for an epidemiology study. Result: The newly developed duplex qPCR methodology showed a sensitivity of 10 copies.L-1 on PEDV and TGEV, which was 100 times higher than that of regular PCR. There were no cross reactions with other common viruses. The inter-and intra-assays had variations on Ct values below 1%. On the 297 specimens, the infection rate of PEDV was 88.89%, that of TGEV 1.01%, and that of both PEDV and TGEV 3.37%. Conclusion: The established duplex qPCR had high sensitivity, specificity, repeatability, and reproducibility for detecting PEDV and TGEV. The 2019-2021 epidemic involving the viruses appeared to be mostly PEDV with low incidents of mixed TGEV and PEDV/TGEV infection.

Focus on laryngeal disease in the cat

This is a title only record which contains no .

Using molecular tools to compare various concentration protocols prior to detecting SARS-CoV-2 RNA in wastewater. (Special Issue: Chemistry and global challenges (part A).)

Background: Reports showed presence of SARS-CoV-2 genetic material in wastewater. Wastewater concentration methods are optimized for detection of non-enveloped viruses so need to be adopted for enveloped viruses and their genetic material. Methods: Conventional (cRT-PCR) and quantitative real time RT-PCR (qRT-PCR) were used as readouts to compare 4 water concentration methods namely, (A) filtration on negatively charged membrane followed by extracting RNA from it, (B) adsorbtion-elution method, (C) flocculation with skimmed milk and (D) polyethylene glycol precipitation, to detect SARS-CoV-2 RNA and 229E human coronavirus (229E-HCoV) as a model for spike-containing enveloped virus from fresh and wastewater. Results: On using cRT-PCR: recovery rate of SARS-CoV-2 RNA was better using method A then B for fresh water and method B then D for wastewater. 229E-HCoV recovery from fresh water was better using method C then A and methods B then D for wastewater. On using qRT-PCR, both methods A and B were better for SARS-CoV-2 RNA recovery from both fresh and wastewater. For the 229E-HCoV methods A was the most efficient for fresh water and method B for wastewater. Conclusion: Method B is recommended for SARS-CoV-2 RNA or whole 229E-HCoV recovery from wastewater.

Isolation, identification and genetic evolution analysis of porcine epidemic diarrhea virus SNJ-P strain in Sichuan Province

Purpose: To investigate the epidemic variation of porcine epidemic diarrhea virus (PEDV) strains in Sichuan Province, and to analyze the causes of poor vaccination effect. Methods: Piglet intestinal samples were collected from a pig farm in Sichuan Province for PCR detection, virus purification, determination of virus titer and virus infection experiments. Whole genome sequencing of isolated strains was determined. The S gene sequence of the isolated strain was compared with the strains from other regions and vaccine strains, and the phylogenetic tree was established. The amino acid site variation of S protein between the isolated strain and the classical vaccine strain CV777 was compared. Results: A PEDV strain was successfully isolated and named as PEDV SNJ-P. The determination of virus titer was 1..107.5/100 L. Animal infection experiments showed that the isolated strain could cause diarrhea, dehydration and other symptoms and lead to death in piglets. Genome sequencing and phylogenetic tree analysis showed that the whole gene of PEDV SNJ-P strain was 28003 bp, and the genotype of the strain was S non-INDEL type. The strains were closely related to the strains of PEDV-WS, CH/JLDH/2016 and CH/HNLH/2015 isolated from China, and were relatively distant with the same type vaccine strain, and were far from the classical vaccine strain. Compared with the classical vaccine strain CV777, the S protein of SNJ-P strain had multiple amino acid mutations, deletions and insertions. Conclusion: Due to the continuous variation of strains, SNJ-P strain is far from the vaccine strain, and the current vaccines cannot provide effective protection. The results of this study are expected to provide reference for the study of PEDV strains and vaccine development in China.

Multiple detection of 13 kinds of viruses in 500 children with acute respiratory tract infection in Hami of Xinjiang province

OBJECTIVE: To investigate and analyze multiple detection of 13 kinds of viruses in 500 children with acute respiratory tract infection in Hami of Xinjiang. METHODS: A total of 500 children with acute respiratory tract infection treated in the hospital between Jan 2018 and Jan 2021 were enrolled. Thirteen kinds of respiratory infection viruses including human respiratory syncytial virus(RSV), human rhinovirus(hRV), respiratory adenovirus(AdV), influenza A and B viruses(Inf A, Inf B), parainfluenza virus(PIV 1/2/3), human enterovirus(hEV), human metapneumovirus(hMPV), human coronavirus(hCoV 229E/OC43) and human Boca virus(hBoV) were detected by multiple reverse transcription polymerase chain reaction(RT-PCR) amplification and capillary electrophoresis. And the results were compared with those by direct sequencing method. RESULTS: Of the 500 samples, 379 samples were positive(75.80%), and the top three detection rates were RSV(19.40%), hRV(16.00%) and Inf B(12.60%). The differences in positive rates of the respiratory virus among <1 year group, 1-3 years group and >3 years group were significant(84.97%, 77.47%, 65.45%)(P<0.05). The detection rate of RSV was the highest in <1 year group, and the detection rates of Inf A and Inf B were the highest in >3 years group. The differences in positive rates of respiratory viruses among the spring group, summer group, autumn group and winter group were significant(74.05%, 63.73%, 77.24%, 84.03%)(P<0.05). The detection rates of RSV, PIV 3, and hMPV were the highest in the winter group, and detection rate of AdV was the highest in spring group. CONCLUSION: RSV is the main infection virus in children with acute respiratory infection in Hami of Xinjiang. The distribution of respiratory viruses is related to age and onset season in children.

Identification of feline corona- virus in breeding catteries by molecular biological methods and shedding persistency

Infectious peritonitis virus (FIPV) causes a fatal disease in cats. This virus occurs both in cats bred in households with optimal welfare and outdoor cats. Feline patients with the effusive form of disease usually survive a few days to weeks from the appearance of the first clinical signs. Cats with the non- effusive form survive for weeks to months. FIPV is caused by a mutation from feline enteric coronavirus (FECV). In our study, we diagnosed feline coronavirus from the feces of 82% of the tested cats. The persistence of the feline coronavirus in the organism is influenced by environmental factors, the genome of the host and the causative agent. Negative environmental conditions that increase the likelihood of FIPV disease are long-term stress, mainly more labile individuals and a high concentration of domesticated cats in one place. In the host, there are important factors such as immune system performance, age, breed and genetic background. In our study, we primarily verified the real time RT-PCR method for identifying the virus from the feces of 71 cats and subsequently gaine the valuable data on the dynamics of feline coronavirus excretion, primarily for epizootological purposes and for the purposes of genetic analyzes of susceptibility to infection.

Current understanding of the airborne transmission of important viral animal pathogens in spreading disease

Current research on airborne transmission of African swine fever virus (ASFV), porcine epidemic diarrhoea virus (PEDV), avian influenza (AIV), porcine reproductive and respiratory syndrome virus (PRRSV), and foot and mouth disease virus (FMDV) was reviewed to evaluate commonalities, knowledge gaps, and methodologies of studying airborne transmission of animal diseases. The reviewed studies were categorised as short-range transmission (within a single facility) and long-range transmission (beyond a single site). Short-range airborne transmission was demonstrated for at least one strain of the above-mentioned pathogens in experimental settings. Most studies reported in the literature concern FMDV, with limited information for ASFV and PEDV, particularly for short-range airborne transmission. Air sampling upwind, downwind, and within infected facilities has been commonly used to demonstrate long-range airborne transmission. The amount of evidence from air sampling for each of the reviewed viruses varies from no evidence on ASFV to evidence from multiple settings for AIV. Computer modelling has been used to study past outbreaks of infectious diseases to assess the contribution of airborne transmission with a multitude of computer models reported in the literature for simulating long-range airborne transmission of FMDV based on past outbreaks. This has resulted in predictive tools for assessing future risk of airborne transmission. Some important computer models are based on epidemiology analysis, weather analysis, and air dispersion. Few models are reported for ASFV, PEDV, and PRRSV. Studies in the literature indicate that airborne transmission is generally affected by virus strain, aerosol type, shedding duration and concentration, environmental conditions, and infectious dose.

Feline infectious peritonitis - an evolving disease

This paper describes the past and current knowledge regarding the physiopathology, clinical signs, diagnosis, treatment and prognosis of feline infectious peritonitis.

PEDV orf3 independently regulates IB kinase beta-mediated NF-B and IFN-beta promoter activities

The Open Reading Frame 3 (ORF3), an accessory protein of porcine epidemic diarrhea virus (PEDV), has been shown to interact with a myriad of cellular proteins, among which include the IB kinase beta (IKBKB). Here, specific IKBKB domains responsible for ORF3-IKBKB interaction were identified. Dysregulation of NF-B and Type I interferon (IFN) in the presence of ORF3 was also demonstrated. We showed that while ORF3 was capable of up-regulating IKBKB-meditated NF-B promoter activity, it surprisingly down-regulated the activation of IKBKB-meditated IFN-beta promoter and expression of IFN-beta mRNA. When overexpressed, ORF3 could suppress Poly I:C mediated type I IFN production and induction. Finally, we demonstrated that IKBKB- and RIG-I-mediated type I IFN induction by ORF3 resulted in different outcomes. Our study is the first to demonstrate the potential and complex roles of ORF3 in the involvement of aberrant immune signaling as well as in the virus-host interaction.