Pyrococcus furiosus Argonaute Based Detection Assays for Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a major cause of diarrhea and diarrhea-related deaths among piglets and results in massive losses to the overall porcine industry. The clinical manifestations of porcine diarrhea brought on by the porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and PDCoV are oddly similar to each other. Hence, the identification of different pathogens through molecular diagnosis and serological techniques is crucial. Three novel detection methods for identifying PDCoV have been developed utilizing recombinase-aided amplification (RAA) or reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with Pyrococcus furiosus Argonaute (PfAgo): RAA-PfAgo, one-pot RT-RAA-PfAgo, and one-pot RT-RAA-PfAgo-LFD. The indicated approaches have a detection limit of around 60 copies/µL of PDCoV and do not cross-react with other viruses including PEDV, TGEV, RVA, PRV, PCV2, or PCV3. The applicability of one-pot RT-RAA-PfAgo and one-pot RT-RAA-PfAgo-LFD were examined using clinical samples and showed a positive rate comparable to the qPCR method. These techniques offer cutting-edge technical assistance for identifying, stopping, and managing PDCoV.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

An isothermal nucleic acid amplification-based enzymatic recombinase amplification method for dual detection of porcine epidemic diarrhea virus and porcine rotavirus A.

Viral diarrhea is the predominant digestive tract sickness in piglings, resulting in substantial profit losses in the porcine industry. Porcine rotavirus A (PoRVA) and porcine epidemic diarrhea virus (PEDV) are the main causes of grave gastroenteritis and massive dysentery, especially in piglets. PoRVA and PEDV have high transmissibility, exhibit similar clinical symptoms, and frequently co-occur. Therefore, to avoid financial losses, a quick, highly efficient, objective diagnostic test for the prevention and detection of these diseases is required. Enzymatic recombinase amplification (ERA) is a novel technology based on isothermal nucleic acid amplification. It demonstrates high sensitivity and excellent specificity, with a short processing time and easy operability, compared with other in vitro nucleic acid amplification technologies. In this study, a dual ERA method to detect and distinguish between PEDV and PoRVA nucleic acids was established. The method shows high sensitivity, as the detection limits were 101 copies/µL for both viruses. To test the usefulness of this method in clinical settings, we tested 64 swine clinical samples. Our results were 100% matched with those acquired using a commercially available kit. Therefore, we have successfully developed a dual diagnostic ERA nucleic acids method for detecting and distinguishing between PEDV and PoRVA.

Pyrococcus furiosus Argonaute-mediated porcine epidemic diarrhea virus detection.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, induces severe vomiting and acute watery diarrhea in unweaned piglets. The pig industry has suffered tremendous financial losses due to the high mortality rate of piglets caused by PEDV. Consequently, a simple and rapid on-site diagnostic technology is crucial for preventing and controlling PEDV. This study established a detection method for PEDV using recombinase-aided amplification (RAA) and Pyrococcus furiosus Argonaute (PfAgo), which can detect 100 copies of PEDV without cross-reactivity with other pathogens. The entire reaction of RAA and PfAgo to detect PEDV does not require sophisticated instruments, and the reaction results can be observed with the naked eye. Overall, this integrated RAA-PfAgo cleavage assay is a practical tool for accurately and quickly detecting PEDV. KEY POINTS: • PfAgo has the potential to serve as a viable molecular diagnostic tool for the detection and diagnosis of viral genomes • The RAA-PfAgo detection technique has a remarkable level of sensitivity and specificity • The RAA-PfAgo detection system can identify PEDV without needing advanced equipment.

An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control.

For rapid and reliable detection of porcine epidemic diarrhea virus (PEDV) from pig clinical samples, a multiplex, real-time, reverse transcription loop-mediated isothermal amplification (mqRT-LAMP) was developed using two sets of primers and assimilating probes specific to the PEDV N gene and the Sus scrofa ß-actin gene, which was used as an endogenous internal positive control (EIPC) to avoid false-negative results. The assay specifically amplified both target genes of PEDV and EIPC in a single reaction without any interference but did not amplify other porcine viral nucleic acids. The limit of detection was 10 copies/µL, 100-fold lower than that of a reverse transcription-polymerase chain reaction (RT-PCR) and equivalent to that of quantitative/real-time RT-PCR (qRT-PCR). This assay has high repeatability and reproducibility with coefficients of variation < 4.0%. The positive signal of the mqRT-LAMP assay was generated within 25 min, demonstrating advantages in rapid detection of PEDV over RT-PCR or qRT-PCR assay, which require at least 2 h turnaround times. In clinical evaluation, the detection rate of PEDV by mqRT-LAMP assay (77.3%) was higher than that of RT-PCR assay (69.7%), and comparable to qRT-PCR (76.8%) with almost 100% concordance (kappa value 0.98). The developed mqRT-LAMP assay can serve as an advanced alternative method for PEDV diagnosis because it has high sensitivity and specificity, rapidity, and reliability even in resource-limited laboratories.

Serological identification of MERS-CoV in camels of Wasit province, Iraq.

Background: Since the first human case of Middle East Respiratory Syndrome (MERS) caused by Coronavirus (MERS-CoV) in 2012, several evidence bases have shown one-humped camels as the main reservoir host, from which infection is transmitted to humans. Aim: Serological investigation of MERS in dromedary camels in Wasit province (Iraq), detection severity of infection, and association to some risk factors. Methods: A total of 455 dromedary camels were selected randomly from two main districts in Wasit province, Iraq, during January and April (2023). Sera of all study camels were examined by enzyme-linked immunosorbent assay (ELISA), and titers of positive study animals were categorized according to their severity. Results: Serological testing yielded 37.58% positive animals for MERS infection. According to the severity of positive ODs (titer), a total of 53.22%, 30.99%, 12.28%, and 3.51% showed mild, moderate, strong, and very strong infections, respectively. Regarding risk factors, significant elevation in seropositivity was seen in camels of >3-6 and >6 years old and reduced in camels of £3 years old with an elevated risk of MERS with increased age. Regionally, seropositivity and relative risk were increased in the camels of Shaykh Sa'd when compared with Al-Numaniyah. Regarding sex, no significant variation was detected between seropositive females and males; however, male camels appeared at higher risk than females. Association between the severity of MERS infection and risk factors revealed that there was a significant increase in mild and moderate infections in female camels of >6 years old; whereas strong and very strong infections were seen in male camels of 33-6 years old. Mild and very strong infections were recorded in Shaykh Sa'd; while moderate and strong infections in Al-Numaniyah. Conclusion: The study indicated a longstanding existence of MERS-CoV in camels of Wasit province; therefore, recent infections or active viral excretion are required for confirmation by molecular approaches.

Complications of the Central Nervous System in Pediatric Patients With Common Cold Coronavirus Infection During 2014-2019.

BACKGROUND: In pediatric patients, the common cold coronavirus (ccCoV) usually causes mild respiratory illness. There are reports of coronavirus causing central nervous system (CNS) infection in experimental animal models. Some immunocompromised patients have also been reported to have fatal CNS infections with ccCoV. The aim of this study was to investigate the clinical characteristics of CNS complications related to ccCoV infection. METHODS: From January 2014 to December 2019, a retrospective analysis was performed of medical records from hospitalized patients under 19 years of age whose ccCoV was detected through polymerase chain reaction in respiratory specimens. The CNS complications were defined as clinically diagnosed seizure, meningitis, encephalopathy, and encephalitis. RESULTS: A total of 436 samples from 420 patients were detected as ccCoV. Among the 420 patients, 269 patients were immunocompetent and 151 patients were immunocompromised. The most common type of ccCoV was OC43 (52% in immunocompetent, 37% in immunocompromised). CNS complications were observed in 9.4% (41/436). The most common type of CNS complication was the fever-provoked seizure under pre-existing neurologic disease (42% in immunocompetent and 60% in immunocompromised patients). Among patients with CNS complications, two immunocompetent patients required intensive care unit admission due to encephalitis. Three patients without underlying neurological disease started anti-seizure medications for the first time at this admission. There was no death related to ccCoV infection. CONCLUSION: ccCoV infection may cause severe clinical manifestations such as CNS complications or neurologic sequelae, even in previously healthy children.

Development of a Recombinant Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies Against Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV), a gammacoronavirus within the Coronaviridae family, is an economically important etiological disease agent in chickens. Both early diagnosis and determination of the immune status of chickens are important for controlling IBV outbreaks in chicken flocks. The N protein is the most abundantly expressed virus-derived protein during IBV infection and can induce a strong immune response by producing antibodies during early infection or immunization. In this study, we found that the amino acid sequences of the N protein between CK/CH/LJL/04I and the other 22 IBVs were conserved, especially in the 1-160 amino acid region. Based on the sequence similarities, the three recombinant proteins, rN160 (amino acid positions 1-160), rN266 (144-409), and rN409 (1-409), were expressed using the Escherichia coli system and subsequently purified. The results demonstrated that the antigenicity and reactivity of rN160 were better than those of rN266 and rN409. As a result, an indirect enzyme-linked immunosorbent assay (ELISA) (rN160 ELISA) was developed to detect the IBV antibody based on the rN160 protein. Using 1,500 clinical field serum samples, the relative sensitivity, specificity, and accuracy of the rN160 ELISA were 98.97%, 92.34%, and 97.93%, respectively, compared to those of a commercial ELISA kit (IDEXX), indicating a strong positive correlation between the two methods. Taken together, these results reveal that the rN160 ELISA is a rapid, simple, and sensitive method for detecting group-specific IBV antibodies for epidemiological investigation and antibody-level monitoring.

A duplex nested RT-PCR method for monitoring porcine epidemic diarrhea virus and porcine delta-coronavirus.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) and porcine delta-coronavirus (PDCoV) are economically important pathogens that cause diarrhea in sows and acute death of newborn piglets. Moreover, the emerging PDCoV was reported to infect children. The current situation is that vaccine prevention has not met expectations, and emergency containment strategies following outbreaks cannot prevent the damages and losses already incurred. Therefore, a more sensitive detection method, that is both convenient and enables accurate and effective sequencing, that will provide early warning of PEDV and PDCoV is necessary. This will enable active, effective, and comprehensive prevention and control, which will possibly reduce disease occurrences. RESULTS: Duplex nested RT-PCR (dnRT-PCR) is an ideal method to achieve early warning and monitoring of PEDV and PDCoV diseases, and to additionally investigate any molecular epidemiological characteristics. In this study, two pairs of primers were designed for each virus based upon the highly conserved N protein sequences of both PEDV and PDCoV strains retrieved from the NCBI Genbank. After optimization of the reaction conditions, the dnRT-PCR assay amplified a 749-bp fragment specific to PEDV and a 344-bp fragment specific to PDCoV. Meanwhile, the specificity and sensitivity of the primers and clinical samples were tested to verify and establish this dnRT-PCR method. The limit of detection (LoD)for both PEDV and PDCoV was 10 copies/µL. The results showed that among 251 samples, 1 sample contained PEDV infection, 19 samples contained a PDCoV infection, and 8 samples were infected with both viruses, following the use of dnRT-PCR. Subsequently, the positive samples were sent for sequencing, and the sequencing results confirmed that they were all positive for the viruses detected using dnRT-PCR, and conventional RT-PCR detection was conducted again after the onset of disease. As these results were consistent with previous results, a detection method for PEDV and PDCoV using dnRT-PCR was successfully established. In conclusion, the dnRT-PCR method established in this study was able to detect both PEDV and PDCoV, concomitantly. CONCLUSIONS: The duplex nested RT-PCR method represents a convenient, reliable, specific, sensitive and anti-interference technique for detecting PEDV and PDCoV, and can additionally be used to simultaneously determine the molecular epidemiological background.

The use of RT-PCR in the diagnosis and differentiation of vaccine strains of chicken infectious bronchitis and Newcastle disease.

Background: Infectious diseases of young and adult birds with respiratory syndrome are a significant deterrent to the development of industrial poultry farming due to decreased productivity and significant mortality. The only effective method of combating viral diseases is timely and targeted vaccination, which largely depends on laboratory diagnostic results. Aim: This article aims to study the real-time reverse transcription polymerase chain reaction, (RT-PCR) which has the prospect of more effective diagnosis of vaccine strains of chicken infectious bronchitis and Newcastle disease. Methods: The fastest and most accurate method for the differential diagnosis of pathogens in an associative viral infection is RT-PCR. The method proposed in the article for selecting primers for amplification made it possible to use this method for the simultaneous interspecies differential diagnosis of two or more viral agents, significantly accelerating their diagnosis. Results: The correlation of the nucleotide sequence obtained from sequencing to a specific virus strain is complicated by the lack of a single nomenclature mechanism for separating genetic groups. Conclusion: The results of this study will allow easy and fast typing of sequences into known and databased virus strains and avoid further confusion in the nomenclature of genetic groups in the future.

Development of an RT-PCR-based RspCas13d system to detect porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an enteropathogen that causes diarrhea in piglets and may undergo cross-species transmission. The prevention and control of PDCoV are complicated, and a sensitive, specific, and accessible method of diagnosis would be advantageous. Whereas qPCR is a standard approach for detecting PDCoV, it is not effectively sensitive. In the present study, we report such a strategy using an RT-PCR-based RspCas13d detection system and its efficacy in clinical sample diagnosis. The detection limit of this method was 4 copies/µL and no cross-reaction with other viruses such as the porcine epidemic diarrhea virus, classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, transmissible gastroenteritis virus and porcine rotavirus. The method was also effective in clinical samples. In summary, we demonstrate that RT-PCR-based RspCas13d detection system is an extremely sensitive and specific nucleic acid-based approach for detecting PDCoV. KEY POINTS: • RspCas13d can be used as a candidate molecular diagnostic tool to diagnose viral genomes. • A novel method is proposed using an RT-PCR-based RspCas13d detection system and its effectiveness in the detection of PDCoV. • The RT-PCR-based RspCas13d detection system has excellent sensitivity and specificity.

Human Activity Recognition Using Hybrid Coronavirus Disease Optimization Algorithm for Internet of Medical Things.

BACKGROUND: In our current digital world, smartphones are no longer limited to communication but are used in various real-world applications. In the healthcare industry, smartphones have sensors that can record data about our daily activities. Such data can be used for many healthcare purposes, such as elderly healthcare services, early disease diagnoses, and archiving patient data for further use. However, the data collected from the various sensors involve high dimensional features, which are not equally helpful in human activity recognition (HAR). METHODS: This paper proposes an algorithm for selecting the most relevant subset of features that will contribute efficiently to the HAR process. The proposed method is based on a hybrid version of the recent Coronavirus Disease Optimization Algorithm (COVIDOA) with Simulated Annealing (SA). SA algorithm is merged with COVIDOA to improve its performance and help escape the local optima problem. RESULTS: The UCI-HAR dataset from the UCI machine learning repository assesses the proposed algorithm's performance. A comparison is conducted with seven well-known feature selection algorithms, including the Arithmetic Optimization Algorithm (AOA), Gray Wolf Optimizer (GWO), Whale Optimization Algorithm (WOA), Reptile Search Algorithm (RSA), Zebra Optimization Algorithm (ZOA), Gradient-Based Optimizer (GBO), Seagull Optimization Algorithm (SOA), and Coyote Optimization Algorithm (COA) regarding fitness, STD, accuracy, size of selected subset, and processing time. CONCLUSIONS: The results proved that the proposed approach outperforms state-of-the-art HAR techniques, achieving an average performance of 97.82% in accuracy and a reduction ratio in feature selection of 52.7%.

Prokaryotic expression of porcine deltacoronavirus S gene truncated segment and establishment of indirect ELISA detection method.

Porcine deltacoronavirus (PDCoV) is an emerging discovered coronavirus that causes significant losses in the global swine industry. This study aimed to establish an indirect ELISA method for detecting PDCoV antibodies using the truncated gene of PDCoV spike protein (S). The purified S protein was used as the coating antigen for the polyclonal antibody. The conditions were optimized to establish an indirect ELISA detection method for PDCoV based on the S protein, which showed good specificity and no cross-reaction with SVV-VP1, ASFV-P72, GETV-E2, PRV-gE, etc. The method has high repeatability, with coefficients of variation within and between batches less than 10%. Compared with the commercial kit, the positive coincidence rate is 86.40%, the negative coincidence rate is 89.43%, and the total coincidence rate is 91.76%. This ELISA can be used for PDCoV serological investigation and antibody evaluation. It can also lay the foundation for further research and development of PDCoV S protein ELISA antibody detection kit.

A Customized Novel Blocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered emerging alphacoronavirus. SADS-CoV shares over 90% genome sequence identity with bat alphacoronavirus HKU2. SADS-CoV was associated with severe diarrhea and high mortality rates in piglets. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. Here, monoclonal antibody (MAb) 6E8 against SADS-CoV N protein accurately recognized SADS-CoV infection. Then, MAb 6E8 was utilized as a blocking antibody to develop blocking ELISA (bELISA). We customized the rN coating antigen with concentration 0.25 µg/mL. According to receiver operator characteristic curve analysis, the cutoff value of the bELISA was determined as 38.19% when the max Youden index was 0.955, and specificity was 100%, and sensitivity was 95.5%. Specificity testing showed that there was no cross-reactivity with other serum positive swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine sapelovirus (PSV). In conclusion, we customized a novel and high-quality blocking ELISA for detection of SADS-CoV infection, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection. IMPORTANCE SADS-CoV was reported to be of high potential for dissemination among various of host species. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. We customed a novel and high-quality bELISA assay for detection of SADS-CoV N protein antibodies, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection.

Establishment and Application of a Triplex Real-Time RT-PCR Assay for Differentiation of PEDV, PoRV, and PDCoV.

Porcine viral diarrhea is very common in clinical practice and has caused huge losses to the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important pathogens of porcine viral diarrhea. Co-infection situations among these three viruses in clinics are common, which increases the difficulty of differential diagnosis. Currently, polymerase chain reaction (PCR) is commonly used to detect pathogens. TaqMan real-time PCR is more sensitive than conventional PCR and has better specificity and accuracy. In this study, a triplex real-time RT-PCR assay based on TaqMan probes was developed for differential detection of PEDV, PoRV, and PDCoV. The triplex real-time RT-PCR assay developed in this study could not detect unrelated pathogens and showed satisfactory specificity, sensitivity, repeatability, and reproducibility with a limit of detection (LOD) of 6.0 × 101 copies/µL. Sixteen clinical samples were used to compare the results of the commercial RT-PCR kit and the triplex RT-PCR for PEDV, PoRV, and PDCoV detection, and the results were completely consistent. A total of 112 piglet diarrhea samples collected from Jiangsu province were next used to study the local prevalence of PEDV, PoRV, and PDCoV. The positive rates of PEDV, PoRV, and PDCoV detected by the triplex real-time RT-PCR were 51.79% (58/112), 59.82% (67/112), and 2.68% (3/112), respectively. The co-infections of PEDV and PoRV were frequent (26/112, 23.21%), followed by the co-infections of PDCoV and PoRV (2/112, 1.79%). This study established a useful tool for simultaneous differentiation of PEDV, PoRV, and PDCoV in practice and provided valuable information on the prevalence of these diarrhea viral pathogens in Jiangsu province.

Assessment and sero-diagnosis for coronaviruses with risk of human spillover.

Zoonotic coronaviruses (CoVs) caused major human outbreaks in the last two decades. One of the biggest challenges during future CoV disease is ensuring rapid detection and diagnosis at the early phase of a zoonotic event, and active surveillance to the zoonotic high-risk CoVs appears the best way at the present time to provide early warnings. However, there is neither an evaluation of spillover potential nor diagnosis tools for the majority of CoVs. Here, we analyzed the viral traits, including population, genetic diversity, receptor and host species for all 40 alpha- and beta-CoV species, where the human-infecting CoVs are from. Our analysis proposed 20 high-risk CoV species, including 6 of which jumped to human, 3 with evidence of spillover but not to human and 11 without evidence of spillover yet, which prediction were further supported by an analysis of the history of CoV zoonosis. We also found three major zoonotic sources: multiple bat-origin CoV species, the rodent-origin sub-genus Embecovirus and the CoV species AlphaCoV1. Moreover, the Rhinolophidae and Hipposideridae bats harbour a significantly higher proportion of human-threatening CoV species, whereas camel, civet, swine and pangolin could be important intermediate hosts during CoV zoonotic transmission. Finally, we established quick and sensitive serologic tools for a list of proposed high-risk CoVs and validated the methods in serum cross-reaction assays using hyper-immune rabbit sera or clinical samples. By comprehensive risk assessment of the potential human-infecting CoVs, our work provides a theoretical or practical basis for future CoV disease preparedness.

The impact of the experimental route of challenge on the host responses and pathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus infection in laying chickens.

Infectious bronchitis virus (IBV) infection can be associated with respiratory, renal, and/or reproductive diseases in chickens. Under natural conditions, conjunctiva, mucosa of upper respiratory tract, and cloaca are the main routes of IBV entry. Experimentally, the study of IBV infection involved various routes of inoculation. This study investigated the impact of adding the trachea as a potential route of viral entry to the oculo-nasal infection on the host responses, pathogenicity, and tissue tropism of the Canadian IBV Delmarva (DMV/1639) strain in laying chickens. Specific-pathogen-free laying chickens were divided into three experimental groups: control group (Con group), oculo-nasal challenged group (ON group), and oculo-nasal/intratracheal challenged group (ON/IT group); all groups were observed for 12 days post-infection (dpi). The clinical signs and reduction in egg production in the ON/IT group started slightly earlier compared to the ON group. At 12 dpi, the gross lesions in the ON/IT group were confined to the ovary, while the ON group showed regressed ovary and atrophied oviduct. Only the ON group showed significantly higher microscopic lesion scores in the lung, kidney, magnum, and uterus compared to the control group at 12 dpi. The oviduct tissues of the ON group showed a significant increase in B cells infiltration compared to ON/IT and control groups. The viral shedding (detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR)), tissue tropism (detected either by qRT-PCR or immunohistochemistry (IHC)), T/natural killer cells infiltration in reproductive tract (detected by IHC), and antibody-mediated immune responses (measured by enzyme-linked immunosorbent assay) showed similar patterns in the ON and ON/IT groups.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.