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

doi:10.21203/rs.3.rs-1749345/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-1749345/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

· 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 porcine delta-coronavirus 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 identify 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 retrieved from the NCBI Genbank PEDV and PDCoV entries. 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/ml. The results showed that among 251 samples, 1 sample contained PEDV infection only, 19 samples contained a PDCoV infection, and 8 samples were infected with both viruses, following the use of dnRT-PCR. Thereafter, 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.

Porcine epidemic diarrhea virus

Porcine delta-coronavirus

Duplex nested RT-PCR

Clinical monitoring

Swine acute diarrhea syndrome（SADS）is one of the most serious diseases affecting the swine industry in China and the rest of the world in recent years[1-3]. Some prevention and control measures have been undertaken to combat this disease[4, 5]. Although these interventions have achieved some successful outcomes, they are far from being effective or even efficient regarding prevention and control. Comprehensive measures are usually taken after an outbreak of PDS, which has caused inevitably significant economic losses[6-8]. Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the main causative agents of PDS, which is presently difficult to prevent and control. The main symptoms of SADS are acute diarrhea in sows and fattening pigs, while in newborn piglets the most common disease outcomes are vomiting, diarrhea and dehydration, and even death which usually occurs within 10 days of birth, with a high incidence at days 3-5 postpartum[9-11]. In addition to diarrhea, sows also exhibit a lack of estrus post weaning and post mating, and increased abortion and premature delivery rates.

PEDV is an acute intestinal infectious disease in pigs characterized by watery diarrhea, vomiting, and dehydration[8, 12], commonly known as winter diarrhea disease. PEDV belongs to the family Coronaviridae, genus Alphacoronavirus, and contains an infectious single-stranded, positive-sense RNA genome measuring about 28 kb[13, 14]. The PEDV genome has six open reading frames (ORFs) and encodes replicase polymeric protein 1ab (pp1ab), spike (S), envelope (E), membrane (M) and nucleocapsid (N) from 5’-3’[15].

PDCoV is also a member of the Coronaviridae family, genus deltacoronavirus[16-20]. Diarrhea outbreaks in newborn piglets have occurred frequently in pig farms across China since early winter 2020. The porcine epidemic diarrhea virus strain was generally believed to be the main pathogenic pathogen in these cases. Subsequently, another novel coronavirus, PDCoV, was found to cause diarrhea in piglets and has become widespread in pig farms in China. As an emerging porcine intestinal coronavirus, PDCoV was first reported in Hong Kong, China in 2012[21], and later in the USA[22], China Mainland[23], South Korea[24], and Thailand[25]. The novel porcine enteric coronavirus disease can cause diarrhea and vomiting in suckling pigs[26], with morbidity and mortality rates as high as 50%-100%, and low mortality post-infection in growing and adult pigs. This has greatly influenced the healthy development of the pigs themselves and impacted the swine industry. In addition, from May 2014 to December 2015, Lednicky collected a total of 369 samples from children presenting to the school clinic with acute undifferentiated febrile illness in Haiti and found three of them were infected with coronavirus strains, which clustered with PDCoV[27]. Therefore, it means that PDCoV also poses a potential threat to human health.

PDCoV is an enveloped virus that has a positive-sense single-stranded RNA (+ssRNA) genome measuring 25.4 kb in length[28, 29]. The PDCoV genome has seven major open reading frames (ORFs). Two overlapping ORFsc(ORF1a and ORF1b) which encode two replication-associated proteins, both are autoproteolytically cleaved into 15 nonstructural proteins (Nsp2 to Nsp16)[30, 31]. The remaining ORF encodes the spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). Additionally, three accessory proteins have been identiﬁed: nonstructural protein 6 (NS6), NS7, and NS7a[32-34].

In both PEDV and PDCoV, the nucleotide sequences of nucleoprotein N are highly conserved compared with nucleotide sequences of envelope (E), spike (S) and membrane (M) in the open reading frame of the two viruses[28], therefore they make suitable diagnostic target genes. At present, laboratory testing is the main diagnostic method employed to detect the two viruses in swine farms. Common RT-PCR and fluorescence quantitative RT-PCR have been used for pathogen detection[35], whilst serological detection methods mainly include virus neutralization (VN) tests[36] and enzyme-linked immunosorbent tests (ELISA)[37].

In view of the urgent situation in the swine industry, a method needs to be established with three main attributes, all of which have been addressed in the present research. First of all, sensitivity of the method must be particularly high. This would enable monitoring, analysis and prediction of the disease prior to outbreak, so as to achieve the role of an early warning system. Secondly, using the two pathogens detailed in this study, both could be monitored and used as an early warning system or for diagnosis simultaneously. Finally, positive samples could ben be sequenced, and this information used immediately to clarify the genotype and molecular evolution characteristics of the infection or outbreak. Although the positive products can be sent for sequencing following the presently used RT-PCR monitoring methods, sensitivity is relatively low. In addition, although the sensitivity of fluorescence quantitative monitoring is relatively good, the process of testing and sequencing is relatively cumbersome. Therefore, in this study, on the basis of conventional RT-PCR, nuclear protein N was used as the diagnostic target gene to establish a dnRT-PCR method that could detect both PEDV and PDCoV simultaneously, so as to improve the sensitivity and specificity of detection. Therefore, the method described in this study has the three basic attributes mentioned above, and additionally satisfies the industry goal of moving from post-onset diagnosis through to pre-onset warning.

As mentioned above, newborn piglets aged 3-5 old have a high incidence of vomiting and diarrhea clinically. It is possible that toxic sow milk leads to cumulative infection of newborn piglets, and if sows have post-mating estrus or an abortion, it may also be related to PEDV and PDCoV. In this study, based on pathogen detection in sow vulva swabs and colostrum via dnRT-PCR, correlations were determined. Therefore, it is possible to use the method outlined in this study to monitor colostrum and vulva swab samples, so as to achieve early warning, and to synchronously identify the etiology of the two pathogens in advance of serious infection and outbreak.

Specificity of the dnRT-PCR

The specificity of the dnRT-PCR was evaluated for target organism using nested primers. First, the specificity was tested using a recombinant positive plasmid. Second, other pathogens associated with diarrheal disease of pigs, including PRRSV, TGEV, PoRV, PRV, Escherichia coli and Staphylococcus aureus, were tested using the dnRT-PCR assay to evaluate its analytical specificity. The detection results showed that there was no amplification product for the negative control or the pathogens in the first reaction, and there was also no band for the second reaction, which proved that the four pairs of primers had good specificity and exhibited high amplification efficiency (Fig.1).

Sensitivity of dnRT-PCR

The sensitivity results from the dnRT-PCR and duplex RT-PRA are shown in Fig. 2. The electrophoresis results from the first PCR amplification showed that the copy number of the gene detected by the outer primer was 10², and the amplification specificity was good. The minimum gene copy number of 10¹ orders of magnitude was detected via double nested RT-PCR amplification. The sensitivity of this method was 1 order of magnitude higher than that of duplex RT-RPA.

Interference test

The detection results showed that all four pairs of primers were able to amplify specific bands from the mixed template, which showed that the primers exhibited good anti-interference performance (Fig. 3).

Evaluation of dnRT-PCR on clinical samples

As shown in Fig. 4, the detection results using the dnRT-PCR method were consistent with those observed in the sequencing and clinical track. Among these samples, one sample was identified as containing a pure infection of PEDV, nineteen samples had only PDCoV present, and eight samples (three colostrum and five vulva) had both PEDV and PDCoV identified using the dnRT-PCR (Table. 4). In contrast, using conventional RT-PCR one positive sample was identified as a PEDV only infection, but only eighteen had PDCoV identified and only seven samples contained both PEDV and PDCoV, the latter of which included three colostrum samples and just 4 vulva samples (Table. 4). The above results indicated that one positive sample of PEDV and two positive samples of PDCoV were not detected using conventional RT-PCR, which may be attributed to the low viral load within those particular samples, indicating an infection of early stage, which is difficult to detect by conventional RT-PCR.

Nucleotide homology analysis of the N gene sequence in PEDV and PDCoV

MegAlign software(MegAlign Pro | Sequence Alignment Software | DNASTAR) was used to compare the nucleotide homology of N gene sequences in 9 sequenced PEDV strains with locus 216-956 from this paper and 7 PEDV reference strains, and the N gene sequence of 19 sequencing PDCoV representative strains with locus 440-784 from this paper and 8 PDCoV reference strains, respectively (Fig. 5). The results showed that the PEDV strains showed high homologies, ranging from 92.0-99.7%, and the homology of the PDCoV strains also exhibited high homologies, ranging from 93.3-99.9%. Therefore, the accuracy of the dnRT-PCR method was further confirmed by the homology analysis undertaken against the sequencing strains in this study.

Under the pressure of African swine fever (ASF), the incidence of related diseases caused by porcine epidemic diarrhea virus (PEDV) and porcine delta-coronavirus (PDCoV) have also been increasing, causing substantial economic losses to the swine industry in China[13, 38]. PEDV and PDCoV can infect pigs of all ages[7, 39]. These two viruses not only cause diarrhea in pigs at different stages, but are also associated with porcine reproductive disorders and respiratory syndrome. Commercial vaccines for PEDV have been marketed and are now widely used in pig farms[40-44]. In the first few years of use, the vaccines offered good prevention and managed to control the viruses to a degree, but the immune effect of these vaccines has become poor in recent years[7, 8, 45, 46], which suggests that the epidemic strain of PEDV may have mutated. Therefore, there is an urgent need to develop and use a highly sensitive monitoring method for PEDV and PDCoV, to give early infection warning. In addition, it is equally important to clarify the molecular epidemiological background of viruses within positive samples and to provide that in a timely manner within the original testing method. To meet the above requirements, four pairs of primers were designed based on the nucleocapsid protein (N) gene fragments within PEDV and PDCoV, and the reaction conditions of the designed primers were optimized. The results showed that the dual-nested RT-PCR (dnRT-PCR) method established in this study exhibited good specificity for detecting the nucleocapsid protein (N) gene fragments of PEDV and PDCoV, and had no cross-reaction with PRRSV, TGEV, PoRV, PRV, E-coli or S. aureus. Repeatability and coincidence rate of this method were 100%. Electrophoresis results of the first PCR amplification showed that the detectable gene copy numbers of the outer primers were 1.2×10²and 1.3×10² respectively. The minimum detection limits of PEDV and PDCoV were 10 copies/ml respectively after two PCR amplifiers. This sensitivity was 10 times higher than that of the duplex RT-RPA method of PEDV and PDCoV established by Li G et al[47], The sensitivity of detection of both PEDV and PDCoV were also 10 times higher than observed using the multi-RT-PCR method against PEDV, TGEV, PoRV and PDCoV established by Jia, S et al., [48]. When compared with the LuminexxTAG multiple detection method of 11 diarrhea viruses established by Shi, Y et al[49], the sensitivity of the method established in this study was 100 times higher for both PEDV and PDCoV, indicating that among the methods currently reported, the sensitivity of the method established in this study was relatively high, it outperformed the other methods. The method also presented with good anti-interference, specificity and repeatability, therefore it is suitable for clinical or normal diagnostic detection and for early warning monitoring of both PEDV and PDCoV. Further, clinical follow-up was performed on pigs that tested negative using conventional RT-PCR but positive by dnRT-PCR, and the corresponding infected pigs showed clinical symptoms of diarrhea in varying degrees after 3-5 days. At this latter time point, both the dnRT-PCR and conventional RT-PCR methods indicated positive results, and sequencing further confirmed that the amplified specific fragments were positive. Therefore, this means a lot of time can be saved during the etiological investigation, costs can be reduced, and the current epidemic situation of PEDV and PDCoV in pig farms under pressure of ASF can be accurately evaluated.

A total of 251 samples were collected from 177 colostrum samples and 74 vulva samples. The positive rate of PDCoV in colostrum was 8.8% (22/251), which was higher than that of PEDV at 1.2% (3/251). This may suggest that PDCoV infects newborn piglets through sow milk. According to the scarce monitoring data available, the role of PDCoV in infecting newborn piglets through this pathway may be more important than that of PEDV. The detection rates of PEDV and PDCoV in vulva swabs were 2.4% (6/251) and 2.0% (5/251), respectively, indicating that a positive vulva swab means infection within the birth canal, which increases the risk of transvaginal infection, to the piglet, with both agents during pregnancy and birth. PEDV and PDCoV infections within the birth canal are also more likely to cause symptoms related to reproductive disorder in sows, this is consistent with the report published by Em-on Olanratmanee et al[50]. In addition, among the 251 samples submitted for examination, the positive rate of PEDV was 3.6% lower than that of PDCoV at 10.8%, which indicated that the diarrhea syndrome was more likely caused by PDCoV infection due to its higher infection rates. There were 8 dual infection samples within the positive samples, including 3 colostrum samples and 5 vulva samples, therefore, mixed infections of PEDV and PDCoV existed together in both colostrum and vulva samples. The probability of double infection within the vulva samples was higher than observed in colostrum, especially given that there were 177(1.7%) colostrum and 74(6.8%) vulva samples, but this further confirmed that the infection of these two viruses may be responsible for causing reproductive disorders in sows.

Viral and bacterial strains

Two viral strains were used as positive controls in this study: a cell cultured adapted “CV-777” strain of PEDV and a cell cultured adapted “CHN-HB” strain of PDCoV, which were identified and stored in our laboratory. To determine the specificity of our methods, the following viruses and bacteria were used: porcine reproductive and respiratory syndrome virus (PRRSV), swine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), Escherichia coli and Staphylococcus aureus, the above strains were also identified and stored in our laboratory.

Sample collection and Genome extraction

In November 2018, 251 samples of colostrum and vulva swabs of sows and newborn piglets were collected from a large-scale pig farm in Hubei, Hunan, Sichuan and Guangdong province. Genomes from the control strains and clinical swabs were collected and centrifuged at 3000 r/min for 15 minutes[51]. Viral RNA was extracted from the samples to be tested using Trizol reagent (AXYGEN Bio., China) according to the manufacturer's instructions, and the concentrations of the extracted RNA were measured for the evaluation of the duplex nested RT-PCR method developed in this study.

Primer design

The nucleotide sequence for PEDV and the PDCoV were collected from GenBank database available in the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/). According to the nucleotide sequence of the N gene, two pairs of specific primers for PEDV and two pairs of specific primers for PDCoV were designed using Primer 5(https://primer-premier-5.software.informer.com/). The conventional RT-PCR primers for PEDV and for PDCoV were designed according to the published reference[9] (Table 1).

Duplex nested RT-PCR

For the first reaction, the amplification was carried out in a total volume of 25 μl of reaction mixture containing 12.5 μl of 2×1 Step Buffer, 1μl of PrimeScript 1 Step Enzyme Mix, 1 μl of PEDV-O-F, PEDV-O-R, PDCoV-O-F and PDCoV-O-R primers at the concentration of 20 μmol/L, respectively and 1 μl of RNA templet. RNase-free water was added bring the total volume to 25 μl. Amplification was performed with a thermocycler (Bioer Co., China) using the following procedure: 30 min at 50℃ for reverse transcription followed by predenaturation at 94°C for 2 min, 32 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 8 min. The negative PCR product from the first reaction was diluted 50 times as the genetic template for the nested PCR.

For the nested PCR, the total volume of the reaction mixture was the same as the first reaction, but the reaction mixture contains 12.5μl of PrimeScript 1 Step Enzyme Mix, 1μl of PEDV-I-F, PEDV-I-R, PDCoV-I-F and PDCoV-I-R primers at the concentration of 20 μmol/L, respectively and 1 μl of templet. RNase-free water was added to bring the volume to 25 μl. The amplification procedure was as follows: predenaturation at 94°C for 2 min, 32 cycles of denaturation at 98°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 50 sec, and a final extension at 72°C for 8 min. The amplified products from two reactions were detected after electrophoresis on 1% agarose gels.

According to the results of the double nested RT-PCR amplification, the target band detected by electrophoresis after the first PCR amplification was reported to be positive (the 996bp band detected was PEDV, and 665bp was PDCoV); or double bands were detected, that is, both viruses were within the sample at the same time), and the sample was considered to have carried a high virulence load. Electrophoresis detected the target band after the second PCR amplification and was also judged to be positive (the 739bp band was PEDV and the 344 bp band was PDCoV, or double bands were detected), but the virulence of the sample was relatively low. If no target bands were detected after two rounds of PCR, the samples were reported to be free of PEDV and PDCoV.

Specificity and sensitivity of the dnRT-PCR primers

The genomes of porcine reproductive and respiratory syndrome (PRRSV), porcine infectious gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine pseudo-rabies virus (PRV), Escherichia coli and Staphylococcus aureus were extracted as templates. The four pairs of primers for PEDV-O and PEDV-I, PDCoV-O and PDCoV-I used in the double nested RT-PCR were tested for specificity to test the four pairs of primers. The positive plasmids obtained above were diluted to a single digit copy number by 10 times ratio, and plasmids with each dilution were selected as templates, and the PEDV-O and PDCoV-O primers were used for the first PCR amplification. The product of the first amplification served as templates for the corresponding gradient, and the PEDV-I and PDCoV-I primers were used for the second PCR amplification. The amplified products from the two reactions were detected following electrophoresis on 1% agarose gels. In addition, the sensitivity of the dnRT-PCR was compared with that of duplex RT-RPA[47].

Interference test

The genome used in the specificity test was mixed with the same amount of the genome, and PEDV-O and PEDV-I, PDCoV-O and PDCoV-I primers were used for PCR amplification respectively to test the anti-interference of the primers and to confirm whether the target band could be amplified from the mixed genome template.

Evaluation of the dnRT-PCR on clinical samples

To evaluate the reliability of the duplex nested RT-PCR on clinical specimens, a total of 251 samples of colostrum and vulva swabs from sows and newborn piglets were collected from Sichuan, Hunan, Hubei, Guangdong provinces and tested with this newly developed assay. The reliability of the detection results was evaluated via sequencing. In addition, the coincidence rate of the dnRT-PCR was compared with results from conventional RT-PCR.

Homology analysis and genetic evolution tree analysis

The sequencing results were analyzed using DNAstar Megalign Software (MegAlign Pro | Sequence Alignment Software | DNASTAR). The PEDV and PDCoV reference strains are shown in Table 2 and Table 3, respectively. The sequences of PEDV and PDCoV generated from this study are shown in Additional file 1 and Additional file 2, respectively.

PEDV: porcine epidemic diarrhea virus; PDCoV: porcine deltacoronavirus; dnRT-PCR: duplex nested RT-PCR.

Additional Files Legends

Additional file 1: The sequences of PEDV generated from this study.

Additional file 2: The sequences of PDCoV generated from this study.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article. All the nucleotide sequences generated from this study have been deposited and are available in the GenBank database.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was financially supported by the Cooperative Innovation Center for Sustainable Pig Production of Huazhong Agricultural University and National Natural Science Foundation of China (Grant No. 31602099) and Hubei GONGFU Farming Co. Itd (GrantNo.34120015).

Authors' contributions

GPL, CQL and LQH designed the research. CQL, LQH, YW, SXC and Tl participate in the filed work. CQL and LQH lead the lab work. CQL and GPL performed date analysis. XLY, LWG and JGZ performed fnal editing and revision. GPL and LXM performed as a laboratory supervisor, funding acquisition, and as corresponding author. All authors read and approved the fnal manuscript.

Acknowledgements

We gratefully acknowledge Dr Guiqin Peng and Zhengfei Liu for their guide of this study, and express our gratitude to EditSprings (https://www.editsprings.cn ) for the expert linguistic services provided.

Authors' information

¹College of Animal Science, Yangtze University, Jingzhou, China

²State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China

³Center for Disease Control and Prevention of Xinzhou Distract, Wuhan, China

⁴Hunan Key Laboratory of Traditional Chinese Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, China

1. Qing Y, Liu J, Huang X, Li Y, Zhang Y, Chen J, et al. Immunogenicity of transmissible gastroenteritis virus (TGEV) M gene delivered by attenuated Salmonella typhimurium in mice. Virus Genes. 2016; 52(2):218-27.

2. Zhou L, Sun Y, Lan T, Wu R, Chen J, Wu Z, et al. Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China. Transbound Emerg Dis. 2019; 66(2):687-95.

3. Zeng S, Zhang H, Ding Z, Luo R, An K, Liu L, et al. Proteome analysis of porcine epidemic diarrhea virus (PEDV)-infected Vero cells. Proteomics. 2015; 15(11):1819-28.

4. Jung K, Saif LJ, Wang Q. Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control. Virus Res. 2020; 286:198045.

5. Wen Z, Xu Z, Zhou Q, Li W, Wu Y, Du Y, et al. Oral administration of coated PEDV-loaded microspheres elicited PEDV-specific immunity in weaned piglets. Vaccine. 2018; 36(45):6803-9.

6. Wang D, Fang L, Xiao S. Porcine epidemic diarrhea in China. Virus Res. 2016; 226:7-13.

7. Yu J, Chai X, Cheng Y, Xing G, Liao A, Du L, et al. Molecular characteristics of the spike gene of porcine epidemic diarrhoea virus strains in Eastern China in 2016. Virus Res. 2018; 247:47-54.

8. Zuo Q, Zhao R, Liu J, Zhao Q, Zhu L, Zhang B, et al. Epidemiology and phylogeny of spike gene of porcine epidemic diarrhea virus from Yunnan, China. Virus Res. 2018; 249:45-51.

9. Ding G, Fu Y, Li B, Chen J, Wang J, Yin B, et al. Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China. Transbound Emerg Dis. 2020; 67(2):678-85.

10. Marthaler D, Raymond L, Jiang Y, Collins J, Rossow K, Rovira A. Rapid Detection, Complete Genome Sequencing, and Phylogenetic Analysis of Porcine Deltacoronavirus. Emerging Infectious Diseases. 2014; 20(8):1347-50.

11. Reveles-Felix S, Carreon-Napoles R, Mendoza-Elvira S, Quintero-Ramirez V, Garcia-Sanchez J, Martinez-Bautista R, et al. Emerging strains of porcine epidemic diarrhoea virus (PEDv) in Mexico. Transbound Emerg Dis. 2019; 67(2):1035-41.

12. Pensaert MB, de Bouck P. A New Coronavirus-Like Partiele Assoeiated With Diarrhea in Swine. Arch Virol. 1978; 58:243-7.

13. Song D, Park B. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes. 2012; 44(2):167-75.

14. Jung K, Saif LJ. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J. 2015; 204(2):134-43.

15. Huang YW, Dickerman AW, Pineyro P, Li L, Fang L, Kiehne R, et al. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio. 2013; 4(5):e00737-13.

16. Hu H, Jung K, Vlasova AN, Chepngeno J, Lu Z, Wang Q, et al. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States. J Clin Microbiol. 2015; 53(5):1537-48.

17. Gong L, Li J, Zhou Q, Xu Z, Chen L, Zhang Y, et al. A New Bat-HKU2-like Coronavirus in Swine, China, 2017. Emerg Infect Dis. 2017; 23(9):1607-9.

18. Pan Y, Tian X, Qin P, Wang B, Zhao P, Yang YL, et al. Discovery of a novel swine enteric alphacoronavirus (SeACoV) in southern China. Vet Microbiol. 2017; 211:15-21.

19. Zhou P, Fan H, Lan T, Yang XL, Shi WF, Zhang W, et al. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin. Nature. 2018; 556(7700):255-8.

20. Liang Q, Zhang H, Li B, Ding Q, Wang Y, Gao W, et al. Susceptibility of Chickens to Porcine Deltacoronavirus Infection. Viruses. 2019; 11(6).

21. Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol. 2012; 86(7):3995-4008.

22. Wang L, Byrum B, Zhang Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014. Emerg Infect Dis. 2014; 20(7):1227-30.

23. Dong N, Fang L, Zeng S, Sun Q, Chen H, Xiao S. Porcine Deltacoronavirus in Mainland China. Emerging Infect Dis. 2015; 21(12):2254-5.

24. Lee JH, Chung HC, Nguyen VG, Moon HJ, Kim HK, Park SJ, et al. Detection and Phylogenetic Analysis of Porcine Deltacoronavirus in Korean Swine Farms, 2015. Transbound Emerg Dis. 2016; 63(3):248-52.

25. Lorsirigool A, Saeng-chuto K, Madapong A, Temeeyasen G, Tripipat T, Kaewprommal P, et al. The genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in Thailand in 2015. Virus Genes. 2017; 53(2):240-8.

26. Chen Q, Gauger P, Stafne M, Thomas J, Arruda P, Burrough E, et al. Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets. Virology. 2015; 482:51-9.

27. Lednicky JA, Tagliamonte MS, White SK, Elbadry MA, Alam MM, Stephenson CJ, et al. Independent infections of porcine deltacoronavirus among Haitian children. Nature. 2021; 600(7887):133-7.

28. Lee S, Lee C. Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus. Virus Res. 2015; 208:136-45.

29. Phan MVT, Ngo Tri T, Hong Anh P, Baker S, Kellam P, Cotten M. Identification and characterization of Coronaviridae genomes from Vietnamese bats and rats based on conserved protein domains. Virus Evol. 2018; 4(2):vey035.

30. Wang L, Su S, Bi Y, Wong G, Gao GF. Bat-Origin Coronaviruses Expand Their Host Range to Pigs. Trends Microbiol. 2018a; 26(6):466-70.

31. Woo PC, Huang Y, Lau SK, Yuen KY. Coronavirus genomics and bioinformatics analysis. Viruses. 2010; 2(8):1804-20.

32. Fang P, Fang L, Hong Y, Liu X, Dong N, Ma P, et al. Discovery of a novel accessory protein NS7a encoded by porcine deltacoronavirus. J Gen Virol. 2017; 98(2):173-8.

33. Fang P, Fang L, Liu X, Hong Y, Wang Y, Dong N, et al. Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6. Virology. 2016; 499:170-7.

34. Luo J, Fang L, Dong N, Fang P, Ding Z, Wang D, et al. Porcine deltacoronavirus (PDCoV) infection suppresses RIG-I-mediated interferon-beta production. Virology. 2016; 495:10-7.

35. Ma L, Cong F, Zhu Y, Wu M, Xu F, Huang R, et al. Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of Theiler's murine encephalomyelitis virus. Molecular and Cellular Probes. 2018; 41:27-31.

36. Panyasing Y, Kedkovid R, Thanawongnuwech R, Kittawornrat A, Ji J, Gimenez-Lirola L, et al. Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities. Vet Microbiol. 2018; 216:72-8.

37. Diel DG, Lawson S, Okda F, Singrey A, Clement T, Fernandes MHV, et al. Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods. Virus Res. 2016; 226:60-70.

38. Mai K, Feng J, Chen G, Li D, Zhou L, Bai Y, et al. The detection and phylogenetic analysis of porcine deltacoronavirus from Guangdong Province in Southern China. Transbound Emerg Dis. 2018; 65(1):166-73.

39. Wang M, Wang Y, Baloch AR, Pan Y, Tian L, Xu F, et al. Detection and genetic characterization of porcine deltacoronavirus in Tibetan pigs surrounding the Qinghai-Tibet Plateau of China. Transbound Emerg Dis. 2018b; 65(2):363-9.

40. Beall A, Yount B, Lin CM, Hou Y, Wang Q, Saif L, et al. Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A. mBio. 2016; 7(1):e01451-15.

41. Deng F, Ye G, Liu Q, Navid MT, Zhong X, Li Y, et al. Identification and Comparison of Receptor Binding Characteristics of the Spike Protein of Two Porcine Epidemic Diarrhea Virus Strains. Viruses. 2016; 8(3):55.

42. Fan B, Yu Z, Pang F, Xu X, Zhang B, Guo R, et al. Characterization of a pathogenic full-length cDNA clone of a virulent porcine epidemic diarrhea virus strain AH2012/12 in China. Virology. 2017; 500:50-61.

43. Hou Y, Lin CM, Yokoyama M, Yount BL, Marthaler D, Douglas AL, et al. Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets. J Virol. 2017; 91(14).

44. Van Diep N, Sueyoshi M, Norimine J, Hirai T, Myint O, Teh APP, et al. Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks. BMC Vet Res. 2018; 14(1):96.

45. Chen N, Li S, Zhou R, Zhu M, He S, Ye M, et al. Two novel porcine epidemic diarrhea virus (PEDV) recombinants from a natural recombinant and distinct subtypes of PEDV variants. Virus Res. 2017; 242:90-5.

46. Chiou H-Y, Huang Y-L, Deng M-C, Chang C-Y, Jeng C-R, Tsai P-S, et al. Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan. Transboundary and Emerging Diseases. 2017; 64(1):157-66.

47. Li G, Wu M, Li J, Cai W, Xie Y, Si G, et al. Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method. J Virol Methods. 2021; 292:114096.

48. Jia S, Feng B, Wang Z, Ma Y, Gao X, Jiang Y, et al. Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea. Mol Cell Probes. 2019; 47:101435.

49. Shi Y, Li B, Tao J, Cheng J, Liu H. The Complex Co-infections of Multiple Porcine Diarrhea Viruses in Local Area Based on the Luminex xTAG Multiplex Detection Method. Front Vet Sci. 2021; 8:602866.

50. Olanratmanee EO, Kunavongkrit A, Tummaruk P. Impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows. Anim Reprod Sci. 2010; 122(1-2):42-51.

51. Wang FF, Liu GP, Zhang F, Li ZM, Yang XL, Yang CD, et al. Natural selenium stress influences the changes of antibiotic resistome in seleniferous forest soils. Environ Microbiome. 2022; 17(1):26.

Table 1: Primers designed for the duplex nested RT-PCR in this study

Virus	Target gene	Primer	Sequence	Position	Product size	Tm		Origin
PEDV	N	PEDV- O- F	5’-GCAAACGGGTGCCATTATCTC-3’	32 - 1027	995bp		63.8	This paper
		PEDV- O- R	5’-GCTCACGAACAGCCACATTAC-3’	32 - 1027	995bp		60.7
		PEDV- I- F	5’-TTGGCATTTCTACTACCTCGGAACA-3’	216 - 956	740bp		65.0
		PEDV- I- R	5’-GCCTGACGCATCAACACCTTT-3’	216 - 956	740bp		64.7
PDCoV	N	PDCoV- O- F	5’-CAGGTCCCAGAGGAAATCTTA-3’	218 - 883	665bp		58.3	This paper
		PDCoV- O- R	5’-TTTGGTAGGTGGCTCATAGGT-3’	218 - 883	665bp		58.6
		PDCoV- I- F	5’-TGCCAAACGCAACCC-3’	440 - 784	344bp		58.5
		PDCoV- I- R	5’-CAGCCATACCCGTCTTCT-3’	440 - 784	344bp		56.1
PEDV	N	PEDV- F	5’-TAGGACTCGTACTGAGGGTGT-3’	26642–26662	600bp		59.4	Ding et al
PEDV	N	PEDV- R	5’-CTATTTTCGCCCTTGGGAATT-3’	27222–27242	600bp		56.6	Ding et al
PDCoV	N	PDCoV- F	5’-GCTGACACTTCTATTAAAC-3’	24301–24319	497bp		48.7	Ding et al
PDCoV	N	PDCoV- R	5’-TTGACTGTGATTGAGTAG-3’	24804-24787	497bp		48.4	Ding et al

Note: PEDV-O-F, PEDV-O-R, PDCoV-O-F and PDCoV-O-R were the primers used in the first round of duplex nested RT-PCR; PEDV-I-F, PEDV-I-R, PDCoV-I-F and PDCoV-I-R were the primers used in the second round of duplex nested RT-PCR.PEDV- F, PEDV-R, PDCoV-F and PDCoV- R were the primers used for conventional RT-PCR.

Table 2: Reference strains for the homology analysis based on the N gene sequences of PEDV

Strain	Source	GenBank
CV777	Switzerland	AF353511.1
FR/001/2014	France	KR011756
USA/Indiana/17846/2013	USA	KF452323.1
GXGG07	China	MK731895.1
JS-HZ2012	China	KC210147.1
AJ1102	China	JX188454.1
CH/YNKM-8/2013	China	KF761675.1

Table 3: Reference strains for the homology analysis based on the N gene sequences of PDCoV

Strain	Source	GenBank
GX03-2019	China	MT648385.1
CHN/Sichuan/2019	China	MK993519.1
USA/Ohio444/2014	USA	KR265862
USA/Iowa459/2014	USA	KR265865
OH1987	USA	KJ46246
YMG/JPN/2014	Japan	KU051641.1
PDCoV/Swine/Thailand/S5015L/2015	Thailand	KU051649
P1_16_BTL_0115/PDCoV/2016/Lao	Lao	KX118627

Table 4: Concordance rate comparisons of the dnRT-PCR with the duplex RT-RPA for the detection of clinical specimens.

Detection method	Sample type	No. of Samples	No. of positive samples (positive %)
Detection method	Sample type	No. of Samples	PEDV	PDCoV	Coinfection
dnRT-PCR	Colostrum	177	3 (1.2)	22 (8. 8)	3 (1. 7)
	Vulva swab	74	6 (2. 4)	5 (2. 0)	5 (2. 0)
	Total	251	9 (3. 6)	27 (10. 8)	8(3. 2)
conventional RT-PCR	Colostrum	177	3 (1. 2)	20 (8. 0)	3 (1. 7)
	Vulva swab	74	5 (2. 0)	5 (2. 0)	4(1. 6)
	Total	251	8 (3. 2)	25 (10. 0)	7(2. 8)

Note: Positive rate = number of positive samples/total number of tested samples.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Background

Results

Discussion

Methods

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1