Pathogenic and metagenomic evaluations reveal the correlations of porcine epidemic diarrhea virus, porcine kobuvirus and porcine astroviruses with neonatal piglet diarrhea.

Porcine epidemic diarrhea virus (PEDV) frequently causes diarrhea outbreaks. However, whether newly discovered enteric viruses such as porcine kobuvirus (PKV) and porcine astroviruses (PAstVs) are also correlated with diarrhea is still unclear. Diarrhea outbreaks were reported in a PEDV-vaccinated pig farm in Xinjiang Uygur Autonomous Region of China from 2019 to 2020. PEDV was a common pathogen detected in fecal samples by routine RT-PCR assays. The PEDV positive fecal sample was used for pathogenic analysis due to the failure isolation of PEDV. The challenged neonatal piglets appeared watery diarrhea within one day post infection (dpi) and all died within 6 dpi. Histopathological and immunohistochemical examinations supported that PEDV is a major pathogen causing intestinal lesions. To further explore enteric viruses associated with neonatal piglet diarrhea, metagenomics sequencing was performed for the diarrheic piglets. Remarkably, PKV was the most abundant virus (58.33%) followed by PEDV (34.45%) and PAstVs (7.22%), which were also confirmed by real-time RT-PCR assays. Significant in vivo replications of PEDV and PKV could only be observed in challenged piglets whilst PAstVs maintained similar virus loads in both challenged and mock infected piglets. Overall, this study provides first pathogenic and metagenomic evidence that significant proliferations of PEDV and PKV are closely associated with severe diarrhea in neonatal piglets, while PAstVs likely play limited roles in neonatal piglet diarrhea.

Transcriptomic profiling reveals different innate immune responses in primary alveolar macrophages infected by two highly homologous porcine reproductive and respiratory syndrome viruses with distinct virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates show high genetic and pathogenic diversity. The mechanisms underlying different virulence of PRRSV isolates are still not fully clarified. Two highly homologous PRRSV isolates (XJ17-5 and JSTZ1712-12) with distinct virulence were identified in our previous study. To evaluate the association between host responses and different virulence, here we investigated the transcriptomic profiles of porcine alveolar macrophages (PAMs) infected with these two isolates. RNA-Seq results showed that there are 1932 differential expression genes (DEGs) between two PRRSV infected groups containing 1067 upregulation and 865 downregulation genes. Compared with the avirulent JSTZ1712-12 infected group, GO analysis identified significant enrichment gene sets not only associated with virus infection but also innate immune response in the virulent XJ17-5 infected group. In addition, KEGG analysis indicated significantly enriched genes associated with NOD-like and RIG-I-like receptor signaling pathways in XJ17-5 vs JSTZ1712-12 group. Furthermore, XJ17-5 isolate induced significantly higher levels of innate immune response associated genes (IL-1ß, CXCL2, S100A8, OAS2, MX1, IFITM3, ISG15 and IFI6) than JSTZ1712-12 isolate, which were further confirmed by real-time PCR. Given that these two isolates share similar replication efficiency in vivo and in vitro, our results indicated that distinct virulence of PRRSV isolates is associated with different host innate immune responses.

Chimeric HP-PRRSV2 containing an ORF2-6 consensus sequence induces antibodies with broadly neutralizing activity and confers cross protection against virulent NADC30-like isolate.

Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.

Systemic Homologous Neutralizing Antibodies Are Inadequate for the Evaluation of Vaccine Protective Efficacy against Coinfection by High Virulent PEDV and PRRSV.

G2 porcine epidemic diarrhea virus (G2 PEDV) and highly pathogenic porcine reproductive and respiratory syndrome virus 2 (HP-PRRSV2) are two of the most prevalent swine pathogens in China's swine herds, and their coinfection occurs commonly. Several PED and PRRS vaccines have been utilized in China for decades, and systemic homologous neutralizing antibodies (shnAbs) in serum are frequently used to evaluate the protective efficacy of PED and PRRS vaccines. To develop a vaccine candidate against G2 PEDV and HP-PRRSV2 coinfection, in this study, we generated a chimeric virus (rJSTZ1712-12-S) expressing S protein of G2 PEDV using an avirulent HP-PRRSV2 rJSTZ1712-12 infectious clone as the viral vector. The rJSTZ1712-12-S strain has similar replication efficacies as the parental rJSTZ1712-12 virus. In addition, animal inoculation indicated that rJSTZ1712-12-S is not pathogenic to piglets and can induce shnAbs against both G2 PEDV and HP-PRRSV2 isolates after prime-boost immunization. However, passive transfer study in neonatal piglets deprived of sow colostrum showed that rJSTZ1712-12-S-induced shnAbs may only decrease PEDV and PRRSV viremia but cannot confer sufficient protection against dual challenge of high virulent G2 PEDV XJ1904-34 strain and HP-PRRSV2 XJ17-5 isolate. Overall, this study provides the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for the evaluation of protective efficacy of PED and PRRS bivalent vaccine (especially for the PED vaccine). IMPORTANCE Porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) coinfection occurs commonly and can synergistically reduce feed intake and pig growth. Vaccination is an effective strategy utilized for PED and PRRS control, and systemic homologous neutralizing antibodies (shnAbs) in serum are commonly used for protective efficacy evaluation of PED and PRRS vaccines. Currently, no commercial vaccine is available against PEDV and PRRSV coinfection. This study generated a chimeric vaccine candidate against the coinfection of prevalent PEDV and PRRSV in China. The chimeric strain can induce satisfied shnAbs against both PEDV and PRRSV after prime-boost inoculation in pigs. But the shnAbs cannot confer sufficient protection against PEDV and PRRSV coinfection in neonatal piglets. To the best of our knowledge, these findings provide the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for evaluating PED and PRRS bivalent vaccine protective efficacy.

Characterization of four types of MLV-derived porcine reproductive and respiratory syndrome viruses isolated in unvaccinated pigs from 2016 to 2020.

Modified live vaccines (MLVs) have been utilized to combat porcine reproductive and respiratory syndrome (PRRS), which raises a serious concern about the MLV-derived PRRS virus (PRRSV) isolates. During the routine investigation of PRRSV in China, four lung samples collected from unvaccinated diseased pigs from 2016 to 2020 were detected as PRRSV positive. The PRRSVs shared high ORF5 identities to CH-1R, JXA1-R, TJM-F92 and RespPRRS MLV vaccines, respectively. The viruses were isolated in Marc-145 cells and denominated as SD1612-1, JS1703-21, JSTZ1907-714 and JSYC20-05-1. Genome comparison confirmed that these isolates share the highest genomic homologies to CH-1R (97.96%), JXA1-R (99.64%), TJM-F92 (99.00%) and RespPRRS MLV (99.57%) than any other known isolates. Genome-based phylogenetic analysis showed that SD1612-1 and CH-1R, JS1703-21 and JXA1-R, JSTZ1907-714 and TJM-F92, JSYC20-05-1 and RespPRRS MLV were grouped in the same branches. In addition, amino acids unique to corresponding vaccine attenuations were also identified in our isolates. Noticeably, amino-acids potentially associated with the virulence revision from MLV strains to parental virulent viruses were also identified in the MLV-derived isolates. Our results confirm that the four types of MLV-derived isolates are circulating and evolving in Chinese swine herds for years, which highlights the necessity for the fair use of PRRS MLVs.

Development and application of a quadruplex real-time PCR assay for differential detection of porcine circoviruses (PCV1 to PCV4) in Jiangsu province of China from 2016 to 2020.

To date, four species of porcine circoviruses (PCVs), including PCV1-4, have been reported to exist in the clinical cases. Fast and effective differential detection is critical to monitor the infection and co-infection status of PCVs for adopting reliable control strategies. However, currently available methods cannot simultaneously differentiate the four species of PCV strains. In this study, a quadruplex real-time PCR assay based on TaqMan probes was developed for differential detection of PCV1-4. The new quadruplex real-time PCR assay exhibited satisfied specificity, sensitivity, repeatability and reproducibility. In addition, the new assay was applied to the detection of 120 clinical samples collected from 2016 to 2020 in Jiangsu province of China and compared with previously reported PCV1-4 singleplex conventional PCR assays. Based on the clinical performance, the results from the quadruplex real-time PCR and conventional PCR assays showed 100% agreement. A total of 47 samples were detected as PCV positive by the quadruplex real-time PCR assay, including 1, 2, 1 samples were co-infected with PCV1 and PCV4, PCV2 and PCV3, PCV2 and PCV4, respectively. Full-length ORF2 sequencing and phylogenetic analysis supported the real-time PCR results that 5, 34, 8 and 4 of the 51 PCV sequences were PCV1, PCV2, PCV3 and PCV4, respectively. This study provides a promising alternative tool for rapid differential detection of PCVs and confirms the coexistence of all species of PCV1-4 strains in Jiangsu province in recent years.

A Systematic Investigation Unveils High Coinfection Status of Porcine Parvovirus Types 1 through 7 in China from 2016 to 2020.

Porcine parvovirus genotype 1 (PPV1) causes reproductive disorder in swine and is prevalent in China. Recently, six new genotypes of PPVs (PPV2 through PPV7) have also been detected in Chinese swine herds. However, the coinfection status of all these seven genotypes of PPVs (PPV1-7) in China was not clarified yet. In this study, we developed a panel of PPV1-7 PCR assays with satisfied specificity, sensitivity and reproducibility and then applied to the detection of PPV1-7 in 435 clinical samples collected from eight provinces of China in 2016-2020. A total of 55.40% samples (241 out of 435) were PPV positive, while PPV2 and PPV3 (both 22.53%) belonging to the genus of Tetraparvovirus were the most prevalent genotypes. Noticeably, PPV1-7 strains were more prevalent in nursery and finishing pigs than in suckling pigs. In addition, coinfection could be detected in all eight provinces and 27.36% (119/435) samples were coinfected with two to five genotypes of PPVs. Meanwhile, the coinfection of PPVs with PCV2 was 22.30% (97/435). Twenty complete genomes of representative PPV1-7 were determined, and phylogenetic analysis confirmed the genotyping results by sequence comparisons and PCR assays. Remarkably, the PPV7 HBTZ20180519-152 strain from domestic pig was recombined from parental JX15-like and JX38-like isolates from wild boars. Selective pressure analysis based on VP2 sequences of PPV1-7 showed that they were predominantly under negative selection, while few positive selection sites could be detected in VP2 of PPV7. Overall, this systematic investigation unveils high prevalence and coinfection of PPV1-7 in China from 2016 to 2020. IMPORTANCE Porcine parvoviruses (PPVs) are prevalent in China associating with reproductive failure in swine. The coinfection of seven genotypes of PPVs (PPV1-7) might have synergistic effects on PPV1 associated SMEDI syndrome. However, the coinfection status of PPV1-7 in China is not clear yet. This study showed that PPV1-7 strains are highly prevalent (55.40%) in China and mainly in nursery and finishing pigs in recent years. In addition, the coinfections of different genotypes of PPVs (27.36%) and PPVs with PCV2 (22.30%) are common. Geographic analysis indicated that different genotypes of PPVs are widely cocirculating in China. Intriguingly, a PPV7 strain from the domestic pig was detected as a recombinant from two wild boar isolates. Selective pressure analyses showed that PPV1-7 are mainly under purifying selection. Our findings provide the first systematic investigation on the prevalence, coinfection, and evolution of PPV1 through PPV7 in Chinese swineherds from 2016 to 2020.

The infectious cDNA clone of commercial HP-PRRS JXA1-R-attenuated vaccine can be a potential effective live vaccine vector.

Multiple commercial porcine reproductive and respiratory syndrome (PRRS) modified live vaccines are currently utilized in Chinese swine herds due to the limited cross-protection of vaccines and coexistence of different PRRS viruses. In this study, an infectious cDNA clone of the highly pathogenic PRRS (HP-PRRS) vaccine JXA1-R strain was generated. We successfully rescued the virus from direct in vitro DNA transfection of rJXA1-R clone, which has similar growth kinetics to the parental JXA1-R virus in Marc-145 cells. To further evaluate the potential use of the cloned rJXA1-R virus as a live vector for foreign gene expression, the enhanced green fluorescent protein (EGFP) was inserted between non-structural and structural genes. Our results showed that the dynamic expression of EGFP can be visualized by live cell imaging system during the infection in Marc-145 cells. The availability of our cloned JXA1-R viruses provides a crucial platform to study the fundamental biology of HP-PRRS virus vaccine and also serves as a potential effective vector for developing live vector vaccines against swine pathogens.

High genetic diversity of Chinese porcine reproductive and respiratory syndrome viruses from 2016 to 2019.

High genetic diversity and limited cross-protection are two major reasons for ineffective control of porcine reproductive and respiratory syndrome virus (PRRSV) infection. Therefore, it's important to dynamically monitor the prevalence of PRRSV for adopting appropriate control strategy. In this study, we analyzed PRRSV infection by detecting 712 clinical samples collected from 2016 to 2019 in China. Totally 100 samples were detected as PRRSV positive, including 2 and 98 samples were infected with PRRSV1 and PRRSV2, respectively. In addition, two out of the 98 PRRSV2 positive samples were co-infected with two distinct viruses. ORF5-based phylogenetic analysis showed that JXA1-like HP-PRRSV2 (lineage 8) and NADC30-like PRRSV2 (lineage 1) isolates are currently predominant, but QYYZ-like PRRSV2, CH-1a-like PRRSV2 and PRRSV1 isolates also co-exist in Chinese swine herds. In addition, two commercial MLV-derived viruses (TJM-F92-like and JXA1-R-like) were frequently detected. GP5 alignment also detected insertion and deletion in the extravirion domain. Our study presents the up-to-date PRRSV infection status and highlights the high genetic diversity of PRRSV currently circulating in China.

Co-infection status of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018.

Classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) are economically important swine viruses that cause reproductive failure and/or respiratory symptoms in pigs. However, the co-infection status of these viruses in Chinese swine herds is not well clarified. In this study, we evaluated the co-infection of these four viruses in 159 pigs collected from 63 herds in eight regions of China from 2016 to 2018. CSFV, PRRSV, PCV2 and PCV3 were detected in 14, 56, 43 and 4 of the pigs, respectively. The percentage of singular infections was 32.71%, while the percentages of dual infections and multiple infections were 15.72% and 3.15%, respectively. The E2 of CSFV, ORF5 of PRRSV, ORF2s of PCV2 and PCV3 from all positive samples were determined and used for phylogenetic analyses. E2-based phylogenetic tree showed that all 14 CSFVs identified in this study belong to 2.1b subtype. ORF5-based phylogenetic tree showed that PRRSV2 is predominant in China while PRRSV1 can also be detected. In addition, 35, 16, 4 and 1 of our PRRSVs are clustered with highly pathogenic PRRSV2, NADC30-like PRRSV2, classical PRRSV2 and PRRSV1, respectively. ORF2-based phylogenetic trees showed that our PCVs are grouped with 2 PCV2 subtypes (PCV2d and PCV2b) and 3 PCV3 subtypes (PCV3a, PCV3b and PCV3c), respectively. Our results provide the latest co-infection status and the diversity of four important swine viruses in Chinese swine herds, which is beneficial for understanding the epidemiology of these viruses.

A novel NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) plays a limited role in the pathogenicity of porcine circoviruses (PCV2 and PCV3) and PRRSV co-infection.

Co-infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co-infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17-36) collected from a diseased pig was co-infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17-36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30-like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17-36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30-like PRRSV SD17-36 isolate is low pathogenic. Our results indicate that the co-infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co-infection.

Development of universal and quadruplex real-time RT-PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA-PRRSV2), highly pathogenic (HP-PRRSV2) and NADC30-like (NL-PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real-time RT-PCR assays using TaqMan-MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real-time RT-PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real-time RT-PCR assays were further validated by comparing with a universal PRRSV conventional RT-PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real-time RT-PCR assays and the conventional RT-PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV-positive, including 2 samples that were determined to be co-infected with NL-PRRSV2 and HP-PRRSV2 isolates by the quadruplex real-time RT-PCR assay. ORF5 sequencing confirmed the real-time RT-PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA-PRRSV2, NL-PRRSV2 and HP-PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.

Identification of Two Porcine Reproductive and Respiratory Syndrome Virus Variants Sharing High Genomic Homology but with Distinct Virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to the global swine industry. Even though several control strategies have been applied, PRRS is still not effectively controlled due to the continuous emergence of new variants and limited cross-protection by current vaccines. During the routine epidemiological investigation in 2017, two PRRSV variants were identified from a severe abortion farm and a clinically healthy farm, respectively. The viruses were isolated and denominated as XJ17-5 and JSTZ1712-12. Genomic sequencing indicated that their genomes are both 14,960 bp in length sharing 99.45% nucleotide identity. Sequence alignments identified a discontinuous 30-amino-acid deletion and a continuous 120-amino-acid deletion in nsp2 of both isolates. Genome-based phylogenetic analysis confirmed that XJ17-5 and JSTZ1712-12 belong to the HP-PRRSV subtype but form a new branch with other isolates containing the same 150-amino-acid deletion in nsp2. Pathogenic analysis showed that XJ17-5 is highly virulent causing 60% mortality, while JSTZ1712-12 is avirulent for piglets. Furthermore, fragment comparisons identified 34-amino-acid differences between XJ17-5 and JSTZ1712-12 that might be associated with the distinct virulence. The identification of highly homologous HP-PRRSV variants with new genetic feature and distinct virulence contributes to further analyze the pathogenesis and evolution of PRRSV in the field.