Lineage 1 PRRSVs infection induces hemorrhagic injury in intestines of piglets: Effects on complement and coagulation cascades.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly transmissible disease of significant concern in the pig industry. Previous studies have demonstrated that the XM-2020 strain (a lineage 1.8 PRRSV IA/2012/NADC30) can induce special hemorrhagic injury in the small intestines. However, the specific mechanism underlying this injurious effect remains incompletely understood. In this study, we examined the pathogenic properties of XM-2020 and YC-2020 strains (a lineage 1.5 PRRSV IA/2014/NADC34) in piglets. Animal pathogenic tests revealed that with either Lineage 1 PRRSVs strains XM-2020 or YC-2020 demonstrated pronounced intestinal hemorrhage and suppression of peripheral immunological organs, comparing to JXA1 infection. Transcriptome analysis of diseased small intestines unveiled that PRRSV infection stimulated oxidative and inflammatory reactions. Remarkably, we also observed activation of the complement system alongside a notable down-regulation of complement and coagulation cascade pathways in the Lineage 1 PRRSVs infection group. Based on these findings, we propose that the primary mechanism driving the hemorrhagic injury of the small intestine caused by Lineage 1 PRRSVs is the suppression of complement and coagulation cascades resulting from immunosuppression. This discovery deepens our understanding of the pathogenicity of PRRSV in the small intestine and provides promising ways out for the development of innovative strategies aimed at controlling PRRSV.

Genetic and pathogenic characterization of an NADC-34-like isolate of porcine reproductive and respiratory syndrome virus.

In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.

Porcine Epidemic Diarrhea Virus: Etiology, Epidemiology, Antigenicity, and Control Strategies in China.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus, which is one of the main causative agents of porcine epidemic diarrhea (PED), with 100% morbidity and 80-100% mortality in neonatal piglets. Since 2010, large-scale PED caused by highly pathogenic variants of PEDV has occurred successively in China and other countries in the world, posing a great threat to the global pig industry. It has been demonstrated in many investigations that the classic attenuated vaccine strain, PEDV CV777, is insufficient to fully protect against the PEDV variants. Moreover, the maternally derived antibodies elicited by inactivated vaccines also cannot completely protect piglets from infection. In addition, feedback feeding poses a risk of periodic PEDV recurrence in pig farms, making it challenging to successfully limit the spread of PEDV in China. This review focuses on the etiology, epidemiology, antigenicity, and control strategies of PEDV in China and provides information for the formulation of effective control measures.

Endogenous Type I-C CRISPR-Cas system of Streptococcus equi subsp. zooepidemicus promotes biofilm formation and pathogenicity.

Streptococcus equi subsp. zooepidemicus (SEZ) is a significant zoonotic pathogen that causes septicemia, meningitis, and mastitis in domestic animals. Recent reports have highlighted high-mortality outbreaks among swine in the United States. Traditionally recognized for its adaptive immune functions, the CRISPR-Cas system has also been implicated in gene regulation, bacterial pathophysiology, virulence, and evolution. The Type I-C CRISPR-Cas system, which is prevalent in SEZ isolates, appears to play a pivotal role in regulating the pathogenicity of SEZ. By constructing a Cas3 mutant strain (ΔCas3) and a CRISPR-deficient strain (ΔCRISPR), we demonstrated that this system significantly promotes biofilm formation and cell adhesion. However, the deficiency in the CRISPR-Cas system did not affect bacterial morphology or capsule production. In vitro studies showed that the CRISPR-Cas system enhances pro-inflammatory responses in RAW264.7 cells. The ΔCas3 and ΔCRISPR mutant strains exhibited reduced mortality rates in mice, accompanied by a decreased bacterial load in specific organs. RNA-seq analysis revealed distinct expression patterns in both mutant strains, with ΔCas3 displaying a broader range of differentially expressed genes, which accounted for over 70% of the differential genes observed in ΔCRISPR. These genes were predominantly linked to lipid metabolism, the ABC transport system, signal transduction, and quorum sensing. These findings enhance our understanding of the complex role of the CRISPR-Cas system in SEZ pathogenesis and provide valuable insights for developing innovative therapeutic strategies to combat infections.

Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

Edwardsiella tarda Causing Septicemia in a Wild Crested Ibis (Nipponia nippon).

An adult Crested Ibis (Nipponia nippon) was found moribund in the Qinling area of China. Postmortem examination and histopathological analysis revealed lung inflammation and multi-organ hemorrhage. Bacterial isolation and whole-genome sequencing confirmed Edwardsiella tarda infection.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties.

Comparing the molecular evolution and recombination patterns of predominant PRRSV-2 lineages co-circulating in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses widespread epidemics in swine herds, yet the drivers underlying lineage replacements/fitness dynamics remain unclear. To delineate the evolutionary trajectories of PRRSV-2 lineages prevalent in China, we performed a comprehensive longitudinal phylodynamic analysis of 822 viral sequences spanning 1991-2022. The objectives encompassed evaluating lineage dynamics, genetic diversity, recombination patterns and glycosylation profiles. A significant shift in the dominance of PRRSV-2 sub-lineages has been observed over the past 3 decades, transitioning from sub-lineage 8.7 to sub-lineage 1.8, followed by extensive diversification. The analysis revealed discordant recombination patterns between the two dominant viral sub-lineages 1.8 and 8.7, underscoring that modular genetic exchanges contribute significantly to their evolutionary shaping. Additionally, a strong association was found between recombination breakpoint locations and transcriptional regulatory sequences (TRSs). Glycosylation patterns also demonstrated considerable variability across sub-lineages and temporally, providing evidence for immune-driven viral evolution. Furthermore, we quantified different evolutionary rates across sub-lineages, with sub-lineage 1.8 uniquely displaying the highest nucleotide substitution rates. Taken together, these findings provide refined insight into the evolutionary mechanisms underpinning cyclic shifts in dominance among regionally circulating PRRSV sub-lineages.

Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells.

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

Bovine parainfluenza virus type 3 infections induce ER stress-mediated autophagy to facilitate virus replication.

Bovine Parainfluenza Virus Type 3 (BPIV3) serves as a crucial pathogen in cattle, adept at triggering severe respiratory symptoms. This investigation explores the intricate interplay of endoplasmic reticulum stress (ER stress), unfolded protein response (UPR), and autophagy upon BPIV3 infection. In this study, we initially confirm a substantial increase in glucose regulatory protein 78 (GRP78) expression, accompanied by noticeable morphological changes and significant expansion of the ER lumen observed through transmission electron microscopy upon BPIV3 infection. Our findings indicate that ER Stress is induced during BPIV3 infection in vitro. Subsequently, we illustrate that BPIV3 triggers ER Stress to facilitate viral replication through heightened autophagy through treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) and utilizing small interfering RNA (siRNA) technology to knock down GRP78. Additionally, we observe that the activation of ER stress initiates the UPR via PERK and ATF6 pathways, with the IRE1 pathway not contributing to the regulation of ER stress-mediated autophagy. Moreover, intervention with the PERK inhibitor GSK2606414, ATF6 inhibitor Ceapin-A7, and siRNA technology successfully reverses BPIV3-induced autophagy. In summary, these findings propose that BPIV3 induces ER stress to enhance viral replication through increased autophagy, with the PERK and ATF6 pathways playing a significant role in ER stress-mediated autophagy.

A NADC30-like PRRSV causes serious intestinal infections and tropism in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China. In this study, a NADC30-Like PRRSV strain was isolated in primary alveolar macrophage (PAM) cells from fecal samples collected from a local pig farm, which suffered severe diarrhea. A pathogenicity comparison study was conducted in 6-week-old piglets by inoculating highly pathogenic HP-PRRSV and NADC30-Like PRRSV isolates. RT-qPCR revealed detection of NADC30-Like PRRSV but not the HP-PRRSV in the intestine. PRRSV infection-related lesions were observed in the intestine were further confirmed by histopathological and immunohistochemical examination (IHC). In addition, severe virus infections were also detected by RT-qPCR. Based on clinical observation and pathogenicity experiments, we confirmed that NADC30-Like PRRSV gained more tissue tropism, especially in the small intestine. This may be the one reason explaining why NADC-Like 30 PRRSV become a major epidemic strain in China since the first outbreak in 2013.

The transcriptional characteristics of NADC34-like PRRSV in porcine alveolar macrophages.

The widespread and endemic circulation of porcine reproductive and respiratory syndrome virus (PRRSV) cause persistent financial losses to the swine industry worldwide. In 2017, NADC34-like PRRSV-2 emerged in northeastern China and spread rapidly. The dynamics analysis of immune perturbations associated with novel PRRSV lineage is still incomplete. This study performed a time-course transcriptome sequencing of NADC34-like PRRSV strain YC-2020-infected porcine alveolar macrophages (PAMs) and compared them with JXA1-infected PAMs. The results illustrated dramatic changes in the host's differentially expressed genes (DEGs) presented at different timepoints after PRRSV infection, and the expression profile of YC-2020 group is distinct from that of JXA1 group. Functional enrichment analysis showed that the expression of many inflammatory cytokines was up-regulated following YC-2020 infection but at a significantly lower magnitude than JXA1 group, in line with the trends for most interferon-stimulated genes (ISGs) and their regulators. Meanwhile, numerous components of histocompatibility complex (MHC) class II and phagosome presented a stronger transcription suppression after the YC-2020 infection. All results imply that YC-2020 may induce milder inflammatory responses, weaker antiviral processes, and more severe disturbance of antigen processing and presentation compared with HP-PRRSV. Additionally, LAPTM4A, GLMP, and LITAF, which were selected from weighted gene co-expression network analysis (WGCNA), could significantly inhibit PRRSV proliferation. This study provides fundamental data for understanding the biological characteristics of NADC34-like PRRSV and new insights into PRRSV evolution and prevention.

A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.

Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.

Nanopore-Based Direct RNA-Sequencing Reveals a High-Resolution Transcriptional Landscape of Porcine Reproductive and Respiratory Syndrome Virus.

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.