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.

Assessment of the Phytochemical Analysis and Antimicrobial Potentials of Zingiber zerumbet.

Antimicrobial resistance (AMR) has arisen as a global concern in recent decades. Plant extracts used in combination with antibiotics are promising against AMR, synergistically. The purpose of this study was to evaluate the component of the bitter ginger (Zingiber zerumbet) extract in different solvents using high-performance liquid chromatography (HPLC), in addition to evaluate the antibacterial activity of these extracts, in combination with their antibiotic potential against four multi-drug resistant (MDR) bacterial strains (Lactobacillus acidophilus, Streptococcus mutans, Enterococcus faecalis and Staphylococcus aureus). Ethanol and the aqueous extracts of bitter ginger were prepared using a conventional solvent extraction method and were evaluated for their phytochemistry using HPLC, qualitatively and quantitatively. Moreover, the antibiotic susceptibility of the pathogenic isolates was determined. A disc diffusion assay was used to obtain the antimicrobial potential of the extracts alone and with antibiotics. Eight components were identified from the separation of the bitter ginger extract by HPLC. For AMR bacteria, the combination of the antibiotic solution with the bitter ginger crude extracts could improve its susceptibility of these antibiotics. This study indicates that the combination of an antibiotic solution with the bitter ginger crude extract exhibits potent antibacterial activities against MDR bacterial strains. Therefore, they can be used for the treatment of various diseases against the microbial pathogen and can be incorporated into medication for antibacterial therapy.

Characterization of Bioactive Compounds and Novel Proteins Derived from Promising Source Citrullus colocynthis along with In-Vitro and In-Vivo Activities.

Herbal products are preferable to synthetic medicines, and the use of traditional medicines is increasing day-by-day. The current study was designed to evaluate the potentials of bioactive compounds from Citrullus colocynthis by performing FTIR, HPLC, and GC-MS analyses, which explore the good concentration of the secondary metabolites, such as gallic acid (74.854 ppm), vanillic acid (122.616 ppm), and ferulic acid (101.045 ppm) with considerable bioactivities. Antimicrobial protein was estimated by performing SDS-PAGE, ranging from 15 to 70 kDa in all protein fractions. The current study also checked the cytotoxicity of the bioactive compounds in the active fraction of C. colocynthis, and to perform this activity, the groups of rats were arranged with 16 rats randomly divided into four groups (three experimental and one control) by administering various dosage of methanolic fractions in dose-dependent manner. Histopathology was conducted on the livers of the rats after 15 days of sacrifice under deep anesthesia. In liver cell slides examined at the maximum dose of 600 mg/kg, minimal morphological changes, such as slight ballooning, nuclear variation, vacuolar degeneration, and hydropic degeneration, were observed. Furthermore, the in silico analysis identified bioactive compounds as potential drug candidates.

Synthesis, Characterization and Biological Activities of Zinc Oxide Nanoparticles Derived from Secondary Metabolites of Lentinula edodes.

Zinc oxide nanoparticles (ZnO NPs) are the second most prevalent metal oxide, owing to their characteristics of low cost, safe, and easily prepared. ZnO NPs have been found to exhibit unique properties which show their potential to be used in various therapies. Numerous techniques have been devised for the manufacture of zinc oxide because it is one of the nanomaterials that has received major research interest. Mushroom sources are proven to be efficient, ecologically friendly, inexpensive, and safe for humankind. In the current study, an aqueous fraction of methanolic extract of Lentinula edodes (L. edoes) was used to synthesize ZnO NPs. The biosynthesis of ZnO NPs was achieved by using the reducing and capping capability of an L. edodes aqueous fraction. Bioactive compounds from mushroom, such as flavonoids and polyphenolic compounds, are used in the green synthesis process to biologically reduce metal ions or metal oxides to metal NPs. Biogenically synthesized ZnO NPs were further characterized by using UV-Vis, FTIR, HPLC, XRD, SEM, EDX, zeta sizer and zeta potential analyses. The FTIR showed the functional group at the spectra in the range 3550-3200 cm-1 indicated the presence of the hydroxyl (OH) group, while bands in the range 1720-1706 cm-1 indicated C=O carboxylic stretches bonds. Furthermore, the XRD pattern of ZnO NPs created in the current study was found to be nanocrystals which are hexagonal. The SEM analysis of ZnO NPs showed spherical shapes and size distributions in the range 90-148 nm. Biologically synthesized ZnO NPs have substantial biological activities including antioxidant, antimicrobial, antipyretic, antidiabetic and anti-inflammatory potential. Biological activities showed significant antioxidant (65.7 ± 1.09), antidiabetic (85.18 ± 0.48), and anti-inflammatory potential (86.45 ± 0.60) at 300 µg inhibition in paw inflammation of (1.1 ± 0.06) and yeast-induced pyrexia (97.4 ± 0.51) at 10 mg in a dose-dependent manner. The outcomes of this research indicated that ZnO NPs significantly reduced inflammation and have the ability to scavenge free radicals and prevent protein denaturation, while also indicating their possible use in food and nutraceutical applications to treat various ailments.

Carbapenem resistance gene crisis in A. baumannii: a computational analysis.

Acinetobacter baumannii (A. baumannii) is one of the members of ESKAPE bacteria which is considered multidrug resistant globally. The objective of this study is to determine the protein docking of different antibiotic resistance gene (ARGs) in A. baumannii. In silico analysis of antibiotic resistance genes against carbapenem are the blaOXA-51, blaOXA-23, blaOXA-58, blaOXA-24, blaOXA-143, NMD-1 and IMP-1 in A. baumannii. The doripenem, imipenem and meropenem were docked to blaOXA-51 and blaOXA-23 using PyRx. The top docking energy was -5.5 kcal/mol by imipenem and doripenem and meropenem showed a binding score of -5. 2 kcal/mol each and blaOXA-23 energy was -4.3 kcal/mol by imipenem and meropenem showed a binding score of -2.3 kcal/mol, while doripenem showed the binding score of -3.4 kcal/mol. Similarly, doripenem imipenem and meropenem were docked to blaOXA-58, IMP-1, Rec A and blaOXA-143, with docking energy was -8.8 kcal/mol by doripenem and meropenem each while imipenem showed a binding score of -4.2 kcal/mol and with IMP-1 demonstrated their binding energies. was -5.7 kcal/mol by meropenem and doripenem showed a binding score of -5.3 kcal/mol, while imipenem showed a binding score of -4.5 kcal/mol. And docking energy was -4.9 kcal/mol by imipenem and meropenem showed binding energy of -3.6 kcal/mol each while doripenem showed a binding score of -3.9 kcal/mol in RecA and with blaOXA-143 docking energy was -3.0 kcal/mol by imipenem and meropenem showed a binding score of -1.9 kcal/mol, while doripenem showed the binding score of -2.5 kcal/mol respectively. Doripenem, imipenem, and meropenem docking findings with blaOXA-24 confirmed their binding energies. Doripenem had the highest docking energy of -5.5 kcal/mol, meropenem had a binding score of -4.0 kcal/mol, and imipenem had a binding score of -3.9 kcal/mol. PyRx was used to dock the doripenem, imipenem, and meropenem to NMD-1. Docking energies for doripenem were all - 4.0 kcal/mol, whereas meropenem had docking energy of -3.3 kcal/mol and imipenem was -1.50 kcal/mol. To the best of our knowledge the underlying mechanism of phenotypic with genotypic resistance molecular docking regarding carbapenem resistance A. baumannii is unclear. Our molecular docking finds the possible protein targeting mechanism for carbapenem-resistant A.baumannii.

Synthesis of Silver Nanoparticles from Extracts of Wild Ginger (Zingiber zerumbet) with Antibacterial Activity against Selective Multidrug Resistant Oral Bacteria.

Antibiotic resistance rate is rising worldwide. Silver nanoparticles (AgNPs) are potent for fighting antimicrobial resistance (AMR), independently or synergistically. The purpose of this study was to prepare AgNPs using wild ginger extracts and to evaluate the antibacterial efficacy of these AgNPs against multidrug-resistant (MDR) Staphylococcus aureus, Streptococcus mutans, and Enterococcus faecalis. AgNPs were synthesized using wild ginger extracts at room temperature through different parameters for optimization, i.e., pH and variable molar concentration. Synthesis of AgNPs was confirmed by UV/visible spectroscopy and further characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy analysis (EDXA), and Fourier-transform infrared spectroscopy (FTIR). Disc and agar well diffusion techniques were utilized to determine the in vitro antibacterial activity of plant extracts and AgNPs. The surface plasmon resonance peaks in absorption spectra for silver suspension showed the absorption maxima in the range of 400-420 nm. Functional biomolecules such as N-H, C-H, O-H, C-O, and C-O-C were present in Zingiber zerumbet (Z. zerumbet) (aqueous and organic extracts) responsible for the AgNP formation characterized by FTIR. The crystalline structure of ZZAE-AgCl-NPs and ZZEE-AgCl-NPs was displayed in the XRD analysis. SEM analysis revealed the surface morphology. The EDXA analysis also confirmed the element of silver. It was revealed that AgNPs were seemingly spherical in morphology. The biosynthesized AgNPs exhibited complete antibacterial activity against the tested MDR bacterial strains. This study indicates that AgNPs of wild ginger extracts exhibit potent antibacterial activity against MDR bacterial strains.

Evaluation of Hematological, Biochemical Profiles and Molecular Detection of Envelope Gene (gp-41) in Human Immunodeficiency Virus (HIV) among Newly Diagnosed Patients.

The Human Immunodeficiency Virus (HIV) is a highly morphic, retrovirus that rapidly evolves through mutation as well as recombination. Because of the immunocompromised status in HIV patients, there is often a higher chance of acquiring different secondary infections followed by liver cirrhosis, hepatitis B & C, and HIV-associated nephropathy. The current study was conducted to see the prevalence of secondary infections, hematological and biochemical markers for liver and renal associated diseases, and to detect the envelope gene (GP41) in newly diagnosed HIV patients. A total of 37 samples were collected from HIV-positive patients registered in different hospital settings under the National AIDS control program. The collected samples were processed for hepatitis B, hepatitis C, hematological analysis, and biochemical analysis. To identify the envelope gene in newly diagnosed HIV patients, polymerase chain reaction (PCR) was performed using four gene-specific primers. The HIV infections were seen more in male as compared to females. A significant decrease in complete blood count was observed in HIV patients when compared to healthy individuals. There was a significant increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine observed in HIV patients. No significant difference was observed in alkaline phosphatase (ALP), total bilirubin, and albumin levels when compared to healthy control. Anemia was observed in 59.4% of HIV patients. A total of three (8.1%) patients were found to be co-infected with hepatitis B and one (2.7 %) was co-infected with hepatitis C. Out of these 37 tested samples, a total of four showed the successful amplification of the envelope gene. This study provides platform for the health care facilitators to regularly monitor the signs, symptoms and clinical biomarkers of HIV-associated infections to prevent toxicity at an early stage to improve the quality of life (QoL) and minimize the mortality rate in HIV patients. Envelope gene mutating frequently results in drug resistance, and thus future research on polymorphism analysis will reveal points of substitutions to improve drug designing.

Biological and physical characterization of bacteriophage JHA against multidrug-resistant Acinetobacter baumannii.

Due to the emergence of antibiotic resistance, bacteriophage therapy appears to be an ideal weapon to utilize against pathogenic bacteria. This study aimed to isolate, identify and characterize the lytic bacteriophage effective against the multidrug-resistant Acinetobacter baumannii clinical isolates. The isolated bacteriophage caused lysis by applying the double-layer agar technique on A. baumannii up to 99% in 18 hours of incubation at 37ºC. The bacterial growth reduction assay exhibited that JHA phage had high adsorption rates and could rapidly inhibit bacterial growth. The pH and thermal stability testing showed that JHA phage was stable in vast ranges of pH from 5 to 9 but its activity was highest at pH7 (1860000±1000 pfu/mL). It was stable in broad ranges of temperatures from 25ºC to 60ºC but the highest activity was found at 37ºC (1300000±30000 pfu/mL). One-step growth test results showed that it has a short latent period, strong lytic ability, high burst size and adsorption rates and was host specific. Scanning electron microscopy (SEM) of JHA phage demonstrated icosahedral heads and tailless particles. Transmission electron microscopy (TEM) revealed JHA phage belongs to Tectiviridae family. All the characteristics of JHA phage possess lytic activity against A. baumannii strains and exhibit novel candidates to use as an alternative competitor to antibiotics in controlling such infections.

Activity of plant essential oils against antibiotic resistant Enterococcus faecalis isolated from diarrheic children.

Activity of plant essential oils and their fractions was evaluated against characterized isolates of antibiotic resistant Enterococcus faecalis recovered from diarrheic children. The isolates were confirmed by polymerase chain reaction (PCR) targeting 16S rRNA gene amplification followed by nucleotide sequencing and accession numbers retrieved were MW349990.1, MW349859.1, MW332122.1, MW356805.1, MW349975.1, MW349988.1, MW356790.1, MW356244.1, MW341593.1 and MW332549.1. These isolates were screened for antibiotic susceptibility to a wide range of antibiotic groups and mean zone of inhibition (ZOI) of all antibiotics were recorded. Antibacterial activity of plant essential oils (n=05) was checked against three antibiotic resistant isolates of E. faecalis. Three plant essential oils having higher ZOI including Cinnamomum verum, Syzygium aromaticum and Nigella sativa were used against resistant E. faecalis isolates to determine minimum inhibitory concentration (MIC). The lowest MIC observed was of S. aromaticum (11.39±3.94 mg mL-1). The S. aromaticum n-hexane plus chloroform fraction displayed higher mean ZOI (16.67±2.51 mm), while the lowest MIC was of n-hexane oil fraction. Based upon gas chromatography-mass spectrometry (GC/MS) analysis, the most effective fatty acid was eugenic acid which is present in higher proportion in both fractions. These fractions of essential oils proved safe for the treatment of antibiotic resistant diarrheic cases of children caused by E. faecalis.

Evaluation of 16S rRNA methyltransferase gene in aminoglycosides resistant isolates of cancer patients.

Aminoglycosides are used in empiric treatment of critically ill patients. Efficacy of aminoglycoside has been reduced due to dissemination of resistance. The aim of this study was to evaluate aminoglycoside resistance in cancer patients with pneumoniae. A total of 150 Bronchoalveolar lavage and Bronchial washing samples were collected from cancer patients. The samples were identified with standard microbiological procedures. Phenotypic susceptibility pattern of the isolates was determined against various groups of antibiotics such as Penicillins, Cephalosporins, Carbapenems, Monobactams, Aminoglycosides, Tetracyclins, Glycopeptides and Sulphonamides. The isolates with phenotypic resistant to aminoglycosides were further evaluated for the presence of armA gene. The strains of E. coli (12.5%), S. aureus (15.6%), Streptococcus (15.6%), Pseudomonas (18.7%) and K. pneumoniae (37.5%) were isolated. The phenotypic resistance profile showed highest resistance against aminoglycosides (Tobramycin, 53.1% Gentamicin and 50% Amikacin) followed by cephalosporins and sulfonamides group. The armA gene was detected in aminoglycoside resistant isolates. The overall genotypic resistance was evaluated as 21.8%. The armA gene was found in K.pneumoniae 23.5%, Pseudomonas 11.8% (4/24) and E. coli 5.9%. High level resistance to aminoglycosides raises therapeutic concern to health care professionals. These findings highlight the importance of effective monitoring and surveillance to the use of broad-spectrum antibiotics.

Identification of VP1 peptides diagnostic of encephalomyocarditis virus from swine.

BACKGROUND: Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in China. EMCV VP1 protein was one of the most important structural proteins and played an important role in the protective immunity. In this study, 10 monoclonal antibodies (McAbs) against EMCV VP1 were screened and identified. RESULTS: Epitope mapping results indicated that McAbs (6E11, 7A7, 7C9) specifically recognized the linear epitopes V(2)ENAEK(7), McAbs (1D1, 2A2, 5A1, 5A11, 5G1) recognized the epitope F(19)VAQPVY(25), and McAbs 1G8 and 3A9 recognized P(42)IGAFTVK(49). Protein sequence alignment of VP1 with 16 EMCV isolates indicated that the epitope F(19)VAQPVY(25) was conserved in all the reference strains. The epitopes P(42)IGAFTVK(49) and V(2)ENAEK(7) only had 1 or 2 variable amino acid among the reference strains. The 3D model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle. CONCLUSIONS: In this study, ten McAbs against EMCV VP1 were developed and three B-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in VP1. All the results herein will promote the future investigations into the function of VP1 of EMCV and development of diagnostic methods of EMCV.

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.

Inhibition of encephalomyocarditis virus replication by shRNA targeting 1D and 3AB genes in vitro and in vivo.

Encephalomyocarditis virus (EMCV) could infect many host species and cause acute myocarditis and sudden death in pre-weaned piglets. It was necessary to develop new antiviral strategies for the treatment of the virus infection. Here, four plasmids expressing shRNA (small hairpin RNA) targeted to 1D or 3AB protein genes of EMCV were constructed and their inhibition efficiency on the replication of EMCV was evaluated in both BHK21 cells and mice. The results showed that three out of those four shRNA constructs could significantly inhibit EMCV replication in BHK21 cells on the levels of viral RNA and protein. Moreover, it was found that the shRNAs could suppress significantly the load of EMCV in the brain tissue of the mice pretreated with the constructs for 6-24 h. The clinical signs and pathological lesions of the mice in the groups inoculated with the shRNA constructed were milder obviously, compared with those in pSUPER-mN3 and challenge control groups. The survival rates of mice inoculated with pSUPER-3AB-1, pSUPER-3AB-2, and pSUPER-1D-1 for 12 h was 100, 80, and 40%, respectively, while, in the control groups it was only 20%. It indicated that the vector-based shRNA targeting to 3AB and 1D genes might be a potential anti-EMCV strategy.

Toxicity evaluation of hydrophilic CdTe quantum dots and CdTe@SiO2 nanoparticles in mice.

Quantum dots have drawn tremendous attention in the field of in vitro and small animal in vivo fluorescence imaging in the last decade. However, concerns over the cytotoxicity of their heavy metal constituents have limited their use in clinical applications. Here, we report our comparative studies on the toxicities of quantum dots (QDs) and silica coated CdTe nanoparticles (NPs) to mice after intravenous injection. The blood cells analysis showed significant increased level of white blood cells (WBCs) in groups treated with CdTe QDs as compared to the control while red blood cells (RBCs) and platelet counts were normal in treated as well as control groups. The concentration of biochemical markers of hepatic damage, alanine amino transferase (ALT) and aspartate aminotransferase (AST) were in the normal range in all the groups. However, renal function analyses of mice showed significantly increased in the concentration of blood urea nitrogen (BUN) and creatinine (CREA) in mice treated with CdTe QDs while remained within normal ranges in both the CdTe@SiO2 NPs and control group. The results of histopathology showed that the CdTe QDs caused mild nephrotoxicity while other organs were normal and no abnormalities were detected in control and CdTe@SiO2 treated group. These findings suggest that the nephrotoxicity could be minimized by silica coating which would be useful for many biomedical applications.

A comparative study of CdTe quantum dots and CdTe@SiO2 nanoparticles: fabrication and cytotoxicity in HEK293 cells.

Quantum Dots have shown remarkable potentials in biomedical research. Herein, we reported the effects of CdTe quantum dots (QDs) and CdTe@SiO2 nanoparticles (NPs) on human embryonic kidney 293 (HEK 293A) cells with the aim of investigating their in vitro cytotoxicity. The CdTe@SiO2 particles were prepared by reverse microemulsion method. The structural morphology of the CdTe and hydrophilic silica-coated CdTe particles were characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) spectrometry and photoluminescence (PL) spectrometry. The in vitro cytotoxicity of CdTe QDs and CdTe@SiO2 nanoparticles was assessed in 293A cells using standard MTT assay, western blot and fluorescent microscopy. The results showed that the CdTe and CdTe@SiO2 particles were relatively uniform with the diameter of about 3.8 nm, 75 nm respectively. The cell viability and the adhesion ability were similar to the control 293A cells. The level of the fibronectin protein expression was decreased with the increasing concentration of CdTe while the no effects were observed on expression of beta-actin in CdTe as well as CdTe@SiO2 treated cells even at highest concentration of 45 microg/mL which demonstrated their good biocompatibility to 293A cells. The results indicate that the CdTe@SiO2 nanoparticles are attractive candidates for biological imaging studies as expected.

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.

Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice.

BACKGROUND: H9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses. METHODS: To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals. RESULTS: Our results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs. CONCLUSION: Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.

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.