Temporal Assessment of Protein Stability in Dried Blood Spots.

The use of protein biomarkers in blood for clinical settings is limited by the cost and accessibility of traditional venipuncture sampling. The dried blood spot (DBS) technique offers a less invasive and more accessible alternative. However, protein stability in DBS has not been well evaluated. Herein, we deployed a quantitative LC-MS/MS system to construct proteomic atlases of whole blood, DBSs, plasma, and blood cells. Approximately 4% of detected proteins' abundance was significantly altered during blood drying into blood spots, with overwhelming disturbances in cytoplasmic fraction. We also reported a novel finding suggesting a decrease in the level of membrane/cytoskeletal proteins (SLC4A1, RHAG, DSC1, DSP, and JUP) and an increase in the level of proteins (ATG3, SEC14L4, and NRBP1) related to intracellular trafficking. Furthermore, we identified 19 temporally dynamic proteins in DBS samples stored at room temperature for up to 6 months. There were three declined cytoskeleton-related proteins (RDX, SH3BGRL3, and MYH9) and four elevated proteins (XPO7, RAN, SLC2A1, and SLC29A1) involved in cytoplasmic transport as representatives. The instability was governed predominantly by hydrophilic proteins and enhanced significantly with an increasing storage time. Our analyses provide comprehensive knowledge of both short- and long-term storage stability of DBS proteins, forming the foundation for the widespread use of DBS in clinical proteomics and other analytical applications.

Investigation into temporal changes in the human bloodstain lipidome.

Bloodstains are crucial pieces of physical evidences found at violent crime scenes, providing valuable information for reconstructing forensic cases. However, there is limited data on how bloodstain lipidomes change over time after deposition. Hence, we deployed a high-throughput high-performance liquid chromatography-mass spectrometry (HPLC-MS) approach to construct lipidomic atlases of bloodstains, whole blood, plasma, and blood cells from 15 healthy adults. A time-course analysis was also performed on bloodstains deposited for up to 6 months at room temperature (~ 25°C). The molecular levels of 60 out of 400 detected lipid species differed dramatically between bloodstain and whole blood samples, with major disturbances observed in membrane glycerophospholipids. More than half of these lipids were prevalent in the cellular and plasmic fractions; approximately 27% and 10% of the identified lipids were uniquely derived from blood cells and plasma, respectively. Furthermore, a subset of 65 temporally dynamic lipid species arose across the 6-month room-temperature deposition period, with decreased triacylglycerols (TAGs) and increased lysophosphatidylcholines (LPCs) as representatives, accounting for approximately 8% of the total investigated lipids. The instability of lipids increased linearly with time, with the most variability observed in the first 10 days. This study sheds light on the impact of air-drying bloodstains on blood components at room temperature and provides a list of potential bloodstain lipid markers for determining the age of bloodstains.

Technical strategy for monozygotic twin discrimination by single-nucleotide variants.

Monozygotic (MZ) twins are theoretically genetically identical. Although they are revealed to accumulate mutations after the zygote splits, discriminating between twin genomes remains a formidable challenge in the field of forensic genetics. Single-nucleotide variants (SNVs) are responsible for a substantial portion of genetic variation, thus potentially serving as promising biomarkers for the identification of MZ twins. In this study, we sequenced the whole genome of a pair of female MZ twins when they were 27 and 33 years old to approximately 30 × coverage using peripheral blood on an Illumina NovaSeq 6000 Sequencing System. Potentially discordant SNVs supported by whole-genome sequencing were validated extensively by amplicon-based targeted deep sequencing and Sanger sequencing. In total, we found nine bona fide post-twinning SNVs, all of which were identified in the younger genomes and found in the older genomes. None of the SNVs occurred within coding exons, three of which were observed in introns, supported by whole-exome sequencing results. A double-blind test was employed, and the reliability of MZ twin discrimination by discordant SNVs was endorsed. All SNVs were successfully detected when input DNA amounts decreased to 0.25 ng, and reliable detection was limited to seven SNVs below 0.075 ng input. This comprehensive analysis confirms that SNVs could serve as cost-effective biomarkers for MZ twin discrimination.

An improved system to generate recombinant canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. METHODS: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. RESULTS: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. CONCLUSION: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

Proteomic Difference Analysis of Whole Blood and Bloodstains.

OBJECTIVES: To study the changes of protein levels in peripheral blood after it dried. METHODS: The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed. RESULTS: A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains. CONCLUSIONS: The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Immunogenicity of recombinant vaccinia virus vaccines co-expressing GP3/GP5 of European PRRSV and Cap protein of PCV2 in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is almost always caused by the North American strain of PRRS virus (PRRSV) in China; the European genotype of PRRSV has emerged in China. The mixed infection of PRRSV and Porcine circovirus type 2 virus (PCV2) are always found in pigs and PRRSV-augmented PCV2 replication and serious clinical symptoms. Current vaccines cannot protect mixed European PRRSV and PCV2 infections. Therefore, the development of a safe and effective new vaccine to prevent and control the mixed infection of European PRRSV and PCV2 is both urgent and necessary. In this study, we developed a recombinant vaccinia vaccine co-expressing the GP3 and GP5 proteins of European PRRSV and the ORF2 protein of PCV2 and evaluated the immunogenicity and its protective effects and its inactivated vaccine in pigs. The recombinant vaccinia vaccine and its inactivated vaccine both elicited significant humoral and cellular immune responses with a higher level of specific antibody responses and T-lymphocyte proliferation than the control group. Furthermore, the pigs inoculated with the recombinant vaccinia vaccine were completely protected against challenge with 105 TCID50 of European PRRSV strain LV. These data suggest that the recombinant vaccinia vaccine is a potential candidate vaccine against European PRRSV and PCV2.

Molecular detection and genomic characterization of porcine circovirus 3 in pigs from Northeast China.

BACKGROUND: First identified in the United States in 2016, porcine circovirus type 3 (PCV3) is a newly emerging porcine circovirus exhibiting a wide range of clinical syndromes, which may be associated with the pathogenicity observed in pigs. RESULTS: The aim of this study was to identify and characterize the full genome sequence of PCV3 strains circulating in Northeast China. Herein, 105 lung samples isolated from sick pigs in Northeast China during 2018 were analyzed for PCV3. Using PCR, the total PCV3-positive rate was 33.3% (35/105), with rates of 17.8% (8/45), 66.7% (10/15), and 37.8% (17/45) in Heilongjiang, Jilin, and Liaoning province, respectively. Additionally, our findings showed that PCV3-positive samples had a high rate of co-infection with PCV2, PPV6, and PPV7. To study the evolution of the PCV3 in Northeast China, we sequenced the entire genome of 13 strains of PCV3. The results of phylogenetic analyses revealed that PCV3 could be divided into two clades, PCV3a and PCV3b. Interestingly, a G deletion at position 1072 was found in the 1999 nt genome of PCV3-CN2018LN-4 (MH277118). The G deletion terminated replicase protein translation and induced a truncated replicase protein. CONCLUSION: These results contribute to the understanding of PCV3 molecular epidemiology and evolution in Northeast China. A new strain of PCV3 with truncated replicase protein was identified.

Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus.

BACKGROUND: The European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) has recently emerged in China. The coexistence of Genotype-I and -II PRRSV strains could cause seriously affect PRRSV diagnosis and management. Current vaccines are not able to protect against PRRSV infection completely and have inherent drawbacks. Thus, genetically engineered vaccines, including DNA vaccine and live vector engineered vaccines, have been developed. This study aimed to determine the enhanced immune responses of mice inoculated with a DNA vaccine coexpressing GP3 and GP5 of a Genotype-I PRRSV. RESULTS: To evaluate the immunogenicity of GP3 and GP5 proteins from European-type PRRSV, three DNA vaccines, pVAX1-EU-ORF3-ORF5, pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed, which were based on a Genotype-I LV strain (GenBank ID: M96262). BALB/c mice were immunized with the DNA vaccines; delivered in the form of chitosan-DNA nanoparticles. To increase the efficiency of the vaccine, Quil A (Quillaja) was used as an adjuvant. GP3 and GP5-specific antibodies, neutralizing antibodies and cytokines (IL-2, IL-4, IL-10 and IFN gamma) from the immunized mice sera, and other immune parameters, were examined, including T-cell proliferation responses and subgroups of spleen T-lymphocytes. The results showed that ORF3 and ORF5 proteins of Genotype-I PRRSV induced GP3 and GP5-specific antibodies that could neutralize the virus. The levels of Cytokines IL-2, IL-4, IL-10, and IFN-γ of the experimental groups were significantly higher than those of control groups after booster vaccination (P < 0.05). The production of CD3+CD4+ and CD3+CD8+ T lymphocyte was also induced. T lymphocyte proliferation assays showed that the PRRSV LV strain virus could stimulate the proliferation of T lymphocytes in mice in the experimental group. CONCLUSIONS: Using Quil A as adjuvant, Genotype-I PRRSV GP3 and GP5 proteins produced good immunogenicity and reactivity. More importantly, better PRRSV-specific neutralizing antibody titers and cell-mediated immune responses were observed in mice immunized with the DNA vaccine co-expressing GP3 and GP5 proteins than in mice immunized with a DNA vaccine expressing either protein singly. The results of this study demonstrated that co-immunization with GP3 and GP5 produced a better immune response in mice.

Development of a RPA-CRISPR/Cas12a based rapid visual detection assay for Porcine Parvovirus 7.

Introduction: Porcine Parvovirus (PPV) is a significant pathogen in the pig industry, with eight genotypes, including PPV7, identified since its emergence in 2016. Co-infections with viruses such as Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) pose serious risks to swine health. Thus, there is an urgent need for rapid, sensitive, and specific detection methods suitable for use in field settings or laboratories with limited resources. Methods: We developed a CRISPR/Cas12a-based assay combined with recombinase polymerase amplification (RPA) for the rapid detection of PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed to target a highly conserved region within the NS1 gene of PPV7. Optimization of crRNA and single-stranded DNA (ssDNA) concentrations was performed to enhance the assay's performance. Results: CrRNA optimization identified crRNA-05 as the optimal candidate for Cas12a-based detection of PPV7, as all synthesized crRNAs demonstrated similar performance. The optimal crRNA concentration was determined to be 200 nM, yielding consistent results across tested concentrations. For ssDNA optimization, the strongest fluorescence signal was achieved with 500 nM of the FAM-BHQ ssDNA receptor. The assay showed a minimal detection limit of 100copies/µl for PPV7, confirmed through fluorescence and lateral flow detection methods. Specificity testing indicated that only PPV7 DNA samples returned positive results, confirming the assay's accuracy. In tests of 50 lung tissue samples from diseased pigs, the RPA-Cas12a assay identified 29 positive samples (58%), surpassing the 22 positive samples (44%) detected by conventional PCR. This highlights the RPA-Cas12a method's enhanced detection capability and its potential utility in clinical surveillance and management of PPV7 in swine populations. Discussion: The RPA-Cas12a assay effectively detects PPV7 in clinical samples, enhancing disease surveillance and control in pigs. Its adaptability to resource-limited settings significantly improves PPV7 management and prevention strategies, thereby supporting the overall health and development of the pig industry.

Progress in PRRSV Infection and Adaptive Immune Response Mechanisms.

Since its discovery, Porcine reproductive and respiratory syndrome (PRRS) has had a huge impact on the farming industry. The virus that causes PRRS is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and because of its genetic diversity and the complexity of the immune response, the eradication of PRRS has been a challenge. To provide scientific references for PRRSV control and vaccine development, this study describes the processes of PRRSV-induced infection and escape, as well as the host adaptive immune response to PRRSV. It also discusses the relationship between PRRSV and the adaptive immune response.

Molecular biology of canine parainfluenza virus V protein and its potential applications in tumor immunotherapy.

Canine parainfluenza virus (CPIV) is a zoonotic virus that is widely distributed and is the main pathogen causing canine infectious respiratory disease (CIRD), also known as "kennel cough," in dogs. The CPIV-V protein is the only nonstructural protein of the virus and plays an important role in multiple stages of the virus life cycle by inhibiting apoptosis, altering the host cell cycle and interfering with the interferon response. In addition, studies have shown that the V protein has potential applications in the field of immunotherapy in oncolytic virus therapy or self-amplifying RNA vaccines. In this review, the biosynthesis, structural characteristics and functions of the CPIV-V protein are reviewed with an emphasis on how it facilitates viral immune escape and its potential applications in the field of immunotherapy. Therefore, this review provides a scientific basis for research into the CPIV-V protein and its potential applications.

Current progress on innate immune evasion mediated by Npro protein of pestiviruses.

Interferon (IFN), the most effective antiviral cytokine, is involved in innate and adaptive immune responses and is essential to the host defense against virus invasion. Once the host was infected by pathogens, the pathogen-associated molecular patterns (PAMPs) were recognized by the host pattern recognition receptors (PRRs), which activates interferon regulatory transcription factors (IRFs) and nuclear factor-kappa B (NF-κB) signal transduction pathway to induce IFN expression. Pathogens have acquired many strategies to escape the IFN-mediated antiviral immune response. Pestiviruses cause massive economic losses in the livestock industry worldwide every year. The immune escape strategies acquired by pestiviruses during evolution are among the major difficulties in its control. Previous experiments indicated that Erns, as an envelope glycoprotein unique to pestiviruses with RNase activity, could cleave viral ss- and dsRNAs, therefore inhibiting the host IFN production induced by viral ss- and dsRNAs. In contrast, Npro, the other envelope glycoprotein unique to pestiviruses, mainly stimulates the degradation of transcription factor IRF-3 to confront the IFN response. This review mainly summarized the current progress on mechanisms mediated by Npro of pestiviruses to antagonize IFN production.

Metagenomic analysis provides bases on individualized shift of colon microbiome affected by delaying colostrum feeding in neonatal calves.

This study investigated the effect of colostrum feeding time on the colon digesta microbiome of 2-day-old dairy calves using whole-genome-based metagenome sequencing, aiming to understand the dynamic changes of the colon microbiome when the colostrum feeding is delayed. In total, 24 male Holstein calves were grouped to different pasteurized colostrum feeding time treatments randomly: TRT0h (45 min after birth, n = 7); TRT6h (6 h after birth, n = 8); and TRT12h (12 h after birth, n = 9). Bacteria, archaea, eukaryotes, and viruses were identified in the colon microbiome, with bacteria (99.20%) being the most predominant domain. Streptococcus, Clostridium, Lactobacillus, Ruminococcus, and Enterococcus were the top five abundant bacteria genera. For colon microbiome functions, 114 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified, with nutrients metabolism-related functions "carbohydrate metabolism," "amino acid metabolism," "metabolism of cofactors and vitamins," "metabolism of terpenoids and polyketides," and "metabolism of other amino acids" being the top five secondary level of KEGG hierarchy functions. When colon microbiomes were compared, they were not affected by delaying first colostrum feeding at both taxonomic and functional levels. However, distinct clusters of colon microbiome profiles were shown based on PERMANOVA analysis despite of different colostrum feeding treatment, suggesting the individualized responses. Moreover, the relative abundance of microbial taxa, microbial functions, and differentially expressed genes was compared between the two distinct clusters, and different relationships were observed among host differentially expressed genes, differential levels of microbial taxa, and microbial functions between the two clusters. Our results suggest that the host may play an important role in shaping the colon microbiome of neonatal dairy calves in response to the early life feeding management. Whether the observed colon microbiome shifts affect gut health and function in the long term requires further research.

Ag85a-S2 Activates cGAS-STING Signaling Pathway in Intestinal Mucosal Cells.

Brucellosis is a zoonotic disease caused by Gram-negative bacteria. Most of the brucellosis vaccines in the application are whole-bacteria vaccines. Live-attenuated vaccines are widely used for brucellosis prevention in sheep, goats, pigs, and cattle. Thus, there is also a need for an adjuvanted vaccine for human brucellosis, because the attenuated Brucella vaccines now utilized in animals cause human illness. Here, we developed a live-attenuated Brucella suis strain 2 vaccine (S2) adjuvanted with Ag85a (Ag85a-S2). We found that Ag85a-S2 activated cGAS-STING pathways both in intestinal mucosal cells in vivo and in the BMDM and U937 cell line in vitro. We demonstrated that the cGAS knockout significantly downregulated the abundance of interferon and other cytokines induced by Ag85a-S2. Moreover, Ag85a-S2 triggered a stronger cellular immune response compared to S2 alone. In sum, Ag85a-S2-mediated enhancement of immune responses was at least partially dependent on the cGAS-STING pathway. Our results provide a new candidate for preventing Brucella pathogens from livestock, which might reduce the dosage and potential toxicity compared to S2.

Positive Regulation of the Antiviral Activity of Interferon-Induced Transmembrane Protein 3 by S-Palmitoylation.

The interferon-induced transmembrane protein 3 (IFITM3), a small molecule transmembrane protein induced by interferon, is generally conserved in vertebrates, which can inhibit infection by a diverse range of pathogenic viruses such as influenza virus. However, the precise antiviral mechanisms of IFITM3 remain unclear. At least four post-translational modifications (PTMs) were found to modulate the antiviral effect of IFITM3. These include positive regulation provided by S-palmitoylation of cysteine and negative regulation provided by lysine ubiquitination, lysine methylation, and tyrosine phosphorylation. IFITM3 S-palmitoylation is an enzymatic addition of a 16-carbon fatty acid on the three cysteine residues within or adjacent to its two hydrophobic domains at positions 71, 72, and 105, that is essential for its proper targeting, stability, and function. As S-palmitoylation is the only PTM known to enhance the antiviral activity of IFITM3, enzymes that add this modification may play important roles in IFN-induced immune responses. This study mainly reviews the research progresses on the antiviral mechanism of IFITM3, the regulation mechanism of S-palmitoylation modification on its subcellular localization, stability, and function, and the enzymes that mediate the S-palmitoylation modification of IFITM3, which may help elucidate the mechanism by which this IFN effector restrict virus replication and thus aid in the design of therapeutics targeted at pathogenic viruses.

Detection and Molecular Characterization of Porcine Parvovirus 7 in Eastern Inner Mongolia Autonomous Region, China.

Porcine parvoviruses (PPV) and porcine circoviruses type 2 (PCV2) are widespread in the pig population. Recently, it was suggested that PPV7 may stimulate PCV2 and PCV3 replication. The present study aimed to make detection and molecular characterization of PPV7 for the first time in eastern Inner Mongolia Autonomous Region, China. Twenty-seven of ninety-four samples (28.72%) and five in eight pig farms were PPV7 positive. Further detection showed that the co-infection rate of PPV7 and PCV2 was 20.21% (19/94), and 9.59% (9/94) for PPV7 and PCV3. In addition, the positive rate of PPV7 in PCV2 positive samples was higher than that in PCV2 negative samples, supporting that PCV2 could act as a co-factor for PPV7 infection. In total, four PPV7 strains were sequenced and designated as NM-14, NM-19, NM-4, and NM-40. The amplified genome sequence of NM-14 and NM-40 were 3,999nt in length, while NM-19 and NM-4 were 3,996nt with a three nucleotides deletion at 3,097-3,099, resulting in an amino acid deletion in the Cap protein. Phylogenetic analysis based on the capsid amino acid (aa) sequences showed that 52 PPV7 strains were divided into two clades, and the four PPV7 strains in this study were all clustered in clade 1. The genome and capsid amino acid sequence of the four PPV7 strains identified in this study shared 80.0-96.9% and 85.9-100% similarity with that of 48 PPV7 reference strains selected in NCBI. Simplot analysis revealed that NM-19 and NM-4 strains were probably produced by recombination of two PPV7 strains from China. The amino acid sequence alignment analysis of capsid revealed that the four PPV7 strains detected in Inner Mongolia had multiple amino acid mutations in the 6 B cell linear epitopes compared with the reference strains, suggesting that the four PPV7 strains may have different characteristics in receptor binding and immunogenicity. In summary, this paper reported the PPV7 infection and molecular characterization in the eastern of Inner Mongolia Autonomous Region for the first time, which is helpful to understand the molecular epidemic characteristics of PPV7.

Immunological evaluation of recombination PRRSV GP3 and GP5 DNA vaccines in vivo.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a threat to the health of pigs worldwide, but commercially available vaccines offer limited protection against PRRSV infection. It is necessary to develop a more effective DNA vaccine. The immunological effects of DNA vaccines with three adjuvants were examined in pigs (Susscrofa domestica) challenged with PRRSV. These DNA vaccines, which encoded PRRSV GP3 and GP5, were formulated with A1, A2, and A3. Serum specific and neutralizing antibodies, IL-4, IFN-γ, IL-2, IL-10, CD4+ and CD8+T-lymphocytes, health status, histopathology, and viral loads were determined. The results showed that the use of adjuvant A3 led to higher levels of neutralizing antibodies and a lower viral load in pigs compared to the other adjuvants. The neutralizing antibody titers of the pVAX-GP35+A1 and pVAX-GP35+A3 groups reached a peak of 1:19 at 35 dpi. The maximum concentration of IL-4 was 136.77 pg/mL in the pVAX-GP35+A3 group. At 35 dpi, the IFN-γ concentration in the pVAX-GP35+A1 group was 227.4 pg/mL. pVAX-GP35+A3 group shows the highest IL-2 and IL-10 expression to the peak of 597.6 pg/mL and 189.1 pg/mL, respectively. We found a formulation demonstrated beneficial immune outcomes. This study provides an alternative vaccine to protect pigs from PRRSV.

Pathogenicity of porcine reproductive and respiratory syndrome virus (ORF5 RFLP 1-7-4 viruses) in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and respiratory problems in piglets. PRRSV infection leads to substantial pig mortality and causing huge economic losses so that disease outbreaks caused by the new PRRSV strain from other regions have caused great concern in China. In this study, we analysed the pathogenicity of the novel ORF5 RFLP 1-7-4-like PRRSV strain, named PRRSV-ZDXYL-China-2018-1 in pigs. The viral challenge test showed that PRRSV-ZDXYL-China-2018-1 infection can cause persistent fever, moderate dyspnoea, serum viraemia and interstitial pneumonia in piglets. The levels of viral loads in serum and PRRSV-specific antigen were also detected in lung tissues were used one-step Taq-Man RT-qPCR and Immunohistochemistry, respectively. At 28dpi, the level of specific antibodies was increased among infected piglets. Importantly, the new virus appeared be a moderately virulent isolate with pathogenicity compared to HP-PRRSV strain LQ (JXA1-like strain). Histological examination revealed severe monocyte haemorrhage and interstitial pneumonia associated with monocyte infiltration in the lung tissue of pigs infected with PRRSV-ZDXYL-China-2018-1 and LQ-JXA1 strains. Immunohistochemistry (IHC) results showed positive brown-red epithelial cells and macrophages in pig lungs. Therefore, it is critical to establish an effective strategy to control the spread of PRRSV in China.

Metagenomic Analysis of Flaviviridae in Mosquito Viromes Isolated From Yunnan Province in China Reveals Genes From Dengue and Zika Viruses.

More than 6,000 mosquitoes of six species from six sites were collected and tested for their virome using metagenomics sequencing and bioinformatic analysis. The identified viral sequences belonged to more than 50 viral families. The results were verified by PCR of selected viruses in all mosquitoes, followed by phylogenetic analysis. In the present study, we identified the partial dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) sequences in mosquitoes. Metagenomic analysis and the PCR amplification revealed three DENV sequences, one of which encodes a partial envelope protein. Two ZIKV sequences both encoding partial nonstructural protein 3 and one JEV sequence encoding the complete envelope protein were identified. There was variability in the viral titers of the newly isolated virus JEV-China/YN2016-1 of different passage viruses. The newly identified Zika virus gene from ZIKV-China/YN2016-1 was an Asian genotype and shared the highest nucleotide sequence identity (97.1%) with a ZIKV sequence from Thailand isolated in 2004. Phylogenetic analysis of ZIKV-China/YN2016-1 and ZIKV-China/YN2016-2 with known Flavivirus genes indicated that ZIKV has propagated in Yunnan province, China.