Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library.

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

HVT-vectored H7 vaccine protects chickens from lethal infection with the highly pathogenic H7N9 Avian influenza virus.

Since mid-2016, the highly pathogenic H7N9 subtype avian influenza virus (AIV) has threatened both public health and the poultry industry. Although a vaccination strategy has been deemed imperative to manage the virus, the most commonly used inactivated vaccines today are susceptible to interference from maternal antibodies and associated with an over-reliance on humoral immunity. In response, we developed a recombination vaccine with the herpesvirus of turkeys (HVT) as the vector to squeeze HPAI H7N9 and assessed its protective efficiency in immunized chickens. By inserting an enhanced green fluorescent protein (EGFP) expression cassette (i.e., MCMV+EGFP+SV40 polyA) into the HVT065 and HVT066 positions, we obtained the recombinant HVT expressing EGFP (i.e., rHVT-EGFP). Electroporation and EGFP tags improved the efficiency of transfection compared with transfection using expression plasmids without any fluorescent labeling and traditional liposomes. Using limiting dilution analysis and ultrasonic cell disruption techniques, we screened and purified a cell-bound herpes virus based on rHVT-EGFP and consequently constructed a recombinant HVT expressing the hemagglutinin (HA) of H7N9 (i.e., rHVT-H7HA), which was able to proliferate similarly to the parental strain, stably pass for at least 15 generations in vitro, and replicate stably in multiple organs in vivo. After chickens were immunized with rHVT-H7HA, the average antibody titers reached up to 3 log2 at 35 d post-vaccination and remained stable. Those results suggest that rHVT-H7HA can protect chickens against H7N9 with a dose-independent immune protection rate of 90% and significantly reduce the lung virus titer 4 d post-challenge.

Multiplex and visual detection of African Swine Fever Virus (ASFV) based on Hive-Chip and direct loop-mediated isothermal amplification.

African swine fever is caused by African swine fever virus (ASFV), and has a mortality rate approaching 100%. It has already caused tremendous economy lost around the world. Without effective vaccine, rapid and accurate on-site detection plays an indispensable role in controlling outbreaks. Herein, by combining Hive-Chip and direct loop-mediated isothermal amplification (LAMP), we establish a multiplex and visual detection platform. LAMP primers targeting five ASFV genes (B646L, B962L, C717R, D1133L, and G1340L) were designed and pre-fixed in Hive-Chip. On-chip LAMP showed the limits of detection (LOD) of ASFV synthetic DNAs and mock samples are 30 and 50 copies per microliter, respectively, and there is no cross-reaction among the target genes. The overall performance of our platform is comparable to that of the commercial kits. From sample preparation to results readout, the entire process takes less than 70 min. Multiplex detection of real samples of ASFV and other swine viruses further demonstrates the high sensitivity and specificity of Hive-Chip. Overall, our platform provides a promising option for on-site, fast and accurate detection of ASFV.

[Antiviral effects of the combination of glycyrrhizin and ribavirin against influenza A H1N1 virus infection in vivo].

Ribavirin is a broad-spectrum antiviral agent and glycyrrhizin has activities of anti-inflammation, immunoregulation and anti-viral infections. To enhance antiviral efficacy and weaken side-effects of ribavirin, antiviral effects of the combination of glycyrrhizin and ribavirin were studied in the present study. Firstly, a mouse model of viral pneumonia was established by inoculation of influenza H1N1 virus. Protective effects of glycyrrhizin and ribavirin used alone or in combination against H1N1 virus infection in mice were evaluated based on the survival rate, lung index and virus titer in lungs of mice. Results showed that the combination of glycyrrhizin and ribavirin significantly inhibited the lung consolidation with a 36% inhibition ratio on the lung swell of infected mice. The combination of the two drugs exhibited synergetic effects on survival of infected mice. The combination of 50 mg · kg(-1) · d(-1) glycyrrhizin and 40 mg · kg(-1) · d(-1) ribavirin resulted a 100% protection for infected mice with a synergetic value of 36, which was significantly higher than the control group and each drug alone. This combination also resulted a significant drop of lung virus titer (P < 0.01), as well as inhibition on the production of proinflammatory cytokines IL-6 (P < 0.01), TNF-α (P < 0.01) and IL-1ß (P < 0.05) induced by virus infection compared to the control. The treatment of ribavirin plus glycyrrhizin was more effective in influenza A infection in mice than either compound used alone, which suggested a potential clinical value of the combination of the two agents.

Complete genome characterization and phylogenetic analysis of three distinct buffalo-origin PCV2 isolates from China.

Complete genome characterization of porcine circovirus type 2 (PCV2) for bovid origins was still unclear in China. Therefore, in this study, PCV2 full-length genome of buffalo-origin was amplified and analyzed using PCR, DNAStar and MEGA 5.1. Genome size of three distinct PCV2 strains (buffalo1, buffalo2 and buffalo3) was 1767 bp (48.56% G+C), 1767 bp (48.67% G+C) and 1768 bp (48.08% G+C), respectively. At the nucleotide level, their identity varied from 95% to 96% for complete genome, from 97% to 97.8% for ORF1, and from 90.6% to 94.4% for ORF2. At the amino acid level, their identity varied from 98.7% to 99% for ORF1, and from 88% to 94.9% for ORF2. Online Blast analysis showed that buffalo1, buffalo2 and buffalo3 had highest nucleotide identity (varied from 99.77% to 99.83%) with porcine-origin PCV2 strains. Moreover, in the phylogenetic tree, they were divided into three different clusters and belonged to the worldwide accepted genotypes of PCV2b, PCV2c and PCV2a, respectively. To summarize, this study first recorded complete genome information of PCV2 for non-porcine origins in China.

Comparative analysis of microRNAs from the lungs and trachea of dogs (Canis familiaris) infected with canine influenza virus.

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs of 18-22-nucleotides in length that regulate gene expression at the post-transcriptional level. The objective of this study was to examine the differences in the miRNA expression profiles of the lungs and trachea of beagle dogs infected with canine influenza virus (CIV). Total RNA was isolated from lung and trachea tissues of beagle dogs infected and non-infected with H3N2 CIV at 4 dpi. A total of 41,512,315 and 39,107,475 reads were obtained from the lung and trachea, respectively. Out of a total 288 dog miRNAs available in miRBase, 227 and 236 miRNAs were detected in the infected (Fg) and the non-infected lungs (Fc), respectively, whereas 242 miRNAs were detected in both the infected (Qg) and the non-infected trachea (Qc). From these, 34 and 45 miRNAs were differentially expressed in the lungs and trachea between the infected and non-infected dogs, respectively. More miRNAs were highly expressed in the non-infected tissues than in the infected tissues. miR-143 was the most abundantly expressed miRNA in the four samples, followed by let-7. In total, 252, 234, 196 and 235 novel miRNAs were identified in the Fc, Fg, Qc, and Qg groups, respectively. To our knowledge, this is the first study examining the miRNA gene expression in CIV infected dogs using the Solexa sequencing approach. We have revealed the existence of a large number miRNAs that are affected by CIV infection as well as identified some potentially new miRNAs. These findings will help us better understand the host-CIV interaction and its relationship to pathogenesis, as well as contribute to the prevention and control of CIV.

Amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1.

BACKGROUND: Few drugs are currently licensed to treat influenza A infection, and new therapies are needed, especially for highly pathogenic strains. Traditional medicinal plants, such as Lycoris radiata, are a potential source of new antiviral agents. OBJECTIVE: To test 15 Amaryllidaceae alkaloids isolated from the bulbs of L. radiata in vitro for antiviral activities against influenza virus type A, A/Chicken/GuangDong/178/2004 (H5N1, 178). METHODS: Antiviral activities of the compounds were tested in time-of-addition assays, hemagglutination inhibition (HI) assays, neuraminidase (NA) activity assays, and viral entry inhibition assays using H5N1-HIV pseudoviruses. Effects of the compounds on localization and activity of the viral ribonucleoprotein (RNP) were determined by immunofluorescence and an RNP minigenome assay, respectively. RESULTS: Among the alkaloids, lycorine (AA1), hippeastrine (AA2), hemanthamine (AA3) and 11-hydroxy vittatine (AA4) exhibited antiviral activities, with EC90 values of 0·52, 82·07, 4·15, and 13·45 µm, respectively. These compounds did not affect the function of the outer membrane proteins or the viral entry process and viral RNP activity. As AA1 and AA3 exhibited stronger antiviral activities, they were further analyzed. Intracellular nucleoprotein (NP) localization showed that AA1 and AA3 inhibited the RNP complex in the nucleus at an early stage of a single-round and multi-round of replication. CONCLUSION: Four Amaryllidaceae alkaloids were first determined that could exert anti-influenza activities after virus entry into cells. Furthermore, AA1 and AA3 could inhibit nuclear-to-cytoplasmic export of the RNP complex of virus replication. Thus, these compounds may be developed further as anti-influenza drug candidates.

Tissue distribution of sialic acid-linked influenza virus receptors in beagle dogs.

Reports of influenza A virus infections in dogs has received considerable attention from veterinarians, virologists, and epidemiologists. Interaction between influenza viral hemagglutinin and cell oligosaccharides containing sialic acid residues results in infection. Sialic acids have an α-2,3-linkage to the penultimate galactose in the avian influenza virus receptor and an α-2,6-linkage in the human receptor. To date, there are no detailed data on the tissue distribution or histological features of either type of sialic acid-linked influenza virus receptors in beagle dogs, which are common laboratory animals and pets. We conducted the current study to visualize the in situ tissue distribution of both sialic acid-linked influenza virus receptors in various organs of beagle dogs using Maackia amurensis lectin II and Sambucus nigra agglutinin. Both α-2,3- and α-2,6-sialic acid-linked receptors were detected in the endothelial cells of the respiratory tract and other organs. Endothelial cells of most gastrointestinal organs were negative for α-2,3-sialic acid-linked receptors in the dogs. Our results suggested that these canine organs may be affected by influenza virus infection. The findings from our study will also help evaluate the occurrence and development of influenza virus infections in dogs.

Complete Genome Sequence of a Novel Avian-Like H3N2 Swine Influenza Virus Discovered in Southern China.

We report here the complete genomic sequence of a novel avian-like H3N2 swine influenza virus containing an H5N1 highly pathogenic avian influenza virus segment that was obtained from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic aquatic birds. The sequence information provided herein suggests that continuing study is required to determine if this virus can be established in the swine population and pose potential threats to public health.

Complete genome sequence of an avian-like H4N8 swine influenza virus discovered in southern China.

We report here the complete genomic sequence of an avian-like H4N8 swine influenza virus containing an H5N1 avian influenza virus segment from swine in southern China. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the Asia lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4N8 influenza virus to domestic pigs under natural conditions.

Na+/H+ exchanger 1 gene expression in tissues of yellow chicken.

The Na(+)/H(+) exchanger 1 (NHE1) transmembrane protein regulates intracellular pH, cell survival, cell growth, cell differentiation and plays a critical role in the progression of some diseases, including the pathogenesis of J avian leukosis. The chicken is an ideal model to study the function of NHE1 because it has developed highly efficient Na(+)-absorptive mechanisms in its small and large intestines. To date, there has been no detailed expression analysis to determine NHE1 expression in various tissues of the chicken. We determined the mRNA and protein expression levels of avian NHE1 by real-time quantitative PCR and immunohistochemical analysis. NHE1 mRNA was detected in all chicken tissues examined. Protein expression levels varied widely among tissues and did not always correlate with mRNA expression. Determining the mRNA and protein of NHE1 expression patterns in chicken should help to delineate the NHE1 role in different tissues and its contribution to physiological and pathological processes. These data provide the basis for examining the distinct function of chicken NHE1 compared with its mammalian counterpart.

Genetic characterization of H1N2 influenza a virus isolated from sick pigs in Southern China in 2010.

In China H3N2 and H1N1 swine influenza viruses have been circulating for many years. In January 2010, before swine were infected with foot and mouth disease in Guangdong, some pigs have shown flu-like symptoms: cough, sneeze, runny nose and fever. We collected the nasopharyngeal swab of all sick pigs as much as possible. One subtype H1N2 influenza viruses were isolated from the pig population. The complete genome of one isolate, designated A/swine/Guangdong/1/2010(H1N2), was sequenced and compared with sequences available in GenBank. The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. HA, NP, M and NS were shown to be closely to swine origin. PB2 and PA were close to avian origin, but NA and PB1were close to human origin. It is a result of a multiple reassortment event. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially before the emergence of highly pathogenic FMDs in pigs in Guangdong.

Effect of compounds on the purification and antibody preparation of the extracellular domain fragment of the receptor CD163.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) has been acknowledged as one of the most important agents affecting swine. The scavenger receptor CD163 is one of the important entry mediators for PRRSV. RESULTS: The tD4 and tD5 CD163 genes were amplified, and the PCR products were cloned into pET-28a(+) (designated pET-28a-tD4 and pET-28a-tD5, respectively). The plasmids pET-28a-tD4 and pET-28a-tD5 were then transformed into the E. coli BL21 (DE3) strain and expressed by adding 1 mmol/L of isopropyl-beta-D-thiogalactopyranoside. The proteins were highly expressed in the supernatant from the tD4- and tD5-producing cells that were incubated with a binding buffer containing the following compounds: ß-mercaptoethanol, urea, Tween 20, glycerol, and SDS, while they were rarely expressed in the supernatant from the tD4- and tD5-producing cells that were incubated with binding buffer without the compounds. The tD4 and tD5 proteins were purified, and BALB/c mice were immunized with the purified proteins. Western blotting analysis showed that the tD4 and tD5 proteins were capable of reacting with tD5 antibodies; the titer of both the tD4 and tD5 antiserums was 1:160 against the tD5 protein, as shown by ELISA. CONCLUSIONS: These studies provide a new way for the purification of proteins expressed in inclusion bodies and the preparation of the corresponding antibodies.

Development and strategies of cell-culture technology for influenza vaccine.

Influenza is a pandemic contagious disease and causes human deaths and huge economic destruction of poultry in the world. In order to control and prevent influenza, mainly type A, influenza vaccine for human and poultry were available since the 1940s and 1920s, respectively. In the development of vaccine production, influenza viruses were cultured originally from chicken embryos to anchorage-dependent cell lines, such as MDCK and Vero. The anchorage-independent lines have also been used to produce influenza virus, such as PER.C6 and engineering modified MDCK and Vero. During the process of influenza vaccine production, the common problem faced by all producers is how to improve the titer of influenza virus. This paper focuses on the developments of cell culture for influenza virus vaccine production, limitations of cell culture, and relative strategies for improvement virus yields in cell-culture systems.

Isolation and Phylogenetic Analysis of H1N1 Swine Influenza Virus from Sick Pigs in Southern China.

Two swine influenza (SI) H1N1 virus was isolated from a pig during a severe outbreak of respiratory disease in south China. The two H1N1 influenza viruses were classical SI virus. A/swine/Guangdong/L6/09 is classical SI virus of recent years, which is of the main SI virus in China. Howere, A/swine/Guangdong/L3/09 was closet to A/swine/Iowa/1931, which was the first isolated SI virus and had demonstrated significant pathogenicity in animal models. The results of phylogenetic analysis of A/swine/Guangdong/L3/09 showed a close relationship with the 1918 pandemic virus. The results suggested that the previous SI virus appeared again. Whether, it brought a new pandemic to pigs deserves more attention.

[Cloning and analysis of full-length genes of a H9N2 subtype avian influenza virus isolated from Guangdong].

Eight full-length genes of an avian influenza virus Chinese isolate of H9N2 subtype, A/Chicken/Guangdong/HL/2006 (H9N2) (abbreviated as Ck/GD/HL/06), were amplified by RT-PCR, including the 5' and 3' non-coding region. All the genes were cloned and sequenced. The phylogenetic analysis results showed the HA gene of Ck/GD/HL/06 was located in the same phylogenetic clade as Dk/HK/Y280/97 (H9N2), while the Dk/HK/Y280/97-like viruses had been predominately isolated from chickens in mainland China. After the analysis of glycosylation sites and receptor-binding sites in the HA, it was shown that the HA of Ck/GD/HL/06 exhibited the common feature of H9 subtype avian influenza virus isolated from China, but the leucine (Leu) residue at the amino acid position 226 indicated the potential of binding with SA alpha,2-6 receptor. The three internal genes of Ck/GD/HL/06 (PB1, PA and NP) had the highest nucleotide identity with A/Viet Nam/1203/2004 (abbreviated A/VN/1203/04) isolate, which was shown to be transmitted from chickens to human and caused lethal infection in human. No analogous H9N2 strains was reported in previous studies. Based on the high similarity of Ck/GD/HL/06 three genes to A/VN/1203/04, it was suggested that the possibility of generating new highly pathogenic H5N1 AIVs by recombination was worthy of our attention. Further studies should be needed for molecular epidemiologic surveillance of H9N2 AIV in the south China for a long time.

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.