Diversity of porcine reproductive and respiratory syndrome virus in Shandong, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant pathogens in swine industry of China. To study infection and genetic variation of PRRSV, 637 tissue samples were collected from diseased pigs in Shandong, and then subjected to detection of PRRSV. The nsp2 and ORF5 genes were sequenced for investigation of variations and phylogenetic analysis. The results showed that positive rate of PRRSV was 9.58% in the collected samples. Phylogenetic analysis of GP5 showed that these strains were clustered into two lineages (1 and 8) indicating different genotypes of PRRSV were circulating in Shandong province. Meanwhile, sequence analysis Of nsp2 showed that the PRRSV strains with 30 amino acids deletions were dominant. Moreover, novel pattern of recombination/deletion and insertion in nsp2 was observed in these strains, indicating that novel PRRSV strains with different patterns of deletions or insertions in nsp2 are emerging in China. All the results suggested that continuous surveillance of PRRSV in China is warranted. Keywords: PRRSV; GP5; nsp2; genetic analysis; Shandong.

Pathological analysis and genetic characterization of the first outbreak H5N8 subtype avian influenza virus isolated from wild swan in Shandong, China.

In January 2021, an outbreak of H5N8 subtype avian influenza was identified from wild mute swans (Cygnus olor) in an overwintering site located within Yellow River Delta Nature Reserve in Shandong, China, for the first time. We sequenced the hemagglutinin (HA) and neuraminidase (NA) genome segments of the virus, named A/mute swan/Shandong/1/2021 (H5N8), and explored the genetic characterization and comparative phylogenetic analysis to track its origin. The wild swans prior to death showed nervous signs accompanied by a twisted neck. The major pathological changes of the heart, lung, trachea, kidney, spleen, and glandular stomach were hemorrhage, and extensive necrotic lesions in the liver and pancreas were also observed. Further analysis of the HA and NA phylogenetic trees generated by H5N8 strains isolated worldwide revealed that the isolate has a close genetic relationship with the H5N8 subtype avian influenza virus (AIV) recently isolated in Korea and Japan from 2020 to 2021, suggesting the potential role of mute swans in the global dissemination of the H5N8 subtype AIVs along the migration routes. Therefore, enhanced active surveillance in wild and domestic birds is required to monitor the introduction and spread of the H5N8 subtype AIV by migratory birds in order to decrease the risk of H5N8 outbreaks.

Genetic characterization, mechanisms and dissemination risk of antibiotic resistance of multidrug-resistant Rothia nasimurium.

Rothia nasimurium is part of the commensal flora of humans and other animals and has recently received increasing attention for its multidrug-resistance (MDR) and pathogenicity. Currently, no systematic reports characterize the genetics, mechanisms, and dissemination risks of antibiotic resistance in MDR R. nasimurium. Here, we present the first report outlining a MDR strain of R. nasimurium, E1706032a, isolated from ducks exhibiting clinical sickness. Phylogenetic analysis indicates that E1706032a mostly likely originated in the commensal bacteria of Amazona aestiva in Florida. E1706032a is resistant to beta-lactams, aminoglycosides, macrolides, sulfonamides, fluoroquinolones, rifamycins, tetracyclines, lincosamides and chloramphenicol. Genetic sequences related to drug resistance were detected, including resistance genes (aac(6')-Ib, ant(3″)-Ia, sul1, dfrA7, erm(X)), efflux pumps (tetZ, qacEΔ1, cmx, phosphate ABC transporter ATP-binding protein), and resistance-related proteins (hydrolase of the metallo-beta-lactamase (MBLs), mycinamicin resistance protein (myrA), DNA-directed RNA polymerase subunit beta (rpoB) variants, etc). E1706032a carries an IS481-like element, IS5564 and IS6-like elements, and IS6100 along with several novel transposases of the IS3 family. E1706032a also carries the class 1 integron gene IntI1, which is downstream adjacent to the gene cassettes aac(6')-Ib, tetZ, dfrA27, ant(3″)-Ia, qacEΔ1, sul1, cmx and upstream adjacent to gene tnpA of IS6100. Genetic analysis suggests that E1706032a carries wide antibiotic resistance and dissemination potential through movable elements and thus has the potential to cause difficult-to-treat infections in animals and humans. The dissemination of E1706032a from parrots in Florida to ducks in eastern China indicates a cross-regional public health infection risk that should be evaluated for risk of global spreading.

Recombinant Lactobacillus acidophilus expressing S1 and S2 domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), which is characterized by a high mortality rate in piglets. Since 2012, a remarkable growth in PED outbreaks occurred in many pig farms in China, landing a heavy blow on the pig industry. In order to develop a new effective vaccine for the current PEDV, oral vaccines were generated by transferring eukaryotic expression recombinant plasmids carrying the S1 and S2 (antigenic sites of the S protein) epitopes of PEDV into a swine-origin Lactobacillus acidophilus (L. acidophilus). After oral immunization of the BALB/c mice, higher levels of anti-PEDV specific IgG and SIgA antibodies and cellular immune responses were detected in mice orally administered with the recombinant L. acidophilus-S1 compared to the L. acidophilus-S2. Furthermore, L. acidophilus-S1 was used to inoculate the pregnant sows orally and the results showed that the recombinant L. acidophilus-S1 could elicit a specific systemic and mucosal immune response. In summary, our study demonstrated that oral immunization with L. acidophilus-S1 could improve the humoral and mucosal immune levels in sows and would be a promising candidate vaccine against PEDV infection in piglets.

Swine Promyelocytic Leukemia Isoform II Inhibits Pseudorabies Virus Infection by Suppressing Viral Gene Transcription in Promyelocytic Leukemia Nuclear Bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against alphaherpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to alphaherpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoforms sPML-II and -IIa, not sPML-I and -IVa, expressed in a sPML knockout cells inhibit PRV infection. Both the unique 7b region of sPML-II and the sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residues 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of alphaherpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an antiviral function but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.

1-{2-[2-(1H-Benzimidazol-1-yl)eth-oxy]eth-yl}-1H-benzimidazol-3-ium hexa-fluoro-phosphate.

In the title salt, C(18)H(19)N(4)O(+)·PF(6) (-), the dihedral angle between the benzimidazolium and benzimidazole ring systems is 16.24 (2)°. In the cation, a π-π inter-action is observed between the imidazolium ring and the benzene ring of the benzimidazole ring system [centroid-centroid distance = 3.5713 (11) Å]. The PF(6) (-) ion is disordered over two sites, with occupancies of 0.895 (2) and 0.105 (2). In the crystal structure, pairs of N-H⋯N hydrogen bonds link the cations, forming centrosymmetric dimers. The dimers are linked via π-π inter-actions [centroid-centroid distance = 3.5606 (11) Å]. In addition, C-H⋯F hydrogen bonds are observed.

Construction of recombinant pseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virus and its safety and immunogenicity.

The bivalent genetic engineering vaccine of Japanese encephalitis (JE) and Aujeszkj disease (AD) was developed to provide a novel approach to prevent and control these two diseases. NS1 gene of Japanese encephalitis virus (JEV) SA14-14-2 strain was produced by reverse transcriptase-mediated PCR (RT-PCR) and was cloned into vector pUSK to form recombinant plasmid (designed as pUSK-NS1). A co-transfection experiment was performed in porcine kidney (PK-15) cells with pUSK-NS1 and the genome of the vector virus (PRV TK(-)/gG(-)/LacZ(+) mutant). By plaque purification, PCR detection and southern hybridization, recombinant pseudorabies virus (PRV) expressing NS1 protein of JEV was acquired and named TK(-)/gG(-)/NS1(+). Western blot analysis and ELISA demonstrated the NS1 protein expression. To evaluate the recombinant virus's potential application, we characterized the safety and immune responses in Balb/c mice and swine. The safety test indicated that, when receiving the recombinant virus at a concentration of 10(6.0)pfu, no virulence of the recombinant virus to the mice, piglets and pregnant sows was observed. The vaccinated animals could acquire protective immunity against lethal challenge of the virulent PRV Ea strain and develop a good humoral and cellular immune response against JEV. The above results revealed that the recombinant virus could be a suitable candidate vaccine strain for developing a novel genetic vaccine to combat pseudorabies and Japanese encephalitis in the pig industry.