Emergence and adaptive evolution of influenza D virus.

As a novel member of the Orthomyxoviridae, influenza D virus (IDV) was firstly isolated from swine. However, cattle were found to serve as its primary reservoir. The study of IDV emergence can shed light into the dynamics of zoonotic infections and interspecies transmission. Although there is an increasing number of strains and sequenced IDV strains, their origin, epidemiology and evolutionary dynamics remain unclear. In this study, we reconstruct the diversity and evolutionary dynamics of IDVs. Molecular detection of swine tissue samples shows that six IDV positive samples were identified in the Eastern China. Phylogenetic analyses suggest three major IDV lineages designated as D/Japan, D/OK and D/660 as well as intermediate lineages. IDVs show strong association with geographical location indicating a high level of local transmission, which suggests IDVs tend to establish a local lineage of in situ evolution. In addition, the D/OK lineage widely circulates in swine in Eastern China, and all of the Chinese virus isolates form a distinct sub-clade (D/China sub-lineage). Furthermore, we identified important amino acids in the HEF gene under positive selection that might affect its receptor binding cavity relevant for its broader cell tropism. The combined results highlight that more attention should be paid to the potential threat of IDV to livestock and farming in China.

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2.

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

Comparative analysis of MicroRNA expression in dog lungs infected with the H3N2 and H5N1 canine influenza viruses.

MicroRNAs, a class of noncoding RNAs 18 to 23 nucleotides (nt) in length, play critical roles in a wide variety of biological processes. The objective of this study was to examine differences in microRNA expression profiles derived from the lungs of beagle dogs infected with the avian-origin H3N2 canine influenza virus (CIV) or the highly pathogenic avian influenza (HPAI) H5N1 virus (canine-origin isolation strain). After dogs were infected with H3N2 or H5N1, microRNA expression in the lungs was assessed using a deep-sequencing approach. To identify the roles of microRNAs in viral pathogenicity and the host immune response, microRNA target genes were predicted, and their functions were analyzed using bioinformatics software. A total of 229 microRNAs were upregulated in the H5N1 infection group compared with those in the H3N2 infection group, and 166 microRNAs were downregulated. MicroRNA target genes in the H5N1 group were more significantly involved in metabolic pathways, such as glycerolipid metabolism and glycerophospholipid metabolism, than those in the H3N2 group. The inhibition of metabolic pathways may lead to appetite loss, weight loss and weakened immunity. Moreover, miR-485, miR-144, miR-133b, miR-4859-5p, miR-6902-3p, miR-7638, miR-1307-3p and miR-1346 were significantly altered microRNAs that potentially led to the inhibition of innate immune pathways and the heightened pathogenicity of H5N1 compared with that of H3N2 in dogs. This study deepens our understanding of the complex relationships among microRNAs, the influenza virus-mediated immune response and immune injury in dogs.

Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Phylogenetic analysis and molecular characteristics of 17 porcine reproductive and respiratory syndrome virus isolates in Southern China from 2010 to 2011.

We analyzed the complete genomic sequences of 17 porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Southern China obtained between 2010 and 2011 and found that four of seven isolates from 2011 were closely related to the JXA1-R strain (vaccine virus of JXA1). This close relationship between field isolates and China domestic vaccine viruses has not been reported to date. The occurrence of vaccine-like viruses potentially creates a threat for the pig breeding industry and brings difficulties for control of this disease.

Regulatory functional role of NLRP3 inflammasome during Mycoplasma hyopneumoniae infection in swine.

Mycoplasma hyopneumoniae causes enzootic pneumonia, a highly contagious respiratory disease in swine that causes significant economic losses worldwide. It is unknown whether the nucleotide oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome regulates the immune response in swine during M. hyopneumoniae infection. The current study utilized an in vivo swine model of M. hyopneumoniae infection to investigate the regulatory functional role of the NLRP3 inflammasome during M. hyopneumoniae infection. Notable histopathological alterations were observed in M. hyopneumoniae-infected swine tissues, which were associated with an inflammatory response and disease progression. Swine M. hyopneumoniae infection was associated with an increase in the expression of the NLRP3 inflammasome, which stimulated pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 18, and interleukin 1 beta (IL-1ß). The impact of the NLRP3 inhibitor, MCC950 on NLRP3 and pro-inflammatory cytokines in M. hyopneumoniae-infected swine was examined to investigate the relationship between the NLRP3 inflammasome and M. hyopneumoniae infection. Taken together, our findings provide strong evidence that the NLRP3 inflammasome plays a critical regulatory functional role in M. hyopneumoniae infection in swine.

Our study highlights the importance of controlling the innate immune defense against respiratory mycoplasma invasion to suppress mycoplasma growth and minimize lung tissue damage. Using an in vivo swine model, we investigated the regulatory functional role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome during acute Mycoplasma hyopneumoniae infection. Furthermore, we also found that NLRP3 expression levels have the potential to serve as a novel diagnostic marker for detecting M. hyopneumoniae infection in the respiratory tract of pigs. The NLRP3 inhibitor, MCC950, was used to investigate how NLRP3 inhibition affects the expression of inflammatory cytokines, and it was found that the NLRP3 inhibitor significantly reduced the mRNA and protein expression of NLRP3, indicating its specific targeting of the NLRP3 inflammasome during M. hyopneumoniae infection in swine. The findings suggest that MCC950 is a promising therapeutic option for treating NLRP3-related disorders, including porcine enzootic pneumonia.

Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China.

To understand the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in South China, we collected 231 clinical samples from pigs with suspected PRRSV infection in Guangdong between 2007 and 2009. We found that 74 of 231 samples were positive by RT-PCR. The PCR products of the ORF5 gene of 35 isolates from different farms were sequenced and their DNA sequences were compared to 23 other PRRSV isolates in the GenBank. We found that the nucleotide similarity among all South China isolates ranged from 87.6% to 100%, and all belonged to the North American genotype. Most of them were classified into subgenotype I, but the rest mapped to subgenotypes III, V or VI. Those in subgenotypes I and III were found to be highly variable in the primary neutralising epitope (PNE) with a specific amino acid mutation (F39/L39→I39), and a few isolates in subgenotypes I and III isolates also had a mutation at L41 (L41→S41). PRRSV isolates in subgenotypes III, V and VI had less potential glycosylation sites than those in subgenotype I. Our data contribute to the understanding of molecular variation of PRRSV in South China.

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.

Genotyping and zoonotic potential of Enterocytozoon bieneusi in cattle farmed in Hainan Province, the southernmost region of China.

Enterocytozoon bieneusi is an intestinal pathogen that infects a wide range of species, including humans. Cattle constitute an important host for E. bieneusi; however, there is a scarcity of information on the prevalence and genotyping of E. bieneusi in cattle in the Hainan Province of China. In this study, PCR analysis of 314 fecal samples from cattle in six cities of Hainan was performed for genotype identification. The average prevalence of E. bieneusi in these animals was 9.9% (31/314), and ranged from 0.0% (0/12) to 20.5% (8/39). Five known genotypes - EbpC (n = 14), BEB4 (n = 12), J (n = 2), I (n = 1), and CHG5 (n = 1) - and a novel genotype: HNC-I (n = 1) - were identified. Genotypes EbpC and HNC-I were placed in zoonotic Group 1, and the remaining four genotypes (BEB4, J, I, and CHG5) were placed in Group 2. Since 93.5% of the genotypes found in the cattle (29/31) (EbpC, BEB4, J, and I) have previously been found in humans, these genotypes are probably involved in the transmission of microsporidiosis to humans.

TITLE: Génotypage et potentiel zoonotique d'Enterocytozoon bieneusi chez les bovins élevés dans la province de Hainan, la région la plus au sud de la Chine. ABSTRACT: Enterocytozoon bieneusi est un pathogène intestinal qui infecte un large éventail d'espèces, y compris les humains. Le bétail constitue un hôte important pour E. bieneusi, mais les informations sur la prévalence et le génotypage d'E. bieneusi chez les bovins de la province de Hainan en Chine sont rares. Dans cette étude, une analyse PCR de 314 échantillons fécaux provenant de bovins dans six villes de Hainan a été réalisée pour l'identification du génotype. La prévalence moyenne d'E. bieneusi chez ces animaux était de 9,9 % (31/314), et variait de 0,0 % (0/12) à 20,5 % (8/39). Cinq génotypes connus, EbpC (n = 14), BEB4 (n = 12), J (n = 2), I (n = 1) et CHG5 (n = 1), et un nouveau génotype, HNC-I (n = 1), ont été identifiés. Les génotypes EbpC et HNC-I sont placés dans le groupe zoonotique 1, et les quatre génotypes restants (BEB4, J, I et CHG5) sont placés dans le groupe 2. Puisque 93,5 % (29/31) (EbpC, BEB4, J et I) des génotypes trouvés chez les bovins ont déjà été trouvés chez l'homme, ces génotypes sont probablement impliqués dans la transmission de la microsporidiose à l'homme.

Genotype identification and phylogenetic analysis of Enterocytozoon bieneusi in farmed black goats (Capra hircus) from China's Hainan Province.

Enterocytozoon bieneusi is an important pathogen commonly found in humans and animals. Farmed animals with close contact to humans are important hosts of E. bieneusi. The role of goats in the transmission of E. bieneusi, however, remains unclear. In this study, 341 fresh fecal samples of black goats were collected from five locations in Hainan Province, China. Enterocytozoon bieneusi was identified and genotyped by sequences of the internal transcribed spacer (ITS) region. Phylogenetic analysis was performed by constructing a neighbor-joining tree of the ITS gene sequences. The average prevalence of E. bieneusi in black goats was 24.0% (82/341) with rates ranging from 6.3% (4/63) to 37.2% (32/86) across the locations (χ2 = 17.252, p < 0.01). Eight genotypes of E. bieneusi were identified, including six known genotypes: CHG5 (n = 47); CHG3 (n = 23); CHG2 (n = 4); CM21 (n = 3); D (n = 2); and AHG1 (n = 1), and two novel genotypes termed HNG-I (n = 1) and HNG-II (n = 1). In the phylogenetic tree, genotype D was clustered into Group 1 and the other identified genotypes were included in Group 2. This represents the first report identifying E. bieneusi in black goats from Hainan Province, with a high prevalence and wide occurrence demonstrated. The two new genotypes identified provide additional insights into the genotypic variations in E. bieneusi. Due to the small percentage of zoonotic genotypes in these animals, there is minimal risk of zoonotic transmission of E. bieneusi.

TITLE: Identification du génotype et analyse phylogénétique d'Enterocytozoon bieneusi chez des chèvres noires (Capra hircus) de la province de Hainan, en Chine. ABSTRACT: Enterocytozoon bieneusi est un agent pathogène important que l'on trouve couramment chez l'homme et les animaux. Les animaux d'élevage, en contact étroit avec l'homme, sont des hôtes importants d'E. bieneusi. Le rôle des chèvres dans la transmission d'E. bieneusi reste toutefois incertain. Dans cette étude, 341 échantillons de fèces fraîches de chèvres noires ont été prélevés dans cinq sites de la province de Hainan, en Chine. Enterocytozoon bieneusi a été identifié et génotypé par des séquences de la région de l'espaceur interne transcrit (ITS). L'analyse phylogénétique a été réalisée en construisant un arbre de jonction voisine des séquences du gène ITS. La prévalence moyenne d'E. bieneusi chez les chèvres noires était de 24,0 % (82/341), avec des taux allant de 6,3 % (4/63) à 37,2 % (32/86) dans tous les sites (χ2 = 17,252, p < 0,01). Huit génotypes d'E. bieneusi ont été identifiés, dont six génotypes connus: CHG5 (n = 47) ; CHG3 (n = 23) ; CHG2 (n = 4) ; CM21 (n = 3) ; D (n = 2) ; AHG1 (n = 1) et deux nouveaux génotypes appelés HNG-I (n = 1) et HNG-II (n = 1). Dans l'arbre phylogénétique, le génotype D appartenait au groupe 1 et les autres génotypes identifiés étaient inclus dans le groupe 2. Il s'agit du premier rapport identifiant E. bieneusi chez des chèvres noires de la province de Hainan, avec une prévalence élevée et une occurrence étendue. Les deux nouveaux génotypes identifiés fournissent des informations supplémentaires sur les variations génotypiques chez E. bieneusi. En raison du faible pourcentage de génotypes zoonotiques chez ces animaux, le risque de transmission zoonotique d'E. bieneusi est minime.

The NS1 protein of H5N6 feline influenza virus inhibits feline beta interferon response by preventing NF-κB and IRF3 activation.

Despite the apparent lack of a feline influenza virus lineage, cats are susceptible to infection by influenza A viruses. Here, we characterized in vitro A/feline/Guangdong/1/2015, an H5N6 avian influenza virus recently isolated from cats. A/feline/Guangdong/1/2015 replicated to high titers and caused CPE in feline kidney cells. We determined that infection with A/feline/Guangdong/1/2015 did not activate the IFN-ß promoter, but inhibited it by blocking the activation of NF-κB and IRF3. We also determined that the viral NS1 protein mediated the block, and that the dsRNA binding domain of NS1 was essential to perform this function. In contrast to treatment after infection, cells pretreated with IFN-ß suppressed viral replication. Our findings provide an example of an H5N6 influenza virus suppressing IFN production, which might be associated with interspecies transmission of avian influenza viruses to cats.

Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research.

Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-ß, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-ß activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks.

Identification of an H6N6 swine influenza virus in southern China.

This is the first report of avian-like H6N6 swine influenza virus from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic ducks. Serological surveillance suggested there had been sporadic H6 swine influenza infections in this area. Continuing study is required to determine if this virus could be established in the swine population and pose potential threats to public health.