Palmitoylation at Residue C221 of Japanese Encephalitis Virus NS2A Protein Contributes to Viral Replication Efficiency and Virulence.

Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.

Two Enterococcus faecium Isolates Demonstrated Modulating Effects on the Dysbiosis of Mice Gut Microbiota Induced by Antibiotic Treatment.

Broad-spectrum antibiotics are frequently used to treat bacteria-induced infections, but the overuse of antibiotics may induce the gut microbiota dysbiosis and disrupt gastrointestinal tract function. Probiotics can be applied to restore disturbed gut microbiota and repair abnormal intestinal metabolism. In the present study, two strains of Enterococcus faecium (named DC-K7 and DC-K9) were isolated and characterized from the fecal samples of infant dogs. The genomic features of E. faecium DC-K7 and DC-K9 were analyzed, the carbohydrate-active enzyme (CAZyme)-encoding genes were predicted, and their abilities to produce short-chain fatty acids (SCFAs) were investigated. The bacteriocin-encoding genes in the genome sequences of E. faecium DC-K7 and DC-K9 were analyzed, and the gene cluster of Enterolysin-A, which encoded a 401-amino-acid peptide, was predicted. Moreover, the modulating effects of E. faecium DC-K7 and DC-K9 on the gut microbiota dysbiosis induced by antibiotics were analyzed. The current results demonstrated that oral administrations of E. faecium DC-K7 and DC-K9 could enhance the relative abundances of beneficial microbes and decrease the relative abundances of harmful microbes. Therefore, the isolated E. faecium DC-K7 and DC-K9 were proven to be able to alter the gut microbiota dysbiosis induced by antibiotic treatment.

Various mobile genetic elements carrying optrA in Enterococcus faecium and Enterococcus faecalis isolates from swine within the same farm.

OBJECTIVES: In this study, the distribution of the oxazolidinone/phenicol resistance gene optrA and the mobile genetic elements involved in its dissemination were analysed among enterococcal isolates from a farrow-to-finish swine farm. METHODS: Enterococcus faecium and Enterococcus faecalis isolates were obtained from all pig production stages in the farm. The optrA-carrying E. faecium and E. faecalis isolates were subjected to PFGE and antimicrobial susceptibility testing. Complete sequences of the genetically unrelated optrA-carrying E. faecium and E. faecalis isolates were determined using Illumina HiSeq and MinION platforms. RESULTS: The optrA gene was present in 12.2% (23/188) of the E. faecium and E. faecalis isolates, most of which originated from nursery and finishing stages. The 23 optrA-positive Enterococcus isolates represented 15 PFGE types. WGS of representative isolates of the 15 PFGE types showed that optrA was carried by diverse genetic elements either located in the chromosomal DNA or on plasmids. A novel optrA-bearing genetic element was identified on two distinct multi-resistance plasmids from E. faecium. Two new hybrid plasmids carrying several resistance genes were found in two E. faecalis isolates. pC25-1-like plasmids and chromosomally integrated Tn6674 and Tn6823-like transposons were prevalent in the remaining Enterococcus isolates. CONCLUSIONS: The gene optrA was found in genetically unrelated E. faecium and E. faecalis isolates from the same farm. Analysis of the genetic contexts of optrA suggested that horizontal transfer including different plasmids and transposons played a key role in the dissemination of optrA in this farm.

p53 promotes ZDHHC1-mediated IFITM3 palmitoylation to inhibit Japanese encephalitis virus replication.

The tumor suppressor p53 as an innate antiviral regulator contributes to restricting Japanese encephalitis virus (JEV) replication, but the mechanism is still unclear. The interferon-induced transmembrane protein 3 (IFITM3) is an intrinsic barrier to a range of virus infection, whether IFITM3 is responsible for the p53-mediated anti-JEV response remains elusive. Here, we found that IFITM3 significantly inhibited JEV replication in a protein-palmitoylation-dependent manner and incorporated into JEV virions to diminish the infectivity of progeny viruses. Palmitoylation was also indispensible for keeping IFITM3 from lysosomal degradation to maintain its protein stability. p53 up-regulated IFITM3 expression at the protein level via enhancing IFITM3 palmitoylation. Screening of palmitoyltransferases revealed that zinc finger DHHC domain-containing protein 1 (ZDHHC1) was transcriptionally up-regulated by p53, and consequently ZDHHC1 interacted with IFITM3 to promote its palmitoylation and stability. Knockdown of IFITM3 significantly impaired the inhibitory role of ZDHHC1 on JEV replication. Meanwhile, knockdown of either ZDHHC1 or IFITM3 expression also compromised the p53-mediated anti-JEV effect. Interestingly, JEV reduced p53 expression to impair ZDHHC1 mediated IFITM3 palmitoylation for viral evasion. Our data suggest the existence of a previously unrecognized p53-ZDHHC1-IFITM3 regulatory pathway with an essential role in restricting JEV infection and provide a novel insight into JEV-host interaction.

NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/ß induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings.

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and ß production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and ß induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and ß production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and ß induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and ß production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and ß production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.

Comparative analysis of the pulmonary microbiome in healthy and diseased pigs.

The lungs possess an effective antimicrobial system and a strong ability to eliminate microorganisms in healthy organisms, and were once considered sterile. With the development of culture-independent sequencing technology, the richness and diversity of porcine lung microbiota have been gaining attention. In order to study the relationship between lung microbiota and porcine respiratory disease complex (PRDC), the lung microbiota in healthy and diseased swine bronchoalveolar lavage fluids were analyzed and compared using the Illumina MiSeq sequencing platform. The predominant microbial communities of healthy and diseased swine were similar at the phylum level, mainly composed of Proteobacteria, Firmicutes, Tenericutes, and Bacteroidetes. However, the bacterial taxonomic communities of healthy and diseased swine differed at the genus level. The higher relative abundances of Lactococcus, Enterococcus, Staphylococcus, and Lactobacillus genera in healthy swine might provide more benefits for lung health, while the enhanced richness of Streptococcus, Haemophilus, Pasteurella, and Bordetella genera in diseased swine might be closely related to pathogen invasion and the occurrence of respiratory disease. In conclusion, the observed differences in the richness and diversity of lung microbiota can provide novel insights into their relationship with PRDC. Analyses of swine lung microbiota communities might produce an effective strategy for the control and prevention of respiratory tract infections.

Mosquito Defensins Enhance Japanese Encephalitis Virus Infection by Facilitating Virus Adsorption and Entry within the Mosquito.

Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death, and disability. Although the Culex mosquito is the primary vector of JEV, little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via the defensin/lipoprotein receptor-related protein 2 (LRP2) axis. Mosquito defensin bound the ED III domain of the viral envelope (E) protein and directly mediated efficient virus adsorption on the target cell surface; the receptor LRP2, which is expressed on the cell surface, affected defensin-dependent adsorption. As a result, mosquito defensin enhanced JEV infection in the salivary gland, increasing the possibility of viral transmission by mosquitoes. These findings demonstrate the novel role of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.IMPORTANCE In this study, we observed the complex roles of mosquito defensin in JEV infection; mosquito defensin exhibited a weak antiviral effect but strongly enhanced binding. In the latter, defensin directly binds the ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. Together, our results indicate that mosquito defensin plays an important role in facilitating JEV infection and potential transmission.

Downregulation of miR-296-3p by highly pathogenic porcine reproductive and respiratory syndrome virus activates the IRF1/TNF-α signaling axis in porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV, species Betaarterivirus suid 1 or 2) is a major pathogen affecting pigs on farms throughout the world. miR-296-3p is a multifunctional microRNA involved in the regulation of the inflammatory response in mice and humans. However, little is known about the biological functions of miR-296-3p in pigs. In this study, we used a highly pathogenic PRRSV-2 (species Betaarterivirus suid 2) strain to show that PRRSV infection robustly downregulates the expression of miR-296-3p in porcine alveolar macrophages (PAMs). Furthermore, we demonstrated that overexpression of miR-296-3p increases the replication of highly pathogenic (HP)-PRRSV in PAMs. Notably, the overexpression of miR-296-3p inhibited the induction of TNF-α, even with increased viral replication, compared with that in the HP-PRRSV-infected control group. We also demonstrated that miR-296-3p targets IRF1-facilitated viral infection and modulates the expression of TNF-α in PAMs during HP-PRRSV infection and that IRF1 regulates the expression of TNF-α by activating the TNF promoter via IRF1 response elements. In summary, these findings show that HP-PRRSV infection activates the IRF1/TNF-α signaling axis in PAMs by downregulating host miR-296-3p. This extends our understanding of the inflammatory response induced by HP-PRRSV infection.

Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay.

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.

Acidity/Alkalinity of Japanese Encephalitis Virus E Protein Residue 138 Alters Neurovirulence in Mice.

The Japanese encephalitis virus (JEV) envelope (E) protein, as one of mediators of virus entry into host cells, plays a critical role in determining virulence. The Glu-to-Lys mutation of residue 138 in E protein (E138) plays an important role in attenuating JEV vaccine strain SA14-14-2. However, it is not clear how E138 attenuates JEV. Here, we demonstrate that the Glu-to-Arg mutation of E138 also determines the attenuation of JEV strain 10S3. Likewise, for its parent strain (HEN0701), a virulence strain, the mutations of E138 are responsible for virulence alteration. Furthermore, we demonstrated that mutations of alkaline residues in E138 contributed to the attenuation of neurovirulence; in contrast, mutations of acidic residues enhanced the neurovirulence of the strains. Moreover, acidity in residue E47 had a similar effect on neurovirulence. Furthermore, the alkaline E138 residue enhanced susceptibility to heparin inhibition in vitro and limited JEV diffusion in mouse brain. These results suggest that the acidity/alkalinity of the E138 residue plays an important role in neurovirulence determination.IMPORTANCE The E protein is the only glycoprotein in mature JEV, and it plays an important role in viral neurovirulence. E protein mutations attenuate JEV neurovirulence through unclear mechanisms. Here, we discovered that E138 is a predominant determinant of JEV neurovirulence. We demonstrated that the alkalinity/acidity of E138 determines JEV neurovirulence. These data contribute to the characterization of the E protein and the rational development of novel JEV vaccines.

T Cell-Restricted Notch Signaling Contributes to Pulmonary Th1 and Th2 Immunity during Cryptococcus neoformans Infection.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen but the cell signaling pathways that drive T cell responses regulating antifungal immunity are incompletely understood. Notch is a key signaling pathway regulating T cell development, and differentiation and functional responses of mature T cells in the periphery. The targeting of Notch signaling within T cells has been proposed as a potential treatment for alloimmune and autoimmune disorders, but it is unknown whether disturbances to T cell immunity may render these patients vulnerable to fungal infections. To elucidate the role of Notch signaling during fungal infections, we infected mice expressing the pan-Notch inhibitor dominant negative mastermind-like within mature T cells with C. neoformans Inhibition of T cell-restricted Notch signaling increased fungal burdens in the lungs and CNS, diminished pulmonary leukocyte recruitment, and simultaneously impaired Th1 and Th2 responses. Pulmonary leukocyte cultures from T cell Notch-deprived mice produced less IFN-γ, IL-5, and IL-13 than wild-type cells. This correlated with lower frequencies of IFN-γ-, IL-5-, and IL-13-producing CD4+ T cells, reduced expression of Th1 and Th2 associated transcription factors, Tbet and GATA3, and reduced production of IFN-γ by CD8+ T cells. In contrast, Th17 responses were largely unaffected by Notch signaling. The changes in T cell responses corresponded with impaired macrophage activation and reduced leukocyte accumulation, leading to diminished fungal control. These results identify Notch signaling as a previously unappreciated regulator of Th1 and Th2 immunity and an important element of antifungal defenses against cryptococcal infection and CNS dissemination.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating ß2-Microglobulin Transcription.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the ß2-microglobulin (ß2M) gene (B2M) and reduced cellular levels of ß2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of ß2M expression. Exogenous expression of Nsp4 downregulated ß2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.

Chemokine receptor antagonist block inflammation and therapy Japanese encephalitis virus infection in mouse model.

Japanese encephalitis (JE) is a viral encephalitis disease caused by infection with the Japanese encephalitis virus (JEV). The virus can cross the blood-brain barrier and cause death or long-term sequela in infected humans or animals. In this study, we first investigated the distribution of JEV infection in brain and further analyzed the dynamic change in inflammation related genes, chemokines, as well as pathological characteristics. Results demonstrated that CCR2 and CCR5 antagonist could significantly inhibit the inflammation. The mice treated with CCR2 and CCR5 antagonists had a higher survival rate between 60% and 70%, respectively. In summary, our study thoroughly illustrated the characteristics of the dynamic change in inflammation related genes and chemokines induced by JEV infection. We further indicated that CCR5 and CCR2 are potential targets for treatment of JE.

Proteomic Analysis of the Secretome of Porcine Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is characterized by reproductive failure and respiratory disorders. The secretome of PRRSV-infected porcine alveolar macrophages (PAMs), which are the primary target cells of PRRSV, was analyzed by label-free quantitative proteomics to gain a profile of proteins secreted during PRRSV infection. A total of 95 secreted proteins with differentially expressed levels between PRRSV- and mock-infected PAMs was screened. Among these, the expression levels of 49 and 46 proteins were up-regulated and down-regulated, respectively, in PRRSV-infected cell supernatants, as compared with mock-infected cell supernatants. Bioinformatic analysis revealed that the differentially expressed proteins were enriched in several signaling pathways related to the immune and inflammatory responses, such as the Toll-like receptor signaling pathway and NF-kappa B signaling pathway, and involved in a great diversity of biological processes, such as protein binding and localization, as well as immune effector processes. In addition, PRRSV-infected cell supernatants induced significant expression of inflammatory cytokines in vascular endothelial cells. These findings suggest that the secreted proteins play potential roles in the host immune and inflammatory responses as well as PRRSV replication, thereby providing new insights into cell-to-cell communication during PRRSV infection.

Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.

A case report of pulmonary tritrichomonosis in a pig.

BACKGROUND: Tritrichomonads like porcine Tritrichomonas foetus (previously named Tritrichomonas suis), can commensally live in nasal cavity of pigs, but it is rare to cause pulmonary tritrichomonosis. CASE PRESENTATION: A 40-day-old piglet was presented for persistent labor breathing and diagnosed with parasite infections in the lung by analysis of bronchoalveolar lavage (BAL) under microscope. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infection. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. FINDINGS: Here, we report a case of pulmonary tritrichomonosis in a pig. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infections. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. CONCLUSION: Our results demonstrate that tritrichomonads like porcine Tritrichomonas foetus can cause lung infections of pigs and reveal that next-generation sequencing is potential to identify rare diseases like pulmonary tritrichomonosis in clinical.

Antimicrobial Resistance of Campylobacter Species Isolated from Broilers in Live Bird Markets in Shanghai, China.

This study was conducted to determine the prevalence of antimicrobial resistance in Campylobacter spp. isolates from broilers in live bird markets (LBMs). A total of 209 Campylobacter spp. isolates (84 Campylobacter jejuni; 125 Campylobacter coli) were recovered from 364 broiler cecum samples collected from five LBMs in Shanghai, China. Minimum inhibitory concentrations of 13 antimicrobials were determined using agar dilution method. More than 96% of the Campylobacter spp. isolates were resistant to quinolones and tetracyclines. A high prevalence of macrolide resistance (erythromycin, 84.0%; azithromycin, 80.8%) was observed in C. coli, but not in C. jejuni (erythromycin, 6.0%; azithromycin, 2.4%). C. coli also showed significantly higher resistance than C. jejuni to clindamycin, gentamicin, and kanamycin. In contrast, C. coli isolates had lower resistance to florfenicol than the C. jejuni isolates. The majority of the C. jejuni (88.1%) and C. coli (97.6%) isolates exhibited multidrug resistance (MDR) to three or more classes of antimicrobials. All of the 208 ciprofloxacin-resistant Campylobacter spp. isolates were positive for the C257T mutation of the gyrA gene. In addition, the tet(O) gene was identified in all of the 202 doxycycline-resistant Campylobacter spp. isolates. Furthermore, 75.7% and 20.4% of the 103 azithromycin-resistant Campylobacter spp. isolates were positive for the A2075G mutation of the 23S rRNA gene and the presence of the erm(B) gene, respectively. Moreover, the cat gene was found in 14.3% (8/56) and 76.8% (73/95) of the chloramphenicol-resistant C. jejuni and C. coli isolates, respectively. To the best of our knowledge, this is the first report of the prevalence of antimicrobial resistance among Campylobacter spp. isolates originating from LBMs. The high prevalence of MDR Campylobacter spp. isolates in LBMs highlights the need to implement efficient intervention measures to control not only Campylobacter contamination in LBMs but also dissemination of antimicrobial resistance among Campylobacter spp. in poultry production.

Identification of a novel bufavirus in domestic pigs by a viral metagenomic approach.

Bufavirus is a single-stranded DNA virus belonging to the genus Protoparvovirus. This study reports the identification and characterization of a porcine bufavirus by a metagenomic approach, and a limited epidemiology investigation of bufavirus in six swine farms. A comparative genome analysis showed a similarity of 93 % to a Hungarian porcine bufavirus. Bayesian and maximum-likelihood analyses of genome sequences showed a close relationship of porcine bufaviruses to human and monkey bufaviruses. Molecular dating of the most recent common ancestors supported a recent introduction of bufaviruses into human and pig populations, respectively. A real-time PCR method was developed to screen 60 faecal samples for the porcine bufavirus DNA, and eight positive samples were found in two neighbouring farms, suggesting a relatively low prevalence (13.3 %). No direct transmission of porcine bufaviruses between two neighbouring farms was found, suggesting that bufaviruses may have spread widely in different geographical regions.