The biological function of a nonstructural protein, NSm, of Akabane virus (AKAV) is unknown. In this study, we generated a series of NSm deletion mutant viruses by reverse genetics and compared their phenotypes. The mutant in which the NSm coding region was almost completely deleted could not be rescued, suggesting that NSm plays a role in virus replication. We next generated mutant viruses possessing various partial deletions in NSm and identified several regions critical for virus infectivity. All rescued mutant viruses produced smaller plaques and grew inefficiently in cell culture, compared to the wild-type virus. Interestingly, although the pathogenicity of NSm deletion mutant viruses varied in mice depending on their deletion regions and sizes, more than half the mice died following infection with any mutant virus and the dead mice exhibited encephalitis as in wild-type virus-inoculated mice, indicating their neuroinvasiveness. Abundant viral antigens were detected in the brain tissues of dead mice, whereas appreciable antigen was not observed in those of surviving mice, suggesting a correlation between virus growth rate in the brain and neuropathogenicity in mice. We conclude that NSm affects AKAV replication in vitro as well as in vivo and that it may function as a virulence factor.

This study aims to determine the microbiological profile and risk factors associated with antimicrobial-resistant bacteria in canine severe corneal ulcers. Thirty-two corneal and conjunctival swabs were collected from dogs with diagnosed severe corneal ulcers that presented to Prasu-Arthorn veterinary teaching hospital in Nakhon Pathom, Thailand from June 2015 to June 2016. Microorganisms were identified by means of genotypic and phenotypic approaches. Of 32 ulcers sampled, 26 (81.3%) yielded culturable microorganisms with 24 bacterial isolates and 7 fungal isolates. The most commonly isolated bacteria were Staphylococcus spp. (45.8%, 11/24) and Pseudomonas aeruginosa (20.8%, 5/24). Out of 11 staphylococcal isolates identified, 10 carried the mecA gene providing methicillin resistance. The extended-spectrum β-lactamase (ESBL) encoding genes blaCTX-M and blaVEB-1 were found in an Acinetobacter lwoffii isolate, and blaSHV was found in a P. aeruginosa isolate. Based on the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint criteria, minimum inhibitory concentrations values showed that all bacteria, except for staphylococci, were susceptible to current ophthalmic antibiotics. More than 50% of staphylococci were resistant to all generations of fluoroquinolones and fusidic acid. Chloramphenicol was highly active against staphylococci (81.3% susceptible). The width (P=0.02) and the depth (P=0.04) of ulcers predicted greater risk of yielding resistant bacteria. The identification of antimicrobial-resistant bacteria prompts practitioners to be prudent when choosing ophthalmic antibiotics for severe corneal ulcers.

Emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from dogs with cutaneous and wound infections has significantly impacted veterinary medicine. This study aimed to isolate S. pseudintermedius from canine pyoderma and investigate the effects of ethanolic extracts of Piper betle (PB), P. sarmentosum (PS), and P. nigrum (PN) on the bacterial growth and biofilm formation of S. pseudintermedius and MRSP. Of the isolated 152 isolates, 53 were identified as S. pseudintermedius using polymerase chain reaction, and 10 isolates (6.58%) were identified as MRSP based on the presence of mecA. Based on phenotype, 90% of MRSPs were multidrug-resistant. All MRSP had moderate (10%, 1/10) and strong (90%, 9/10) biofilm production ability. PB extracts were the most effective in inhibiting planktonic cells, and the minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC50) was 256 µg/mL (256-1024 µg/mL) for S. pseudintermedius isolates and 512 µg/mL (256-1024 µg/mL) for MRSP isolates. The MIC90 for S. pseudintermedius and MRSP was 512 µg/mL. In XTT assay, PB at 4× MIC showed an inhibition rate of 39.66-68.90% and 45.58-59.13% for S. pseudintermedius and MRSP, respectively, in inhibiting biofilm formation. For PB at 8× MIC, the inhibition rates for S. pseudintermedius and MRSP were 50.74-81.66% and 59.57-78.33%, respectively. Further, 18 compounds were identified in PB using gas chromatography-mass spectrometry, and hydroxychavicol (36.02%) was the major constituent. These results indicated that PB could inhibit bacteria growth of and biofilm formation by S. pseudintermedius and MRSP isolated from canine pyoderma in a concentration-dependent manner. Therefore, PB is a potential candidate for the treatment of MRSP infection and biofilm formation in veterinary medicine.

Fifty-two bats captured during July 2008 in the Philippines were tested by reverse transcription-PCR to detect bat coronavirus (CoV) RNA. The overall prevalence of virus RNA was 55.8%. We found 2 groups of sequences that belonged to group 1 (genus Alphacoronavirus) and group 2 (genus Betacoronavirus) CoVs. Phylogenetic analysis of the RNA-dependent RNA polymerase gene showed that groups 1 and 2 CoVs were similar to Bat-CoV/China/A515/2005 (95% nt sequence identity) and Bat-CoV/HKU9-1/China/2007 (83% identity), respectively. To propagate group 2 CoVs obtained from a lesser dog-faced fruit bat (Cynopterus brachyotis), we administered intestine samples orally to Leschenault rousette bats (Rousettus leschenaulti) maintained in our laboratory. After virus replication in the bats was confirmed, an additional passage of the virus was made in Leschenault rousette bats, and bat pathogenesis was investigated. Fruit bats infected with virus did not show clinical signs of infection.

The entry mechanisms of Akabane virus (AKAV), Bunyaviridae family, have not yet been determined. In this study, chemical inhibitors were used to analyze endocytic mechanisms during AKAV infection of mammalian cell lines. The analyses using drug treatments followed by quantitative measurement of viral RNA and N protein revealed that AKAV enters non-bovine-derived cell lines (Vero, HmLu-1 and BHK cells) in a manner indicative of clathrin endocytosis. By contrast, AKAV infection in bovine-derived cell lines (LB9.K and MDBK cells) is independent of this pathway. Further analyses indicated that AKAV entry into bovine cell lines involves a non-clathrin, non-caveolae endocytic pathway that is dependent on dynamin. We conclude that although both cell types require a low pH for AKAV penetration, AKAV utilizes alternative entry pathways into mammalian cell lines.

The AIM2 inflammasome is activated by DNA, leading to caspase-1 activation and release of pro-inflammatory cytokines interleukin 1β (IL-1β) and IL-18, which are critical mediators in host innate immune responses against various pathogens. Some viruses employ strategies to counteract inflammasome-mediated induction of pro-inflammatory cytokines, but their in vivo relevance is less well understood. Here we show that the herpes simplex virus 1 (HSV-1) tegument protein VP22 inhibits AIM2-dependent inflammasome activation. VP22 interacts with AIM2 and prevents its oligomerization, an initial step in AIM2 inflammasome activation. A mutant virus lacking VP22 (HSV-1ΔVP22) activates AIM2 and induces IL-1β and IL-18 secretion, but these responses are lost in the absence of AIM2. Additionally, HSV-1ΔVP22 infection results in diminished viral yields in vivo, but HSV-1ΔVP22 replication is largely restored in AIM2-deficient mice. Collectively, these findings reveal a mechanism of HSV-1 evasion of the host immune response that enables efficient viral replication in vivo.