ObjectiveTo compare the histological staining performance of four various tissue fixatives, including a self-developed fixative, for preparing paraffin sections of rat eyeball tissue. MethodsTwenty 5-week-old male Sprague-Dawley (SD) rats were randomly divided into 4 groups (n=5 per group). After anesthesia by intraperitoneal injection of Zoletil™ 50 at a dose of 45 mg/kg body weight, the rats were euthanized by exsanguination via the abdominal aorta. Bilateral eyeballs were carefully extracted intact and fixed for 72 h in 10% formaldehyde fixative, glutaraldehyde-formaldehyde mixed fixative, Davidson's fixative, and self-developed fixative, respectively. After fixation, the eyeballs were longitudinally sectioned along the optic nerve, with the portions containing the optic nerve preserved. The tissues were then dehydrated, embedded, and sectioned. Following hematoxylin and eosin (HE) staining, histological staining quality was compared among ocular structures including the cornea, lens, and retina. ResultsThe overall appearance of rat eyeballs showed marked shrinkage in the 10% formaldehyde group and the glutaraldehyde-formaldehyde group, whereas the eye morphology remained round and intact in the modified Davidson's fixative group and self-developed fixative group. The corneal stroma exhibited obvious rupture, and the cells were arranged in folded arrangement in the modified Davidson's fixative group, 10% formaldehyde group, and self-developed fixative group. In contrast, the corneal cells in the glutaraldehyde-formaldehyde group were neatly arranged, showing no rupture or folding, and exhibited clear staining, indicating the highest quality of corneal sectioning among all groups. In the 10% formaldehyde group, cracks were observed in the equatorial and cortical regions of the lens, but the lens fiber structure remained intact. The lenses in the modified Davidson's fixative group showed extensive rupture and detachment. The glutaraldehyde-formaldehyde group displayed only slight cracks at the equator. In the self-developed fixative group, mild red folding was limited to the peripheral lens region, with the remaining structures intact and unbroken. These findings indicated that the glutaraldehyde-formaldehyde and self-developed fixative groups achieved the best lens sectioning quality. The retina was severely detached from the choroid/sclera layer, with extensive rupture of each cellular layer in the 10% formaldehyde fixative group. In the glutaraldehyde-formaldehyde group, partial detachment between the retina and choroid/sclera was observed. The outer plexiform layer and nerve fiber layer showed separation with edema, while the cells in all layers were neatly arranged. In both the Davidson's fixative and the self-developed fixative groups, the retina remained intact without rupture, and no structural separation was observed in any layer. Both demonstrated advantages in preserving the integrity and orderly arrangement of all retinal layers; however, the self-developed fixative group exhibited greater contrast. ConclusionThe choice of fixative significantly affects the morphological preservation of various structures in the rat eye. The self-developed fixative demonstrates the best overall performance in maintaining overall eye morphology, the structural integrity of the lens, and retinal adhesion. For studies focusing solely on the corneal structure, the glutaraldehyde-formaldehyde mixed fixative is recommended. The 10% formaldehyde fixative exhibits unsatisfactory fixation effects for all the aforementioned ocular structures and is not recommended for detailed morphological studies of eyeball tissues.

Objective To isolate the Japanese encephalitis virus carried by Culex tritaeniorhynchus in Dongchuan District of Yunnan Province and analyze its molecular characteristics, so as to provide insights into the prevention and control of Japanese encephalitis in Yunnan Province. Methods Mosquito specimens were collected using mosquito-trapping lamps from pig farms in Batang Village and Xiaoxin Village, Dongchuan District, Kunming City, Yunnan Province in July 2016, and the mosquito species was identified according to the mosquito morphology. Then, 60 to 100 mosquitoes of each species served as a group and were ground. Baby hamster kidney-21 (BHK-21) cells and Aedes albopictus clone C6/36 cells were used for virus isolation, and positive isolates were identified using flavivirus primers. The positive isolates were amplified using reverse transcription polymerase chain reaction (RT-PCR) assay with 15 pairs of specific primers covering the full length of the genotype I Japanese encephalitis virus, and DNA sequence assembly was performed using the software SeqMan in the DNASTAR package. The obtained sequences were aligned with the complete sequences of 38 Japanese encephalitis virus downloaded from the GenBank with the software MegAlign, and the nucleotide and amino acid homology analyses of the obtained sequences were performed. The difference in amino acid sites was analyzed with the software GeneDoc, and phylogenetic trees were created based on the sequences of the coding region and E protein of the isolated Japanese encephalitis virus with the software Mega X. In addition, the secondary and tertiary structures of the E protein of the Japanese encephalitis virus were predicted using the online tool SOPMA and the software Swiss-Model. Results A total of 5 820 mosquitoes were collected and 3 843 Cx. tritaeniorhynchus (66.03%) were identified according to the mosquito morphology. A positive virus isolate, termed YNDC55-33, was isolated from Cx. tritaeniorhynchoides following batches of virus isolation from mosquito specimens, and cytopathic effect was observed following inoculation into BHK-21 and C6/36 cells. The YNDC55-33 virus isolate was successfully amplified with the flavivirus primes, and a long sequence containing 300 nucleotides was obtained. Following sequence alignment using the BLAST tool, the sequence of the YNDC55-33 virus isolate had high homology with that of the genotype I Japanese encephalitis virus. A long sequence with 10 845 nucleotides in length, which encoded 3 432 amino acids, was obtained by splicing the full sequence of the YNDC55-33 virus isolate. Phylogenetic analysis based on the whole-genome sequence and E gene sequence of the YNDC55-33 virus isolate showed that the new YNDC55-33 virus isolate was most closely related to the genotype I Guizhou isolate (GenBank accession number: HM366552), with nucleotide homology of 98.5% and amino acid homology of 99.4%, and the YNDC55-33 virus isolate shared 97.96% ± 0.33% nucleotide homology and 99.35% ± 0.08% amino acid homology with other genotype I Japanese encephalitis virus isolates, and < 90% nucleotide homology and < 98% amino acid homology with other genotypes of Japanese encephalitis virus. The YNDC55-33 virus isolate and the live attenuated virus vaccine candidate SA14-14-2 isolate differed at 16 amino acid sites on E gene, and 7 out of 8 key amino acid sites related to neurovirulence. The secondary and tertiary structures of the E protein of the YNDC55-33 virus isolate were predicted to be characterized by random coils. Conclusions A genotype I Japanese encephalitis virus was isolated from Cx. tritaeniorhynchus in Dongchuan District, Kunming City. This virus isolate and the live attenuated virus vaccine candidate SA14-14-2 isolate does not differ at antigenic epitopes-related key amino acid sites, and the major protein structure of the virus isolate is random coils. This study adds new data for the epidemiological distribution of Japanese encephalitis virus in Yunnan Province, which may provide insights into the prevention and control of Japanese encephalitis in the province.