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Murine coronavirus (CoV) is a beta-CoV that infects mice by binding to carcinoembryonic antigen-related cell adhesion molecule 1. Intraperitoneal infection with the murine CoV strain JHM (JHMV) induces acute mild hepatitis in mice. While both innate and acquired immune responses play a significant role in the protection against murine CoV infection in mice, CD8 + cytotoxic T lymphocytes (CTLs) and interferon-γ are essential for viral clearance in JHMV-induced hepatitis. In addition, CoVs are characterized by high diversity, caused by mutations, recombination, and gene gain/loss. 25V16G is an immune-escape JHMV variant, which lacks a dominant CTL epitope. By evading immune responses, 25V16G establishes persistent infections, leading to granulomatous serositis in interferon-γ-deficient mice. These examples of CoV-associated pathogenesis in mice might provide useful information on other CoV infections, including coronavirus disease 2019 (COVID-19).

Bats are the second diversity species of mammals and widely distributed in the world. They are thought to be reservoir and vectors of zoonotic pathogens. However, there is scarce report of the evidence of pathogenic bacteria kept in bats. The precise knowledge of the pathogenic bacteria in bat microbiota is important for zoonosis control. Thus, metagenomic analysis targeting the V3-V4 region of the 16S rRNA of the rectal microbiota in Rousettus amplexicaudatus was performed using high throughput sequencing. The results revealed that 103 genera of bacteria including Camplyobacter were detected. Campylobacter was second predominant genus, and Campylobacter coli and Campylobacter jejuni were identified in microbiome of R. amplexicaudatus. Campylobacteriosis is one of the serious bacterial diarrhea in human, and the most often implicated species as the causative agent of campylobacteriosis is C. jejuni. Therefore, we investigated the prevalence of C. jejuni in 91 wild bats with PCR. As a result of PCR assay targeted on 16S-23S intergenic spacer, partial genome of C. jejuni was detected only in five R. amplexicaudatus. This is the first report that C. jejuni was detected in bat rectal swab samples. C. jejuni is the most common cause of campylobacteriosis in humans, transmitted through water and contact with livestock animals. This result indicated that R. amplexicaudatus may be a carrier of C. jejuni.

In this study, cDNA of Toll-like receptors (TLR) 3, 7 and 9 were synthesized and completely sequenced. The coding regions of cDNA for bat TLR3, TLR7 and TLR9 were 2,718, 3,150 and 3,090 bp in length, respectively. The open reading frames encoded 905, 1,049 and 1,029 amino acids for TLR3, TLR7 and TLR9, respectively. The nucleotide sequences, predicted amino acid sequences and predicted domain structures of the three bat TLRs had high homology with those of other mammals. In addition, the expression profiles of each TLR in main organs were analyzed. Expression of TLR3 was highest in the liver, whereas the expressions of TLR7 and TLR9 were highest in the spleen.

This is the first report on the cDNA sequences of bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 p40, and tumor necrosis factor (TNF)-alpha. The cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-alpha comprise 459, 405, 624, 537, 990, and 699 base pairs respectively. Moreover, each of the cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-alpha contain a single open reading frames encoding 152, 134, 207, 178, 329, and 232 amino acids, respectively. The comparison of bat cytokines with Perrissodactyla (horse), Carnivora (dog and cat), and Cetartiodactyla (cattle and pig) orthologs revealed a high degree of homology. Although the N-terminal amino acids and cysteine residues are highly conserved in each mature cytokine, the deduced N-linked glycosylation sites vary across species.

After intraperitoneal infection with mouse hepatitis virus, strain JHM (JHMV), JHMV replicated in the spleen of C57BL/6 mice for a few days but cleared within a week. The acute viral clearance coincided with moderate expansion of CD8+T cells and modest expansion of CD4+T cells, and was impaired moderately in mice depleted of CD8+T cells and completely in mice depleted of both CD4+ and CD8+T cell subsets. Flow cytometric analysis showed that expression of cell surface markers on the spleen T cells changed during JHMV infection. CD8+T cells expressing increased amounts of CD11a, CD43, CD44 and CD49d, and those expressing decreased levels of T cell receptor alpha beta, CD8, CD45RB and L-selectin were expanded in the spleen after JHMV infection. However, they did not express CD11b, CD25 or NK1.1. They used highly heterogenous V beta chains for their T cell receptors. In addition to CD11ahighCD8+T cells, CD11ahighCD4+T cells were detected transiently after JHMV infection. The virus-specific cytotoxic T lymphocyte (CTL) activity was observed in both CD4+ and CD8+ spleen T cells from mice 7 days post-infection. The present study shows the dynamics of CD8+ and CD4+T cells in the spleen during JHMV infection in mice and suggests that CD11ahighT cells may be involved in JHMV clearance in vivo because their appearance was temporally correlated with T cell-mediated viral clearance in vivo and antiviral CTL activity in vitro.

The mechanism of CD11ahighCD8+ T cell induction after mouse hepatitis virus infection, which has been suggested to play a key role in the elimination of infectious virus from the spleen in C57BL/6 mice, was studied. In CD4+ T cell-depleted mice, CD11ahighCD8+ T cells were induced in the spleen and spleen cells showed virus-specific cytotoxic T lymphocyte activity after mouse hepatitis virus infection. The same results were obtained in adult thymectomized mice. These results indicate that CD11ahighCD8+ T cells can be generated after mouse hepatitis virus infection in the absence of either CD4+ T cells or the thymus.