Abstract
<title>Abstract</title> <p> Background The tick microbiome comprises diverse microorganisms, many of which may affect the transmission of medically important tick-borne pathogens and elicit immune responses in tick vectors. As interactions during simultaneous viral and bacterial infections remain poorly characterized, we aimed to determine the growth and replication dynamics of select Gram-negative ( <italic>Escherichia coli</italic> ), Gram-positive ( <italic>Enterococcus faecalis</italic> and <italic>Enterococcus faecium</italic> ) bacteria, and tick-borne encephalitis virus (TBEV) in coinfected ticks, to assess the responses of the Toll, IMD, and JAK/STAT signaling pathways. Methods Laboratory-bred nymphal <italic>I. ricinus</italic> ticks were intrarectally coinfected with standard strains of <italic>E. coli</italic> , <italic>E. faecalis</italic> , or <italic>E. faecium</italic> and TBEV strain Hypr. Surface-decontaminated unfed ticks at 1, 3, and 7 dpi were used for (i) culturing on selection agar media and (ii) qPCR analysis of viral load and immune markers of Toll ( <italic>toll 4</italic> , <italic>spätzle 1</italic> , <italic>myd88</italic> , <italic>cactus 2</italic> , <italic>dorsal</italic> ), IMD ( <italic>uev1a</italic> , <italic>bendless</italic> , <italic>caspar</italic> , <italic>relish</italic> ), and JAK/STAT ( <italic>domeless</italic> , <italic>stat</italic> ) signaling pathways. Results Bacterial growth in dormant ticks incubated for up to 7 days, with or without TBEV coinfection, was comparable. However, analyses revealed significantly reduced growth of <italic>E. faecium</italic> compared with <italic>E. faecalis</italic> , whereas <italic>E. coli</italic> multiplied at a significantly higher rate. On the other hand, while <italic>E. coli</italic> coinfection promoted viral growth on day 7; <italic>E. faecium</italic> had a significantly negative impact on viral replication rate. Interestingly, <italic>E. faecalis</italic> did not affect TBEV replication. Immune responses in TBEV + <italic>E. coli</italic> - or TBEV + <italic>E. faecium</italic> -coinfected ticks were characterized by stimulation of all tested pathways, with the most increased gene expression levels of <italic>cactus 2</italic> and <italic>myd88</italic> (Toll), <italic>caspar</italic> and <italic>relish</italic> (IMD), as well as <italic>domeless</italic> (JAK/STAT). In contrast, TBEV + <italic>E. faecalis</italic> coinfected ticks responded primarily through elevated <italic>domeless</italic> expression of JAK/STAT. Standalone virus infection in ticks triggered primarily the IMD pathway, reflected in increased <italic>caspar</italic> and significant <italic>relish</italic> upregulation, which served as one of the central antimicrobial defenses. Conclusions Our findings reveal species-specific microbial influences on viral replication and tick immune induction, advancing understanding of complex interactions shaping tick-borne pathogen dynamics. </p>