Black carp ATG16L1 negatively regulates STING-mediated antiviral innate immune response.

The precise control of interferon (IFN) production is indispensable for the host to eliminate invading viruses and maintain a homeostatic state. In mammals, stimulator of interferon genes (STING) is a prominent adaptor involved in antiviral immune signaling pathways. However, the regulatory mechanism of piscine STING has not been thoroughly investigated. Here, we report that autophagy related 16 like 1 (bcATG16L1) of black carp (Mylopharyngodon piceus) is a negative regulator in black carp STING (bcSTING)-mediated signaling pathway. Initially, we substantiated that knockdown of bcATG16L1 increased the transcription of IFN and ISGs and enhanced the antiviral activity of the host cells. Subsequently, we identified that bcATG16L1 inhibited the bcSTING-mediated IFN promoter activation and proved that bcATG16L1 suppressed bcSTING-mediated antiviral ability. Furthermore, we revealed that bcATG16L1 interacted with bcSTING and the two proteins shared a similar subcellular distribution. Mechanically, we found that bcATG16L1 attenuated the oligomerization of bcSTING, which was a key step for bcSTING activation. Taken together, our results indicate that bcATG16L1 interacts with bcSTING, dampens the oligomerization of bcSTING, and negatively regulates bcSTING-mediated antiviral activity.

STAT2 negatively regulates RIG-I in the antiviral innate immunity of black carp.

The signal transducer and activator of transcription 2 (STAT2), a downstream factor of type I interferons (IFNs), is a key component of the cellular antiviral immunity response. However, the role of STAT2 in the upstream of IFN signaling, such as the regulation of pattern recognition receptors (PRRs), remains unknown. In this study, STAT2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The open reading frame (ORF) of bcSTAT2 comprises 2523 nucleotides and encodes 841 amino acids, which presents the conserved structure to that of mammalian STAT2. The dual-luciferase reporter assay and the plaque assay showed that bcSTAT2 possessed certain IFN-inducing ability and antiviral ability against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Interestingly, we detected the association between bcSTAT2 and bcRIG-I through co-immunoprecipitation (co-IP) assay. Moreover, when bcSTAT2 was co-expressed with bcRIG-I, bcSTAT2 obviously suppressed bcRIG-I-induced IFN expression and antiviral activity. The subsequent co-IP assay and immunoblotting (IB) assay further demonstrated that bcSTAT2 inhibited K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I, however, did not affect the oligomerization of bcRIG-I. Thus, our data conclude that black carp STAT2 negatively regulates RIG-I through attenuates its K63-linked ubiquitination, which sheds a new light on the regulation of the antiviral innate immunity cascade in vertebrates.

GSTP1 is a negative regulator of MAVS in the antiviral signaling against SVCV infection.

Glutathione S-transferase P1 (GSTP1), the most ubiquitous member of the GST superfamily, plays vital roles in the detoxification, antioxidant defense, and modulation of inflammatory responses. However, limited studies have been conducted on the function of GSTP1 in antiviral innate immunity. In this study, we have cloned the homolog of GSTP1 in triploid hybrid crucian carp (3nGSTP1) and investigated its regulatory role in the interferon signaling pathway. The open reading frame of 3nGSTP1 is composed of 627 nucleotides, encoding 209 amino acids. In response to spring viremia of carp virus (SVCV) infection, the mRNA level of 3nGSTP1 was up-regulated in the liver, kidney, and caudal fin cell lines (3 nF C) of triploid fish. The knockdown of 3nGSTP1 in 3 nF C improved host cell's antiviral capacity and attenuated SVCV replication. Additionally, overexpression of 3nGSTP1 inhibited the activation of IFN promoters induced by SVCV infection, poly (I:C) stimulation, or the RLR signaling factors. The co-immunoprecipitation assays further revealed that 3nGSTP1 interacts with 3nMAVS. In addition, 3nGSTP1 dose-dependently inhibited 3nMAVS-mediated antiviral activity and reduced 3nMAVS protein level. Mechanistically, 3nGSTP1 promoted ubiquitin-proteasome degradation of MAVS by promoting its K48-linked polyubiquitination. To conclude, our results indicate that GSTP1 acts as a novel inhibitor of MAVS, which negatively regulates the IFN signaling.

Natural product honokiol exhibits antiviral effects against Micropterus salmoides rhabdovirus (MSRV) both in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is a formidable pathogen, presenting a grave menace to juvenile largemouth bass. This viral infection frequently leads to epidemic outbreaks, resulting in substantial economic losses within the aquaculture industry. Unfortunately, at present, there are no commercially available vaccines or pharmaceutical treatments to combat this threat. In order to address the urgent need for therapeutic strategy to resist MSRV infection, the antiviral activity of natural product honokiol against MSRV was explored in this study. Firstly, cellular morphology was directly observed in an inverted microscope when treated with honokiol after MSRV infection. The results clarified that honokiol significantly lessened cytopathic effect (CPE) induced by MSRV and protected the integrity of GCO cells. Furthermore, the viral nucleic acid expression (G gene) was detected by reverse transcription real-time quantitative PCR (RT-qPCR) and the results indicated that honokiol significantly decreased the viral loads of MSRV in a concentration-dependent manner, and honokiol showed a high antiviral activity with IC50 of 2.92 µM. Besides, honokiol significantly decreased the viral titre and suppressed apoptosis caused by MSRV. Mechanistically, honokiol primarily inhibited the initial replication of MSRV and discharge of progeny virus to exert anti-MSRV activity. More importantly, in vivo experiments suggested that honokiol (40 mg/kg) expressed a fine antiviral activity against MSRV when administrated with intraperitoneal injection, which led to a notable 40% improvement in the survival rate among infected largemouth bass. In addition, it also resulted in significant reduction in the viral nucleic acid expression within liver, spleen and kidney at 2, 4 and 6 days following infection. What is more, 100 mg/kg honokiol with oral administration also showed certain antiviral efficacy in MSRV-infected largemouth bass via improving the survival rate by 10.0%, and decreasing significantly the viral nucleic acid expression in liver, spleen and kidney of largemouth bass on day 2. In summary, natural product honokiol is a good candidate to resist MSRV infection and has promising application prospects in aquaculture.

Separation of spring viraemia of carp virus from large-volume samples using immunomagnetic beads.

A method for separation of spring viraemia of carp virus (SVCV) from large-volume samples using immunomagnetic beads (IMBs) coated with a polyclonal antibody against SVCV was developed. The optimum amount of IMBs was 2 mg in 100 mL. After IMB treatment, the detection limit of SVCV in reverse transcription quantitative PCR (RT-qPCR) was 103 times the 50% tissue culture infectious dose per mL in 100-mL samples. The concentration of viral RNA extracted from SVCV that had been separated using IMBs was 5.18 × 103-fold higher than that of the unseparated SVCV. When fish samples were tested, the concordance rates of the IMBs/RT-qPCR and RT-qPCR were 100% and 67.5%, respectively.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus.

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Evaluation of Taxonomic Characteristics of Matlo and Phala Bat Rabies-Related Lyssaviruses Identified in South Africa.

We report the genetic characterization of two potentially novel rabies-related lyssaviruses identified from bats in Limpopo province, South Africa. Matlo bat lyssavirus (MBLV) was identified in two Miniopterus natalensis (Natal long-fingered) bats in 2015 and 2016, and Phala bat lyssavirus (PBLV) was identified in a Nycticeinops schlieffeni (Schlieffen's) bat in 2021. The distribution of both of these bat species is largely confined to parts of Africa, with limited reports from the Arabian Peninsula. MBLV and PBLV were demonstrated to group with the unassigned and phylogroup I lyssaviruses, respectively. MBLV was most closely related to Lyssavirus caucasicus (WCBV), whereas PBLV was most closely related to Lyssavirus formosa (TWBLV-1) and Taiwan bat lyssavirus 2 (TWBLV-2), based on analysis of the N and G genes, the concatenated N + P + M + G + L coding sequence, and the complete genome sequence. Based on our analysis, MBLV and WCBV appeared to constitute a phylogroup separate from Lyssavirus lleida (LLEBV) and Lyssavirus ikoma (IKOV). Analysis of the antigenic sites suggests that PBLV will likely be serologically distinguishable from established lyssaviruses in virus-neutralization tests, whereas MBLV appeared to be antigenically highly similar to WCBV. Taken together, the findings suggested that, while PBLV is likely a new lyssavirus species, MBLV is likely related to WCBV.

Generation of Recombinant Snakehead Rhabdovirus (SHRV) Expressing Artificial MicroRNA Targeting Spring Viremia of Carp Virus (SVCV) P Gene and In Vivo Therapeutic Use Against SVCV Infection.

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish (Danio rerio) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.

Communicating the risks of handling bats: analysing approaches used by Australian stakeholders in the context of Australian bat lyssavirus.

Australian bat lyssavirus (ABLV) is a member of the Lyssavirus genus of the Rhabdoviridae family and is found in Australian bat species. It is of public health concern because of the rabies-like syndrome it causes in humans, resulting in government health and wildlife agencies using varied communication approaches to inform targeted audiences about zoonotic risks associated with handling bats. Despite these warnings, the number of reports of human-bat interactions remains high. This paper details a survey conducted to analyse the approaches utilised by a range of stakeholders to educate and communicate warnings to their target audiences. The survey focused on identifying the target audiences, communication methods used, along with the message frequency, content, and perceived effectiveness. Analysis of the top three messages delivered by stakeholders revealed that over half were information-focused messages and over a third, instruction-focused. Stakeholders identified the need to balance messaging about bat handling risks with information regarding the vulnerable status of bats and their environmental significance. Whilst the most common and (perceived) effective method of communication was one-on-one discussions, it was also identified to be ineffective for targeting mass audiences leading stakeholders to recognise the need to adapt to more efficient means of communication. The outcomes of this study may be useful to improve risk communication strategies regarding ABLV in Australia.

Untargeted LC-MS metabolomics reveals the metabolic responses in olive flounder subjected to hirame rhabdovirus infection.

Hirame novirhabdovirus (HIRRV), which mainly infects the olive flounder (Paralichthys olivaceus), is considered to be one of the most serious viral pathogens threatening the global fish culture industry. However, little is known about the mechanism of host-pathogen interactions at the metabolomic level. In this study, in order to explore the metabolic response of olive flounder to HIRRV infection, liquid chromatography mass spectrometry (LC-MS) was used to detect the changes of endogenous compounds of the olive flounder after HIRRV infection. A total of 954 unique masses were obtained, including 495 metabolites and 459 lipids. Among them, 7 and 173 qualified differential metabolites were identified at 2 days and 7 days post-infection, respectively. Distinct metabolic profiles were observed along with viral infection. At the early stage of infection, only a few metabolites were perturbed. Among them, the level of inosine and carnosine were increased and the potential antiviral ability of these two metabolites was further confirmed by exogenous addition experiment. At the late stage of HIRRV infection, the metabolic profiles changed remarkably. The changes in amino acids and nucleotides especially the 7-methylguanine also accelerated the amplification of viral particles. And the down-regulation of glutathione (GSH) implied an elevated level of ROS (reactive oxygen species) that attenuated the immune system of flounders. HIRRV also induced the accumulation of purine and reduction of pyrimidine, and elevated LPC and LPE levels. The unbalanced purine/pyrimidine and altered lipid profile may be beneficial for the replication and infection of HIRRV at the late stage of infection. These findings provide new insights into the pathogenic mechanism of HIRRV infection in olive flounder.

Phylogenetic analysis of spring viraemia of carp virus isolated in Serbia.

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the Rhabdovirus carpio or SVC virus (SVCV), a member of the Sprivivirus genus, within the Rhabdoviridae family. Phylogenetically, SVCV is divided into four genogroups (SVCV a, SVCV b, SVCV c and SVCV d), which have a reasonable correlation with the geographical distribution of the virus. In the late twentieth century, the disease was widespread in Serbian aquaculture and caused massive deaths in common carp. This study aimed to molecularly characterize the circulating SVCV isolates in Serbia over a 17-year period. The genetic relationships between 21 SVCV isolates from common carp and rainbow trout in Serbia between 1992 and 2009 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). The phylogenetic analysis showed that the dominant SVCV isolates in Serbia belong to the SVCV d genogroup, with only one isolate belonging to genogroup SVCV b. The SVCV strains circulating in Serbia exhibited high homogeneity, as several isolates shared 100% similarity within these genogroups. Most Serbian isolates belonged to SVCV d1 and d2 subgroups, with one isolate notably different and included in a new subgroup SVCV d5. Understanding the SVCV genetic variants circulating in Serbia would be helpful in future epizootic investigations.

Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines - viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated.

Emergence, Tropism, Disease, and Treatment of Australian Bat Lyssavirus Infections in Humans.

Australian bat lyssavirus (ABLV) is a negative-sense, single-stranded RNA rhabdovirus capable of causing fatal acute encephalitis in humans with similar pathogenesis to its closest serologic relative, rabies virus (RABV). In this review, we describe emergence and classification of ABLV, its known virology, reservoirs, and hosts, as well as both the pathogenesis and treatment approaches currently employed for presumed infections. ABLV was first identified in New South Wales, Australia in 1996 and emerged in humans months later in Queensland, Australia. Only five known bat reservoirs, all of which fall within the Pteropus and Saccolaimus genera, have been identified to date. Although ABLV antigens have been identified in bats located outside of Australia, the three known human ABLV infections to date have occurred within Australia. As such, there remains a potential for ABLV to expand its presence within and beyond Australia. ABLV infections are currently treated as if they were RABV infections by administering neutralizing antibodies against RABV at the site of the wound and employing the rabies vaccine upon possible exposures. Due to its recent emergence, there is still much left unknown about ABLV, posing concerns with how to safely and effectively address current and future ABLV infections.

4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin: The first inhibitor of fish rhabdovirus glycoprotein.

Spring viraemia of carp virus (SVCV), a highly pathogenic rhabdovirus, could cause spring viraemia of carp (SVC) with up to 90% lethality. Like other rhabdoviruses, the entry of SVCV into susceptible cells was mediated by a single envelope glycoprotein G. Specific inhibitors targeting the glycoprotein were the most effective means to alleviate the epidemic. The programs including SWISS-MODEL, I-TASSER, Phyre2 and AlphaFold2 were used to build a three-dimensional structural model of glycoprotein. The structural comparison between SVCV-G and homology protein VSV-G revealed that the SVCV glycoprotein ectodomain (residues 19 to 466) folded into four distinct domains. Based on the potential small molecule binding sites on glycoprotein surfaces, virtual screening of the anti-SVCV drug libraries was performed using Autodock software and 4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin (MOA) with a high binding affinity was identified. The solubility enhancer tags including trigger factor and maltose binding protein were fused with the ectodomain of glycoprotein, and the target protein with a purity of about 90% was successfully obtained. The interaction confirmation tests revealed that the fluorescence intensity of a characteristic peak induced by the endogenous chromophores in glycoprotein was decreased with the addition of MOA, indicating changes in the microenvironment of glycoprotein. Moreover, the interaction could cause a slight conformational change in glycoprotein, as shown by the content of ß-turn, ß-folding, and random coil of protein all increased with the decrease of α-helix content after the addition of MOA compound. These results demonstrated that MOA could act as a novel drug against fish rhabdovirus via direct targeting of glycoprotein.

Herbal active small molecule as an immunomodulator for potential application on resistance of common carp against SVCV infection.

Herbal immunomodulators are an important part of prevention and control on viral diseases in aquaculture because of their propensity to improve immunity in fish. The present study was conducted to evaluate the immunomodulatory effect and antiviral activity of a synthesized derivative (serial number: LML1022) against spring viremia of carp virus (SVCV) infection in vitro and in vivo. The antiviral data suggested that LML1022 at 100 µM significantly inhibited the virus replication in epithelioma papulosum cyprini (EPC) cells, and may completely inhibit the infectivity of SVCV virion particles to fish cells by affecting the viral internalization. The results in the related stability of water environments also demonstrated that LML1022 had an inhibitory half-life of 2.3 d at 15 °C, which would facilitate rapid degradation of LML1022 in aquaculture application. For in vivo study, the survival rate of SVCV-infected common carp was increased 30% at least under continuous oral injection of LML1022 at 2.0 mg/kg for 7 d treatment. Additionally, pretreatment of LML1022 on fish prior to SVCV infection also obviously reduced the viral loads in vivo as well as an improved survival rate, showing that LML1022 was potential as an immunomodulator. As an immune response, LML1022 significantly upregulated the immune-related gene expression including IFN-γ2b, IFN-I, ISG15 and Mx1, indicating that its dietary administration may improve the resistance of common carp against SVCV infection.

Pathogenicity of two lineages of infectious hematopoietic necrosis virus (IHNV) to farmed rainbow trout (Oncorhynchus mykiss) in South Korea.

In May 2015, a high mortality event in farmed rainbow trout occurred in Jeollabuk-do province in Korea. Histopathological analysis revealed necrosis in the kidney, liver, branchial arch, and gills of moribund fish, and infectious hematopoietic necrosis virus (IHNV) was detected in the lesions by immunohistochemistry. Cytopathic effects were observed in EPC, FHM, and RTG-2 cell lines after inoculation with kidney and spleen tissues and IHNV was detected by reverse transcription polymerase chain reaction (PCR). The amplified PCR product was sequenced, and phylogenetic analysis placed IHNV in the JRt Nagano group. Both in vivo and in vitro trials were performed to compare the virulence properties between RtWanju15 isolate, which causes 100% mortality in imported fry, and a previous isolate RtWanju09 of the JRt Shizuoka group isolated from eggs of healthy broodfish. In vivo challenge with high dose on specific pathogen free (SPF) rainbow trout fry performed in Denmark with isolates RtWanju09, RtWanju15 and DF04/99 isolates showed a survival rates of 60%, 37.5% and 52.5% (average), respectively without statistical difference. The replication efficiency of the two isolates in the in vitro challenge was similar.

Expression characteristics of non-virion protein of Hirame novirhabdovirus and its transfection induced response in hirame natural embryo cells.

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process.

Transmission dynamics of lyssavirus in Myotis myotis: mechanistic modelling study based on longitudinal seroprevalence data.

We investigated the transmission dynamics of lyssavirus in Myotis myotis and Myotis blythii, using serological, virological, demographic and ecological data collected between 2015 and 2022 from two maternity colonies in northern Italian churches. Despite no lyssavirus detection in 556 bats sampled over 11 events by reverse transcription-polymerase chain reaction (RT-PCR), 36.3% of 837 bats sampled over 27 events showed neutralizing antibodies to European bat lyssavirus 1, with a significant increase in summers. By fitting sets of mechanistic models to seroprevalence data, we investigated factors that influenced lyssavirus transmission within and between years. Five models were selected as a group of final models: in one model, a proportion of exposed bats (median model estimate: 5.8%) became infectious and died while the other exposed bats recovered with immunity without becoming infectious; in the other four models, all exposed bats became infectious and recovered with immunity. The final models supported that the two colonies experienced seasonal outbreaks driven by: (i) immunity loss particularly during hibernation, (ii) density-dependent transmission, and (iii) a high transmission rate after synchronous birthing. These findings highlight the importance of understanding ecological factors, including colony size and synchronous birthing timing, and potential infection heterogeneities to enable more robust assessments of lyssavirus spillover risk.

Comparison of Pan-Lyssavirus RT-PCRs and Development of an Improved Protocol for Surveillance of Non-RABV Lyssaviruses.

Rabies is a zoonotic and fatal encephalitis caused by members of the Lyssavirus genus. Among them, the most relevant species is Lyssavirus rabies, which is estimated to cause 60,000 human and most mammal rabies deaths annually worldwide. Nevertheless, all lyssaviruses can invariably cause rabies, and therefore their impact on animal and public health should not be neglected. For accurate and reliable surveillance, diagnosis should rely on broad-spectrum tests able to detect all known lyssaviruses, including the most divergent ones. In the present study, we evaluated four different pan-lyssavirus protocols widely used at an international level, including two real-time RT-PCR assays (namely LN34 and JW12/N165-146), a hemi-nested RT-PCR and a one-step RT-PCR. Additionally, an improved version of the LN34 assay ((n) LN34) was developed to increase primer-template complementarity with respect to all lyssavirus species. All protocols were evaluated in silico, and their performance was compared in vitro employing 18 lyssavirus RNAs (encompassing 15 species). The (n) LN34 assay showed enhanced sensitivity in detecting most lyssavirus species, with limits of detection ranging from 10 to 100 RNA copies/µL depending on the strain, while retaining high sensitivity against Lyssavirus rabies. The development of this protocol represents a step forward towards improved surveillance of the entire Lyssavirus genus.