Viruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), a fatal disease of boid snakes with an economic and ecological impact, as it affects both captive and wild constrictor snakes. The clinical picture of BIBD is highly variable but often only limited. Intracytoplasmic inclusion bodies (IB), which develop in most cell types including blood cells, are the pathognomonic hallmark of BIBD; their detection represents the diagnostic gold standard of the disease. However, IBs are not consistently present in clinically healthy reptarenavirus carriers, which can, if undetected, lead to and maintain the spread of the disease within and between snake populations. Sensitive viral detection tools are required for screening and control purposes; however, the genetic diversity of reptarenaviruses hampers the reverse transcription (RT) PCR-based diagnostics. Here, we describe a multiplex RT-PCR approach for the molecular diagnosis of reptarenavirus infection in blood samples. The method allows the detection of a wide range of reptarenaviruses with the detection limit reaching 40 copies per microliter of blood. Using 245 blood samples with a reference RT-PCR result, we show that the technique performs as well as the segment-specific RT-PCRs in our earlier studies. It can identify virus carriers and serve to limit reptarenavirus spreading in captive snake collections.

Reptarenaviruses cause Boid inclusion body disease (BIBD), a lethal disease primarily affecting captive boa constrictors. The presence of cytoplasmic inclusion bodies (IBs), mainly composed of viral nucleoprotein (NP), in various cell types is characteristic to and a diagnostic criterion of BIBD. We have previously reported that reptarenavirus replication and IB formation are efficient in cell cultures that are maintained at 27–30 °C but not in cells that are kept at 37 °C, the temperature commonly used for mammalian cell cultures. Here, considering the poikilothermic nature of snakes, we studied the ideal temperature(s) for reptarenavirus propagation and the expression of reptarenavirus NP. We incubated Boa constrictor kidney-derived I/1Ki cells at different temperatures (24–36 °C), inoculated them with University of Giessen virus 1 (UGV-1) and monitored both cell growth and virus proliferation. Cell growth was optimal at 30–34 °C and was not significantly affected by UGV-1 infection. Viral RNA release per cell was highest at ambient temperatures between 28 and 32 °C, as determined by qRT-PCR. However, the cells passaged at day 15 post-inoculation released viral RNA at comparable levels even when kept at slightly lower temperatures (24–26 °C). Morphometric analyses undertaken on sections of cell pellets immunostained for reptarenavirus NP found the expression to be most intense at 32 and 34 °C in freshly inoculated cells, and at 28–32 °C in passaged cells. The NP expression positively correlated with the amount of viral RNA released per cell. Our results indicate that the optimal temperature ranges for boid cell growth and reptarenavirus replication (as judged based on antigen expression and RNA release) overlap at about 32 °C. They also suggest that environmental temperature modulation could represent a strategy to impair reptarenavirus replication and, potentially, the spread of reptarenaviruses within and between snake collections.

Hypertrophic cardiomyopathy (HCM) is the most prevalent acquired heart disease in cats and shares many clinical, phenotypical and pathological features with human HCM. Despite its relevance, knowledge on the pathomechanisms underlying the disease is limited. The present study aimed to characterize the molecular phenotypic changes in cardiomyocytes in feline HCM (fHCM) to better understand their contribution to the pathogenesis. To achieve this, the myocardium of the left ventricular free wall of 15 cats with confirmed fHCM and 30 control cats (two age groups: 16 cats 18-month-old, and 14 older adult cats without cardiac disease) were subjected to RT-qPCRs for markers representative of cardiomyocyte function. Overall, all markers were expressed at the highest level in young control cats, and increasing age correlated with decreased expression, regardless of sex. The comparison between the older adult control cats and those with HCM showed increased transcription levels for most markers associated with the disease, and higher expression of all markers in affected male cats compared to females. The constitutive transcription of all markers provides evidence of continuous myocardial adaptation throughout cats’ life. The high transcription values in the myocardium of young healthy cats and male cats affected by HCM suggest a particularly high myocardial responsiveness early in life and with HCM and reveal sex as relevant factor in the disease process. These results support the relevance of age and sex in the cardiac response to HCM in feline hearts.

Boid inclusion body disease (BIBD) is a reptarenavirus-induced fatal disease of boid snakes that, while diagnosed with ease due to the pathognomonic intracytoplasmic inclusion bodies (IB), is not yet fully characterized pathomorphologically and with regards to its pathogenesis. The present study retrospectively investigated a random cohort of 50 captive male and female boa constrictors of variable age (neonate to 25 years) for histopathological changes and in situ viral protein (nucleoprotein, NP, and glycoprotein 2, GP2) expression patterns. IB formation and NP expression were scored semiquantitatively. The vast majority of snakes (42/50; 84%) exhibited histopathological changes; these mainly (39/42; 93%) represented inflammatory processes that most frequently affected the gastrointestinal tract. Overall, IB and NP scores were significantly higher in the more recent cases (2020-2025) compared to the older cases (2000-2003). NP scores did not differ between age groups or sex, but were lower in animals with clinical signs and higher in those with histopathological changes. No statistically significant association was found between NP scores and inflammatory processes, neither in general nor in individual organs. GP2 expression, as an indicator of virion assembly, was less consistent than NP expression and generally restricted to individual cells with and without IBs. There was no histological evidence of overt damage in infected cells. Jointly, the results indicate different phases of restrictive and systemic viral replication that are not directly associated with pathological processes and/or clinical signs.