A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

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.

Reptarenavirus S Segment RNA Levels Correlate with the Presence of Inclusion Bodies and the Number of L Segments in Snakes with Reptarenavirus Infection-Lessons Learned from a Large Breeding Colony.

Reptarenaviruses cause boid inclusion body disease (BIBD), a fatal disease particularly impacting captive boa constrictor collections. The development of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus nucleoprotein (NP) in many cell types of affected snakes is characteristic of BIBD. However, snakes can harbor reptarenaviruses without showing IBs, hence representing carriers and a potential source of transmission. The RNA genome of reptarenaviruses comprises a small (S) and a large (L) segment, and the snakes with BIBD commonly carry a swarm of reptarenavirus segments. To design sensitive and reliable tools for the diagnosis of reptarenavirus infection in snake colonies, we used metatranscriptomics to determine the reptarenavirus segments present in a large boa constrictor breeding colony. The analysis identified one reptarenavirus S segment and three L segments in the colony. The sequence data served to design real-time reverse transcription-PCR (RT-PCR) targeting the found S segment. This allowed us to identify all infected animals and to quantify the S segment RNA levels, which we found to correlate with the presence of IBs. We further found a positive correlation between the number of L segments and the S segment RNA level, which could suggest that L segment excess also contributes to the IB formation. Information on cohousing of the snakes showed a clear association of reptarenavirus infection with cohousing in general and cohousing with infected animals. Information on breeding and offspring confirmed that vertical transmission occurred. Furthermore, our data suggest that some animals might be able to clear the infection or at least exhibit transient or intermittent viremia. IMPORTANCE Boid inclusion body disease (BIBD) is caused by reptarenavirus infection, and while reptarenavirus nucleoprotein is the main component of the inclusion bodies (IBs) characteristic of BIBD, not all reptarenavirus-infected snakes demonstrate IBs in their cells. Identification of infected individuals is critical for controlling the spread of the disease; however, the genetic divergence of reptarenaviruses complicates reverse transcription-PCR (RT-PCR)-based diagnostics. Here, we tested a next-generation-sequencing-based approach to establish a tailored "colony-specific" set of diagnostic tools for the detection of reptarenavirus small (S) and large (L) genome segments. With this approach, we could demonstrate that an S-segment-specific RT-PCR is highly effective in identifying the infected individuals. We further found the S segment RNA level to positively correlate with the presence of IBs and the number of L segments, which could direct future studies to identify the BIBD pathogenetic mechanisms.

Neisseria gonorrhoeae Coinfection during Chlamydia muridarum Genital Latency Does Not Modulate Murine Vaginal Bacterial Shedding.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most frequently reported agents of bacterial sexually transmitted disease worldwide. Nonetheless, C. trachomatis/N. gonorrhoeae coinfection remains understudied. C. trachomatis/N. gonorrhoeae coinfections are more common than expected by chance, suggesting C. trachomatis/N. gonorrhoeae interaction, and N. gonorrhoeae infection may reactivate genital chlamydial shedding in women with latent (quiescent) chlamydial infection. We hypothesized that N. gonorrhoeae would reactivate latent genital Chlamydia muridarum infection in mice. Two groups of C. muridarum-infected mice were allowed to transition into genital latency. One group was then vaginally inoculated with N. gonorrhoeae; a third group received N. gonorrhoeae alone. C. muridarum and N. gonorrhoeae vaginal shedding was measured over time in the coinfected and singly infected groups. Viable C. muridarum was absent from vaginal swabs but detected in rectal swabs, confirming C. muridarum genital latency and consistent with the intestinal tract as a C. muridarum reservoir. C. muridarum inclusions were observed in large intestinal, but not genital, tissues during latency. Oviduct dilation was associated with C. muridarum infection, as expected. Contradicting our hypothesis, N. gonorrhoeae coinfection did not reactivate latent C. muridarum vaginal shedding. In addition, latent C. muridarum infection did not modulate recovery of vaginal viable N. gonorrhoeae. Evidence for N. gonorrhoeae-dependent increased C. muridarum infectivity has thus not been demonstrated in murine coinfection, and the ability of C. muridarum coinfection to potentiate N. gonorrhoeae infectivity may depend on actively replicating vaginal C. muridarum. The proportion of mice with increased vaginal neutrophils (PMNs) was higher in N. gonorrhoeae-infected than in C. muridarum-infected mice, as expected, while that of C. muridarum/N. gonorrhoeae-coinfected mice was intermediate to the singly infected groups, suggesting latent C. muridarum murine infection may limit PMN response to subsequent N. gonorrhoeae infection. IMPORTANCE Our work builds upon the limited understanding of C. muridarum/N. gonorrhoeae coinfection. Previously, N. gonorrhoeae infection of mice with acute (actively replicating) vaginal C. muridarum infection was shown to increase recovery of viable vaginal N. gonorrhoeae and vaginal PMNs, with no effect on C. muridarum vaginal shedding (R. A. Vonck et al., Infect Immun 79:1566-1577, 2011). It has also been shown that chlamydial infection of human and murine PMNs prevents normal PMN responses, including the response to N. gonorrhoeae (K. Rajeeve et al., Nat Microbiol 3:824-835, 2018). Our findings show no effect of latent genital C. muridarum infection on the recovery of viable N. gonorrhoeae, in contrast to the previously reported effect of acute C. muridarum infection, and suggesting that acute versus latent C. muridarum infection may have distinct effects on PMN function in mice. Together, these studies to date provide evidence that Chlamydia/N. gonorrhoeae synergistic interactions may depend on the presence of replicating Chlamydia in the genital tract, while chlamydial effects on vaginal PMNs may extend beyond acute infection.

First Report of TTSuV1 in Domestic Swiss Pigs.

Serum prevalence of Torque teno sus viruses (TTSuV1 and k2; family Anelloviridae) is known to be high in the porcine population worldwide but pathogenesis and associated pathomorphological lesions remain to be elucidated. In this study, quantitative real-time PCR for detection of TTSuV1 was performed in 101 porcine samples of brain tissue, with animals showing inflammatory lesions or no histological changes. Additionally, a pathomorphological and immunohistochemical characterization of possible lesions was carried out. Selected cases were screened by TTSuV1 in situ hybridization. Furthermore, TTSuV1 quantitative real-time PCR in splenic and pulmonary tissue and in situ hybridization (ISH) in spleen, lungs, mesenteric lymph node, heart, kidney, and liver were performed in 22 animals. TTSuV1 was detected by PCR not only in spleen and lung but also in brain tissue (71.3%); however, in general, spleen and lung tissue displayed lower Ct values than the brain. Positive TTSuV1 results were frequently associated with the morphological diagnosis of non-suppurative encephalitis. Single TTSuV1-positive lymphocytes were detected by ISH in the brain but also in lungs, spleen, mesenteric lymph node and in two cases of non-suppurative myocarditis. A pathogenetic role of a TTSuV1 infection as a co-factor for non-suppurative encephalitides cannot be ruled out.

Chlamydia caviae in Swiss and Dutch Guinea Pigs-Occurrence and Genetic Diversity.

Chlamydia (C.) caviae is a known pathogen in guinea pigs, causing conjunctivitis, respiratory infections and abortions. Recently, a C. caviae-induced zoonotic link was identified as the etiology of severe community-acquired pneumonia in humans. Here, 784 conjunctival and rectal swabs originating from 260 guinea pigs and 110 rabbits from 64 husbandries in Switzerland, as well as 200 composite conjunctival swabs originating from 878 guinea pigs from 37 husbandries in The Netherlands were examined by real-time PCR followed by conventional PCR and sequencing. Chlamydiaceae were detected in 2.3% (18/784) and 12.5% (25/200) of all Swiss and Dutch samples, respectively. An overall C. caviae occurrence was detected in 2.7% (7/260) and 8.9% (78/878) of all Swiss and Dutch guinea pigs, respectively. OmpA genotyping of 64 C. caviae-positive samples resulted in 33 sequences sharing 100% nucleotide identity with the strains isolated from the zoonotic transmission cases in The Netherlands. However, all ompA sequences of this study were distinct from the C. caviae GPIC reference strain. C. caviae was not detected in rabbits but C. psittaci genotype A was identified in guinea pigs and rabbits, raising concerns about the importance of these animal species as novel zoonotic sources for C. psittaci.

Falcons From the United Arab Emirates Infected With Chlamydia psittaci/C abortus Intermediates Specified as Chlamydia buteonis by Polymerase Chain Reaction.

Chlamydiaceae are obligate intracellular bacteria with a broad host range. Several studies have found chlamydial species that are genetically intermediate between Chlamydia psittaci and Chlamydia abortus in various avian species. One of these intermediate Chlamydia species, found in a red-shouldered hawk (Buteo lineatus), was recently classified as a new species Chlamydia buteonis. This newly described Chlamydia species has, so far, only been reported in hawks exhibiting clinical signs of conjunctivitis, dyspnea, and diarrhea. In the present study, fecal samples of 5 gyrfalcons (Falco rusticolus), 3 gyr/peregrine falcon hybrids (Falco rusticolus × Falco peregrinus), and 15 falcons of unknown species presented to falcon clinics on the Arabian Peninsula were shipped to the Vetsuisse Faculty, University of Zurich (Zurich, Switzerland), for examination for the presence of Chlamydiaceae. A step-wise diagnostic approach was performed to identify the chlamydial species involved. Chlamydiaceae were detected in 21/23 falcons by a family-specific real-time quantitative PCR (qPCR). Further identification with a 23S ribosomal RNA-based microarray assay and 16S conventional PCR and sequencing yielded inconclusive results, indicating the presence of an intermediate Chlamydia species. Because none of the falcons tested positive for Chlamydia psittaci by specific qPCR, all 23 samples were subjected to a Chlamydia buteonis-specific qPCR, which was positive in 16/23 samples. Detailed information regarding clinical history was available for 8 falcons admitted to a falcon clinic in Dubai, United Arab Emirates. Six of those birds that were presented to the clinic because of loss of performance and poor general condition, including vomiting and diarrhea, were positive for C buteonis. In 2 birds without clinical disease signs admitted for a routine health examination, 1 was positive for C buteonis, and 1 was negative. It is yet unknown whether Chlamydia buteonis causes disease in birds, but the findings in this study indicate that Chlamydia buteonis may be an infectious pathogen in falcon species.

Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants.

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the ß-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Chlamydia suis is associated with intestinal NF-κB activation in experimentally infected gnotobiotic piglets.

Chlamydia suis intestinal infection of single-animal experimental groups of gnotobiotic newborn piglets was previously reported to cause severe, temporary small intestinal epithelium damage. We investigated archived intestinal samples for pro-inflammatory nuclear factor kappa B (NF-κB) activation, Interleukin (IL)-6 and IL-8 production and immune cell influx. Samples were collected 2, 4 and 7 days post-inoculation with C. suis strain S45/6 or mock inoculum (control). Increased nuclear localization of epithelial NF-κB, representative of activation, in the jejunum and ileum of C. suis-infected animals, compared to uninfected controls, began by 2 days post-infection (dpi) and persisted through 7 dpi. Infected animals showed increased production of IL-8, peaking at 2 dpi, compared to controls. Infection-mediated CD45-positive immune cell influx into the jejunal lamina propria peaked at 7 dpi, when epithelial damage was largely resolved. Activation of NF-κB appears to be a key early event in the innate response of the unprimed porcine immune system challenged with C. suis. This results in an acute phase, coinciding with the most severe clinical symptoms, diarrhea and weight loss. Immune cells recruited shortly after infection remain present in the lamina propria during the recovery phase, which is characterized by reduced chlamydial shedding and restored intestinal epithelium integrity.

Serpentoviruses: More than Respiratory Pathogens.

In recent years, nidoviruses have emerged as important respiratory pathogens of reptiles, affecting captive python populations. In pythons, nidovirus (recently reclassified as serpentovirus) infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe, often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full postmortem examinations on 30 serpentovirus reverse transcription-PCR (RT-PCR)-positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether this not yet reported wider distribution of lesions is associated with previously unknown serpentoviruses or changes in the serpentovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and obtain virus isolates. Immunohistochemistry and immunofluorescence staining against serpentovirus nucleoprotein demonstrated that the virus infects not only a broad spectrum of epithelia (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts, etc.), but also intravascular monocytes, intralesional macrophages, and endothelial cells. With next-generation sequencing we obtained a full-length genome for a novel serpentovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing serpentovirus infection-associated respiratory or systemic disease did not reveal sequence association to phenotypes; however, functional studies with different strains are needed to confirm this observation. The results indicate that serpentoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated viral systemic spread.IMPORTANCE During the last years, python nidoviruses (now reclassified as serpentoviruses) have become a primary cause of fatal disease in pythons. Serpentoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previous evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that comprises important zoonotic pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. Serpentoviruses belong to the same order as the above-mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order Nidovirales.

Survey on Chlamydiaceae in cloacal swabs from Swiss turkeys demonstrates absence of Chlamydia psittaci and low occurrence of Chlamydia gallinacean.

In Switzerland, domestic turkey meat is a niche product. Turkeys are fattened on mixed family-based farms scattered across the country, with most providing access to an uncovered outdoor pasture for the birds. Swiss fattening turkeys may therefore get infected with Chlamydiaceae via wild birds or their faeces, potentially shedding these bacteria at a later stage. The aim of the present study was to acquire baseline data about the shedding of Chlamydiaceae in clinically unremarkable Swiss fattening turkeys at slaughter, potentially exposing slaughterhouse workers to infection. In this large-scale study, 1008 cloacal swabs of Swiss turkeys out of 53 flocks from 28 different grow-out farms with uncovered outdoor pasture were collected over the course of 14 months and examined for the occurrence of Chlamydiaceae by a family-specific 23S-rRNA real-time PCR. Positive samples were further analyzed by Chlamydia psittaci (C. psittaci)-specific real-time PCR and the Arraymate DNA Microarray for species identification. All samples were negative for C. psittaci, but seven swabs out of one flock were tested positive for Chlamydia gallinacea (0.7%). Although turkeys with access to pasture may have contact with Chlamydiaceae-harbouring wild birds or their faeces, the infection rate in Swiss turkeys was shown to be low.

The influence of centrifugation and incubation temperatures on various veterinary and human chlamydial species.

The Chlamydiaceae are Gram-negative bacteria causing diseases in humans and in both, endothermic (mammals and birds) and poikilothermic (e.g. reptiles, amphibians) animals. As most chlamydial species described today were isolated from humans and endothermic animals, the commonly used culturing temperature in vitro is 37 °C, although the centrifugation temperature during experimental infection, a technique necessary to improve the infection rate, may vary from 25 to 37 °C. The aim of this study was to investigate the influence of different centrifugation (28° or 33 °C) and incubation temperatures (28 °C or 37 °C) on the average inclusion size, infectivity and ultrastructural morphology of human and animal chlamydial strains, as well as two recently described species originating from snakes, C. poikilothermis and C. serpentis, in LLC-MK2 cells at 48 h post infection. Infectivity and average inclusion size was reduced at an incubation temperature of 28 °C compared to 37 °C for all strains including C. poikilothermis, although the latter formed larger, fully matured inclusions at 28 °C in comparison to the other investigated Chlamydia species. C.psittaci displayed a shorter developmental cycle than the other species confirming previous studies. Higher centrifugation temperature increased the subsequent inclusion size of C. trachomatis, C. abortus and C. suis but not their infectivity, while the incubation temperature had no discernable effect on the morphology, inclusion size and infectivity of the other chlamydial strains. In conclusion, we found that all Chlamydia species are viable and can grow at low incubation temperatures, although all strains grew better and more rapidly at 37 °C compared to 28 °C.

Identification of a Novel Deltavirus in Boa Constrictors.

Hepatitis D virus (HDV) forms the genus Deltavirus unassigned to any virus family. HDV is a satellite virus and needs hepatitis B virus (HBV) to make infectious particles. Deltaviruses are thought to have evolved in humans, since for a long time, they had not been identified elsewhere. Herein we report, prompted by the recent discovery of an HDV-like agent in birds, the identification of a deltavirus in snakes (Boa constrictor) designated snake HDV (sHDV). The circular 1,711-nt RNA genome of sHDV resembles human HDV (hHDV) in its coding strategy and size. We discovered sHDV during a metatranscriptomic study of brain samples of a Boa constrictor breeding pair with central nervous system signs. Applying next-generation sequencing (NGS) to brain, blood, and liver samples from both snakes, we did not find reads matching hepadnaviruses. Sequence comparison showed the snake delta antigen (sHDAg) to be 55% and 37% identical to its human and avian counterparts. Antiserum raised against recombinant sHDAg was used in immunohistology and demonstrated a broad viral target cell spectrum, including neurons, epithelial cells, and leukocytes. Using RT-PCR, we also detected sHDV RNA in two juvenile offspring and in a water python (Liasis macklotisavuensis) in the same snake colony, potentially indicating vertical and horizontal transmission. Screening of 20 randomly selected boas from another breeder by RT-PCR revealed sHDV infection in three additional snakes. The observed broad tissue tropism and the failure to detect accompanying hepadnavirus suggest that sHDV could be a satellite virus of a currently unknown enveloped virus.IMPORTANCE So far, the only known example of deltaviruses is the hepatitis delta virus (HDV). HDV is speculated to have evolved in humans, since deltaviruses were until very recently found only in humans. Using a metatranscriptomic sequencing approach, we found a circular RNA, which resembles that of HDV in size and coding strategy, in a snake. The identification of similar deltaviruses in distantly related species other than humans indicates that the previously suggested hypotheses on the origins of deltaviruses need to be updated. It is still possible that the ancestor of deltaviruses emerged from cellular RNAs; however, it likely would have happened much earlier in evolution than previously thought. These findings open up completely new avenues in evolution and pathogenesis studies of deltaviruses.

Bovine Fetal Placenta During Pregnancy and the Postpartum Period.

Bovine abortion is a worldwide problem, but despite extensive histopathologic and molecular investigations, the cause of abortion remains unclear in about 70% of cases. Cellular debris is a commonly observed histopathologic finding in the fetal placenta and is often interpreted as necrosis. In this study, the nature of this cellular debris was characterized, and histologic changes in the normal fetal placenta during pregnancy and after delivery were assessed. In addition, the presence of the most common abortifacient pathogens in Switzerland ( Chlamydiaceae, Coxiella burnetii, Neospora caninum) was tested by polymerase chain reaction. We collected 51 placentomes and 235 cotyledons from 41 and from 50 cows, respectively. In total, cellular debris was present in 48 of 51 (94%) placentomes and in 225 of 235 (96%) cotyledons, inflammation occurred in 1 of 51 (2%) placentomes and in 46 of 235 (20%) cotyledons, vasculitis was seen in 1 of 51 (2%) placentomes and 46 of 235 (20%) cotyledons, and 18 of 51 (35%) placentomes and 181 of 235 (77%) cotyledons had mineralization. The amount of cellular debris correlated with areas of positive signals for cleaved caspase 3 and lamin A. Therefore, this finding was interpreted as an apoptotic process. In total, 10 of 50 cotyledons (20%) were positive for C. burnetii DNA, most likely representing subclinical infections. The results of our study indicate that histologic features in the fetal placenta such as cellular debris, inflammation, vasculitis, and mineralization must be considered physiological processes during pregnancy and after delivery. Therefore, their presence in placentae of aborted fetuses must be interpreted with caution and might not be necessarily linked to an infectious cause of abortion.

Chlamydiae in human intestinal biopsy samples.

Chlamydia trachomatis is frequently detected in anorectal specimens from men and women. A recent hypothesis suggests that C. trachomatis is a natural commensal organism asymptomatically colonizing the gastrointestinal tract. In this study, we investigated the presence of chlamydial DNA and antigen in intestinal biopsy samples taken during colonoscopy. Cases (n = 32) were patients whose histopathology reports included the term 'chlamydia', suggesting a possible history of infection. Control patients (n = 234) did not have chlamydia mentioned in their histopathology report and all tested negative for Chlamydiaceae DNA by 23S ribosomal RNA-based real-time PCR. Amongst the cases, C. trachomatis DNA was detected in the appendix and colon of two female and one male patients. Chlamydia abortus DNA was present in the colon of a fourth female patient. Thus, chlamydial DNA could be demonstrated in intestinal biopsy samples proximal to the anorectal site and inclusions were identified in rectum or appendix of two of these patients by immunohistochemistry. However, the findings in two cases were compatible with sexually acquired C. trachomatis. The identification of C. trachomatis DNA/antigen does not prove the presence of active infection with replicating bacteria. Larger prospective studies on fresh tissue samples are required to confirm the data obtained in this study.

Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses-The representatives of genus Hartmanivirus, family Arenaviridae.

The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular inclusion bodies typical for the reptarenavirus-induced boid inclusion body disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed.

Co-composting of biowaste and wood ash, influence on a microbially driven-process.

A trial at semi-industrial scale was conducted to evaluate the effect of wood ash amendment on communal biowaste in a composting process and on the final composts produced. For this purpose, three treatments including an unamended control (C0) and composts with additions of 6% (C6), and 12% (C12) of wood ash (w/w) were studied, and physico-chemical parameters as well as microbial activity and community composition were investigated. At the end of the process, composts were tested for toxicity and quality, and microbial physiological activity. The influence of ash addition on compost temperature, pH, microbial activity and composition was stronger during the early composting stages and diminished with time, whereby composts became more similar. Using the COMPOCHIP microarray, a reduction in the pathogenic genera Listeria and Clostridium was observed, which together with the temperature increases of the composting process helped in the hygienisation of composts. Lactobacillus species were also affected, such that reduced hybridisation signals were observed with increased ash addition, due to the increased pH values in amended composts. Organic matter mineralisation was also increased through ash addition, and no negative effects on the composting process were observed. The nutrient content of the final products was increased through the addition of ash, and no toxic effects were observed. Nonetheless, greater concentrations of heavy metals were found in composts amended with more ash, which resulted in a downgrading of the compost quality according to the Austrian Compost Ordinance. Thus, regulation of both input materials and end-product quality is essential in optimising composting processes.