Detection of PRRSV-1 in tongue fluids under experimental and field conditions and comparison of different sampling material for PRRSV sow herd monitoring.

BACKGROUND: Infection with porcine reproductive and respiratory syndrome virus (PRRSV) leads to significant economic losses worldwide. One of the initial measures following an outbreak is to stabilise the herd and to prevent vertical transmission of PRRSV. The objective of this study was to detect PRRSV in different sampling material, both in an experimental model and on a commercial piglet producing farm, with a focus on evaluating the suitability of tongue fluid samples. RESULTS: In the experimental model, PRRSV negative pregnant gilts were infected with PRRSV-1 AUT15-33 on gestation day 85 and necropsy of gilts and foetuses was performed three weeks later. 38.3% of individual foetal serum and 39.4% of individual foetal thymus samples were considered PRRSV RT-qPCR positive. Tongue fluids from individual foetuses showed a 33.0% positivity rate. PRRSV RNA was detected in all but one sample of litter-wise pooled processing fluids and tongue fluids. In the field study, the investigated farm remained PRRSV positive and unstable for five consecutive farrowing groups after the start of the sampling process. Tongue fluid samples pooled by litter in the first investigated farrowing group had a 54.5% positivity rate, with the overall highest viral load obtained in the field study. In this farrowing group, 33.3% of investigated litter-wise pooled processing fluid samples and all investigated serum samples (pools of 4-6 individuals, two piglets per litter) were considered positive. Across all investigated farrowing groups, tongue fluid samples consistently showed the highest viral load. Moreover, tongue fluid samples contained the virus in moderate amounts for the longest time compared to the other investigated sampling material. CONCLUSION: It can be concluded that the viral load in individual foetuses is higher in serum or thymus compared to tongue fluid samples. However, litter-wise pooled tongue fluid samples are well-suited for detecting vertical transmission within the herd, even when the suspected prevalence of vertical transmission events is low.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths.

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at - 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Retrospective Analysis of the Detection of Pathogens Associated with the Porcine Respiratory Disease Complex in Routine Diagnostic Samples from Austrian Swine Stocks.

The diagnostic workup of respiratory disease in pigs is complex due to coinfections and non-infectious causes. The detection of pathogens associated with respiratory disease is a pivotal part of the diagnostic workup for respiratory disease. We aimed to report how frequently certain viruses and bacteria were detected in samples from pigs with respiratory symptoms in the course of routine diagnostic procedures. Altogether, 1975 routine diagnostic samples from pigs in Austrian swine stocks between 2016 and 2021 were analysed. PCR was performed to detect various pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) (n = 921), influenza A virus (n = 479), porcine circovirus type 2 (PCV2) (n = 518), Mycoplasma (M.) hyopneumoniae (n = 713), Actinobacillus pleuropneumoniae (n = 198), Glaesserella (G.) parasuis (n = 165) and M. hyorhinis (n = 180). M. hyorhinis (55.1%) had the highest detection rate, followed by PCV2 (38.0%) and Streptococcus (S.) suis (30.6%). PRRSV was detected most frequently in a pool of lung, tonsil and tracheobronchial lymph node (36.2%). G. parasuis was isolated more frequently from samples taken after euthanasia compared to field samples. PRRSV-positive samples were more likely to be positive for PCV2 (p = 0.001), M. hyopneumoniae (p = 0.032) and Pasteurella multocida (p < 0.001). M. hyopneumoniae-positive samples were more likely to be positive for P. multocida (p < 0.001) and S. suis (p = 0.046), but less likely for M. hyorhinis (p = 0.004). In conclusion, our data provide evidence that lung samples that were positive for a primary pathogenic agent were more likely to be positive for a secondary pathogenic agent.

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic.

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation.

Litters of Various-Sized Mummies (LVSM) and Stillborns after Porcine Reproductive and Respiratory Syndrome Virus Type 1 Infection-A Case Report.

Diverse origins and causes are described for papyraceous mummifications of porcine foetuses, but the porcine reproductive and respiratory syndrome virus (PRRSV) is not one of them. In contrast, PRRSV is unlikely to cause mid-term placental transmission but may cause late-term abortions and weakness of piglets. This case report describes a sudden occurrence of mummified foetuses of various sizes and stillborns and delayed birth (>115 days) in more than 50% of sows from one farrowing batch, while newborn piglets were mostly vital. Neither increased embryonic death nor infertility was reported. Three litters with mummies, autolysed piglets and stillborn piglets were investigated, and infections with porcine parvoviruses, porcine teschoviruses, porcine circoviruses, encephalomyocarditis virus, Leptospira spp. and Chlamydia spp. were excluded. Instead, high viral loads of PRRSV were detected in the thymus pools of piglets at all developmental stages, even in piglets with a crown-rump length between 80 and 150 mm, suggesting a potential mid-term in utero transmission of the virus. Genomic regions encoding structural proteins (ORF2-7) of the virus were sequenced and identified the virulent PRRSV-1 strain AUT15-33 as the closest relative. This case report confirms the diversity of PRRSV and its potential involvement in foetal death in mid-gestation.

A single, episodic event of unilateral/bilateral scrotal swelling in a group of adult boars at an Austrian boar stud.

BACKGROUND: Scrotal swelling is a clinical situation which can be caused by different aetiologies. In this case report, we describe a multi-week episode of unilateral and bilateral scrotal swelling in boars at an Austrian boar stud and its diagnostic work-up. CASE PRESENTATION: In the summer of 2020, the herd veterinarian of an Austrian boar stud reported that over a period of six weeks, five out of 70 boars presented with unilateral severe swelling of the left scrotum and three out of 70 boars with bilateral severe swelling of the left and moderate swelling of the right scrotum, respectively. A complete history was obtained and an on-site evaluation of the facility was done. Five boars were necropsied, and a variety of samples harvested for further diagnostic investigations. Infectious differential diagnoses associated with unilateral swelling of the scrotum or the testis were excluded through serological and tissue testing. In three of the five boars, histopathology revealed complete acute haemorrhagic necrosis of the left testis concurrent with strongly congested blood vessels. Review of the collected information with a group of experts in the field of boar stud management resulted with consensus that, most likely, trauma was the etiologic event causing the clinical signs and pathology. Coincident with discussion of implementing video recording cameras in the boar housing area, no further clinical cases followed. As this case occurred during the first lockdown of the COVID-19 pandemic, we propose that the distress and travelling restrictions may have contributed to frustration among boar stud workers, which was consequently expressed as misbehaviour against boars. CONCLUSIONS: Once all known infectious causes of unilateral swelling of the scrotum were excluded, a critical diagnostic work-up focused on non-infectious causes. Non-infectious causes, such as trauma, need to be carefully evaluated, as it may also include human misbehaviour against boars. Summarizing all findings of this case report, the authors hypothesize that a blunt trauma was the reason for the series of mainly unilateral swelling of the scrota of boars.

A Conserved Stem-Loop Structure within ORF5 Is a Frequent Recombination Hotspot for Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) with a Particular Modified Live Virus (MLV) Strain.

The emergence of recombinant PRRSV strains has been observed for more than a decade. These recombinant viruses are characterized by a genome that contains genetic material from at least two different parental strains. Due to the advanced sequencing techniques and a growing number of data bank entries, the role of PRRSV recombinants has become increasingly important since they are sometimes associated with clinical outbreaks. Chimeric viruses observed more recently are products of PRRSV wild-type and vaccine strains. Here, we report on three PRRSV-1 isolates from geographically distant farms with differing clinical manifestations. A sequencing and recombination analysis revealed that these strains are crossovers between different wild-type strains and the same modified live virus vaccine strain. Interestingly, the recombination breakpoint of all analyzed isolates appears at the beginning of open reading frame 5 (ORF5). RNA structure predictions indicate a conserved stem loop in close proximity to the recombination hotspot, which is a plausible cause of a polymerase template switch during RNA replication. Further research into the mechanisms of the stem loop is needed to help understand the PRRSV recombination process and the role of MLVs as parental strains.

Successful treatment of coagulation disorders and hypoalbuminaemia in a puppy with Infectious Canine Hepatitis.

Due to high vaccination coverage of the dog population in Western and Middle Europe, veterinarians are usually not familiar with clinical signs and treatment of Infectious Canine Hepatitis (ICH). This case report describes a 4-month-old female mixed breed dog that was imported from Bulgaria. According to the history, the puppy was presented with lethargy, pyrexia, icterus and melaena. On clinical examination, the dog additionally exhibited a painful abdomen and bleeding tendency at the venous puncture sites. Blood analysis revealed anaemia, left shift without leucocytosis, increased liver enzymes and prolonged coagulation times. Polymerase Chain Reaction (PCR) and subsequently sequence analysis performed out of urine confirmed Canine Adenovirus 1 (CAV-1) as the causative agent of the disease. Peripheral oedema developed on the dog´s head and limbs during the progression of the disease due to severe hypoalbuminaemia. Initial treatment of the puppy included transfusion of whole blood and fresh frozen plasma. Hypoalbuminaemia was treated by transfusion using human albumin. On day eight after starting the treatment, the dog was released from the hospital due to an unremarkable clinically condition. This case report indicates that ICH might become a re-emerging disease by means of rising dog imports. Especially, the severe form of ICH can be associated with several life-threatening complications that require hospitalisation and intensive care treatment.

Reproductive failure in an Austrian piglet-producing farm due to porcine circovirus genotype 2d.

Infections of pigs with porcine circovirus type 2 (PCV2) can lead to various clinical conditions including reproductive disorders (PCV2-RD). In general, a transplacental infection of fetuses leads to mummification and stillbirth. So far, PCV2-RD has mainly been described in specific-pathogen-free (SPF) herds or farms with a high proportion of gilts. From December 2018 to February 2019, a high abundance of mummified fetuses (15.5%) was observed in two farrowing groups in an Austrian piglet-producing farm. PCV2 DNA was detected using qPCR in organs of all six investigated fetuses (2.07 × 108-1.09 × 1012 PCV2) genome equivalents/g tissue and via in situ hybridisation in organs from five fetuses, while histologic lesions were not observed in a single fetal heart. All isolates were sequenced and identified as PCV2d. After the implementation of a regular vaccination of all sows against PCV2, the abundance of mummified fetuses dropped to 3.5% in May 2019. In contrast to previous reports about PCV2-RD, this farm was neither an SPF herd nor a start-up herd with a high proportion of gilts. The implementation of regular PCV2 vaccination helped to reduce the abundance of mummified fetuses substantially.

Porcine Circoviruses and Herpesviruses Are Prevalent in an Austrian Game Population.

During the annual hunt in a privately owned Austrian game population in fall 2019 and 2020, 64 red deer (Cervus elaphus), 5 fallow deer (Dama dama), 6 mouflon (Ovis gmelini musimon), and 95 wild boars (Sus scrofa) were shot and sampled for PCR testing. Pools of spleen, lung, and tonsillar swabs were screened for specific nucleic acids of porcine circoviruses. Wild ruminants were additionally tested for herpesviruses and pestiviruses, and wild boars were screened for pseudorabies virus (PrV) and porcine lymphotropic herpesviruses (PLHV-1-3). PCV2 was detectable in 5% (3 of 64) of red deer and 75% (71 of 95) of wild boar samples. In addition, 24 wild boar samples (25%) but none of the ruminants tested positive for PCV3 specific nucleic acids. Herpesviruses were detected in 15 (20%) ruminant samples. Sequence analyses showed the closest relationships to fallow deer herpesvirus and elk gammaherpesvirus. In wild boars, PLHV-1 was detectable in 10 (11%), PLHV-2 in 44 (46%), and PLHV-3 in 66 (69%) of animals, including 36 double and 3 triple infections. No pestiviruses were detectable in any ruminant samples, and all wild boar samples were negative in PrV-PCR. Our data demonstrate a high prevalence of PCV2 and PLHVs in an Austrian game population, confirm the presence of PCV3 in Austrian wild boars, and indicate a low risk of spillover of notifiable animal diseases into the domestic animal population.

New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria.

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population.

ADAM17 Is an Essential Factor for the Infection of Bovine Cells with Pestiviruses.

The entry of BVDV into bovine cells was studied using CRIB cells (cells resistant to infection with bovine viral diarrhea virus [BVDV]) that have evolved from MDBK cells by a spontaneous loss of susceptibility to BVDV. Recently, larger genetic deletions were reported but no correlation of the affected genes and the resistance to BVDV infection could be established. The metalloprotease ADAM17 was reported as an essential attachment factor for the related classical swine fever virus (CSFV). To assess whether ADAM17 might be involved in the resistance of CRIB-1 cells to pestiviruses, we analyzed its expression in CRIB-1 and MDBK cells. While ADAM17 protein was detectable in MBDK cells, it was absent from CRIB-1 cells. No functional full-length ADAM17 mRNA could be detected in CRIB cells and genetic analysis revealed the presence of two defective alleles. Transcomplementation of functional ADAM17 derived from MDBK cells in CRIB-1 cells resulted in a nearly complete reversion of their resistance to pestiviral infection. Our results demonstrate that ADAM17 is a key cellular factor for the pestivirus resistance of CRIB-1 cells and establishes its essential role for a broader range of pestiviruses.

Active equine parvovirus-hepatitis infection is most frequently detected in Austrian horses of advanced age.

BACKGROUND: Equine parvovirus-hepatitis (EqPV-H) research is in its infancy. Information regarding prevalence, geographical distribution, genetic diversity, pathogenesis and risk factors enhances understanding of this potentially fatal infection. OBJECTIVES: Determining the prevalence of EqPV-H in Austrian equids. Investigating factors increasing probability of infection, liver-associated biochemistry parameters, concurrent equine hepacivirus (EqHV) infection and phylogenetic analysis of Austrian EqPV-H variants. STUDY DESIGN: Cross-sectional study. METHODS: Sera from 259 horses and 13 donkeys in Austria were analysed for anti-EqPV-H VP1-specific antibodies by luciferase immunoprecipitation system (LIPS) and EqPV-H DNA by nested polymerase chain reaction (PCR). Associations between infection status, sex and age were described. Glutamate dehydrogenase (GLDH), gamma-glutamyl transferase (GGT), bile acids and albumin concentrations were compared between horses with active infection and PCR-negative horses. PCR targeting partial EqPV-H NS1 was performed and phylogenetic analysis of Austrian EqPV-H variants was conducted. Complete coding sequences (CDS) of four Austrian variants were determined by next-generation sequencing (NGS) and compared with published sequences. RESULTS: Horses' EqPV-H seroprevalence was 30.1% and DNA prevalence was 8.9%. One horse was co-infected with EqHV. Significantly, higher probability of active EqPV-H infection was identified in 16- to 31-year-old horses, compared with 1- to 8-year-old horses (P = 0.002; OR = 8.19; 95% CI = 1.79 to 37.50) and 9- to 15-year-old horses (P = 0.03; OR = 2.96; 95% CI = 1.08 to 8.17). Liver-associated plasma parameters were not significantly different between horses with active infection and controls. Austrian EqPV-H variants revealed high similarity to sequences worldwide. No evidence of EqPV-H was detected in donkeys. MAIN LIMITATIONS: Equids' inclusion depended upon owner consent. There was only one sampling point per animal and the sample of donkeys was small. CONCLUSIONS: EqPV-H antibodies and DNA are frequently detected in Austrian horses, without associated hepatitis in horses with active infection. The risk of active EqPV-H infection increases with increasing age. Phylogenetic evidence supports close relation of EqPV-H variants globally, including Austrian variants.

[Challenges in Influenza diagnostics in a swine herd - a case report]. / Herausforderungen bei der Influenzadiagnostik in einem Schweinebetrieb ­ ein Fallbericht.

In a gilt producing farm in Lower Austria, respiratory diseases occurred over the previous years in self-reared gilts after being introduced into the sow herd. In addition, fertility disorders in terms of late abortions and re-breeders were observed in the fall of 2019. Nasal swabs of 3 gilts with respiratory signs and fever were tested positive for influenza A virus (IAV) subtype H1avN1 by PCR. However, examination of serum samples from these animals at 2 different time points did not detect antibodies using the standard hemagglutination inhibition (HI) test of the laboratory. Examination of additional age groups likewise failed to detect H1avN1 antibody titers. In consequence to the extension of the diagnostic panel of the HI test by 7 additional H1avN1 test antigens, a clear seroconversion of the PCR positive sows against 2 different H1avN1 isolates could be measured. In addition, high antibody titers against these 2 H1avN1 strains were also detectable in the majority of the remaining age groups tested. Following the administration of the trivalent influenza vaccine, which has been approved throughout Europe, a significant improvement of the clinical presentation in the herd was achieved. The present case report illustrates that direct and indirect pathogen detection should be used in combination for targeted influenza diagnostics. In addition, it was shown that the continuous adaptation of test antigens to the isolates circulating in the field would be extremely crucial for the significance of the HI test.

Discovery of a phylogenetically distinct poxvirus in diseased Crocodilurus amazonicus (family Teiidae).

A novel poxvirus was discovered in Crocodilurus amazonicus (Teiidae) presenting with a debilitating skin disease. The generated first genome sequence of a reptilian poxvirus revealed the closest phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.

The Stable Fly (Stomoxys calcitrans) as a Possible Vector Transmitting Pathogens in Austrian Pig Farms.

This pilot study aimed to investigate stable flies from Austrian pig farms for the presence of defined swine pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2), hemotrophic mycoplasmas in ingested blood and/or body parts and bacteria on the surface of the flies. Furthermore, the use of stable flies as a diagnostic matrix for the detection of pathogens in the ingested pig blood should be investigated. In total, 69 different microorganisms could be found on the surface of tested S. calcitrans from 20 different pig farms. Escherichia coli was the most common bacterium and could be found on flies from seven farms. In seven farms, hemotrophic mycoplasmas were detected in stable flies. PRRSV could not be found in any of the samples of these 20 farms but PCV2 was detected in six farms. Whether the stable fly can be used as a matrix to monitor the health status cannot be accurately determined through this study, especially in regard to PRRSV. Nevertheless, it might be possible to use the stable fly as diagnostic material for defined pathogens like Mycoplasma suis and PCV2.

The first RT-qPCR confirmed case of tick-borne encephalitis in a dog in Scandinavia.

BACKGROUND: Tick-borne encephalitis (TBE) is a zoonotic neurological disease caused by tick-borne encephalitis virus (TBEV), a flavivirus endemic in parts of Europe and Asia. Seroconversion without signs of clinical disease is common in dogs and most of the cases previously described have been tentatively diagnosed by combining neurologic signs with serum antibody titres. Here, the first Scandinavian RT-qPCR-confirmed clinical case of TBE in a dog is reported. CASE PRESENTATION: A 4-year old castrated male Pointer Labrador cross was presented with acute-onset ataxia. During hospitalisation, the dog developed seizures. Despite aggressive treatment with steroids, antimicrobials and sedation/anaesthesia, there was continued deterioration during the following 24 h after admission and the dog was euthanised and submitted for necropsy. Histopathological changes in the brain were consistent with lymphoplasmacytic and histiocytic meningoencephalomyelitis. RT-qPCR examination of the brain was positive for TBEV, confirming infection. CONCLUSIONS: Meningoencephalomyelitis caused by TBEV should be a diagnostic consideration in dogs presenting with clinical signs of central nervous system disease such as acute-onset ataxia and seizures in areas where TBEV-positive ticks are endemic. Clinical TBE may be underdiagnosed in dogs due to lack of specific testing.

Development and validation of a portable, point-of-care canine distemper virus qPCR test.

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife.

No Evidence of Mosquito Involvement in the Transmission of Equine Hepacivirus (Flaviviridae) in an Epidemiological Survey of Austrian Horses.

Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Concurrently, 386 serum samples from horses in eastern Austria were analyzed for EqHV-specific antibodies by luciferase immunoprecipitation system (LIPS) and for EqHV RNA by RT-qPCR. Additionally, liver-specific biochemistry parameters were compared between EqHV RNA-positive horses and EqHV RNA-negative horses. Phylogenetic analysis was conducted in comparison to previously published sequences from various origins. No EqHV RNA was detected in mosquito pools. Serum samples yielded an EqHV antibody prevalence of 45.9% (177/386) and RNA prevalence of 4.15% (16/386). EqHV RNA-positive horses had significantly higher glutamate dehydrogenase (GLDH) levels (p = 0.013) than control horses. Phylogenetic analysis showed high similarity between nucleotide sequences of EqHV in Austrian horses and EqHV circulating in other regions. Despite frequently detected evidence of EqHV infection in Austrian horses, no viral RNA was found in mosquitoes. It is therefore unlikely that mosquitoes are vectors of this flavivirus.