Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases.

In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.

Prevalence of feline leukaemia virus and feline immunodeficiency virus in domestic cats in Ireland.

Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting felid species worldwide. A study was performed over a period of 5 months in Ireland with the aim to get an updated and more realistic prevalence of these retroviruses. A total of 183 EDTA-anticoagulated whole-blood samples were collected from cats distributed between 10 clinics. The samples were tested using both point-of-care enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Basic clinical data and vaccination history were also recorded for the sampled cats. The results of ELISA tests showed a prevalence of 10.4 and 3.3% for FIV and FeLV, respectively, and an apparent prevalence of 9.3% for FIV and 11.6% for FeLV with PCR. Phylogenetic analysis of the partial polymerase (pol) gene sequences obtained from 8 FIV-positive strains showed that all but one of the Irish strains belonged to FIV subtype A, and one to subtype B. The overall mean genetic similarity between the analysed strains was 91.15%.

A Diagnostic Device for In-Situ Detection of Swine Viral Diseases: The SWINOSTICS Project.

In this paper, we present the concept of a novel diagnostic device for on-site analyses, based on the use of advanced bio-sensing and photonics technologies to tackle emerging and endemic viruses causing swine epidemics and significant economic damage in farms. The device is currently under development in the framework of the EU Commission co-funded project. The overall concept behind the project is to develop a method for an early and fast on field detection of selected swine viruses by non-specialized personnel. The technology is able to detect pathogens in different types of biological samples, such as oral fluids, faeces, blood or nasal swabs. The device will allow for an immediate on-site threat assessment. In this work, we present the overall concept of the device, its architecture with the technical requirements, and all the used innovative technologies that contribute to the advancements of the current state of the art.

First Report of Porcine Parvovirus 8 in Europe: Widespread Detection and Genetic Characterization on Commercial Pig Farms in Hungary and Slovakia.

Porcine parvovirus 8 (PPV8), a novel virus in the Parvoviridae family, was first identified in 2022 in lung samples of domestic pigs from China. Retrospective analyses showed that it had been circulating in China since 1998, but no other countries had reported its presence so far. A recent study conducted in South America did not detect any PPV8-positive samples in that region. Here, we report the detection of PPV8 in Hungarian and Slovakian pig farms and the estimated prevalence of the virus in Hungary. Altogether, 2230 serum, 233 oral fluid, and 115 processing fluid samples were systematically collected from 23 Hungarian and 2 Slovakian pig farms between 2020 and 2023. A real-time quantitative PCR method was developed to detect the viral genome. Our results revealed the presence of PPV8 on 65% of the Hungarian farms and both Slovakian farms included in our study, marking its first detection in Europe. Oral fluid samples showed the highest positivity rates, reaching up to 100% in some herds. The viral genome was successfully detected in serum and processing fluid samples too, but with significantly lower prevalence rates of 4% and 5%, respectively. Genetic analysis of 11 partial VP2 sequences demonstrated high similarity to the original Chinese strain but with unique amino acid mutations, suggesting possible local evolution of the virus. Our study presents the first scientific evidence of PPV8 infection outside of China and offers a comprehensive assessment of its prevalence in the Hungarian pig population. Further research is required to understand its potential impact on swine health.

Label-Free Detection of African Swine Fever and Classical Swine Fever in the Point-of-Care Setting Using Photonic Integrated Circuits Integrated in a Microfluidic Device.

Swine viral diseases have the capacity to cause significant losses and affect the sector's sustainability, a situation further exacerbated by the lack of antiviral drugs and the limited availability of effective vaccines. In this context, a novel point-of-care (POC) diagnostic device incorporating photonic integrated circuits (PICs), microfluidics and information, and communication technology into a single platform was developed for the field diagnosis of African swine fever (ASF) and classical swine fever (CSF). The device targets viral particles and has been validated using oral fluid and serum samples. Sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device, and PCR was the reference method employed. Its sensitivities were 80.97% and 79%, specificities were 88.46% and 79.07%, and DOR values were 32.25 and 14.21 for ASF and CSF, respectively. The proposed POC device and PIC sensors can be employed for the pen-side detection of ASF and CSF, thus introducing novel technological advancements in the field of animal diagnostics. The need for proper validation studies of POC devices is highlighted to optimize animal biosecurity.

Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry.

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin-biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30-50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

Evaluation of the Virulence of Low Pathogenic H9N2 Avian Influenza Virus Strains in Broiler Chickens.

Our study aimed to investigate the virulence of three recent H9N2 LPAIV strains belonging to the G1 lineage, isolated from field infections in North Africa and the Middle East. Three-week-old commercial broiler chickens (in total 62) were included and randomly allocated into three infected test groups and one control group. Each test group was inoculated intranasally/intratracheally with one of the three H9N2 isolates at a dose of 108 EID50 virus. The control group received phosphate-buffered saline (PBS) via the same route of application. The pathogenicity was evaluated based on clinical signs and gross pathological and histopathological lesions, the viral antigen load was assessed through immunohistochemistry staining (IHC), and a semi-quantitative detection of the genetic material was conducted via a real-time PCR. Our findings confirmed the obvious respiratory tract tropism of the virus strains with variable renal tropism. In contrast to the highly pathogenic AIVs, the tested H9N2 strains did not show replication in the central nervous system. The virus presence and lesions, mainly in the respiratory tract, were predominant on dpi 5 and significantly reduced or disappeared by dpi 11. A clear difference was demonstrated among the three isolates: the A/chicken/Morocco/2021/2016 strain proved to be significantly more virulent than the Egyptian and Saudi Arabian ones, which showed no remarkable difference.

Detection and localization of atypical porcine pestivirus in the testicles of naturally infected, congenital tremor affected piglets.

Atypical porcine pestivirus (APPV) belongs to the genus Pestivirus within the family Flaviviridae. Recently, APPV has been identified as the causative agent of congenital tremor (CT) type AII. The disease is a neurological disorder that affects newborn piglets and is characterized by generalized trembling of the animals and often splay legs. CT is well known worldwide, and the virus seems to be highly prevalent in major swine producing areas. However, little is known about the epidemiology of the infection, transmission and spread of the virus between herds. Here, we show the high prevalence of APPV in processing fluid samples collected from Hungarian pig herds which led us to investigate the cellular targets of the virus in the testicles of newborn piglets affected by CT. By the development of an RNA in situ hybridization assay and the use of immunohistochemistry on consecutive slides, we identified the target cells of APPV in the testicle: interstitial Leydig cells, peritubular myoid cells and smooth muscle cells of medium-sized arteries. Previous studies have shown that APPV can be found in the semen of sexually mature boars suggesting the role of infected boars and their semen in the transmission of the virus similar to many other members of the Flaviviridae family. As in our case, the virus has not been identified in cells beyond the Sertoli cell barrier, further studies on infected adult boars' testicles and other reproductive glands are needed to analyze the possible changes in the cell tropism of APPV that might contribute to its prolonged extraction by the semen beyond the period of viraemia.

Detection of Porcine Respirovirus 1 (PRV1) in Poland: Incidence of Co-Infections with Influenza A Virus (IAV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Herds with a Respiratory Disease.

Porcine respirovirus 1 (PRV1) is also known as porcine parainfluenza virus 1 (PPIV1). The prevalence and the role of PRV1 infections for pig health is largely unknown. In order to assess the PRV1 prevalence in Poland, nasal swabs and oral fluids collected from pigs from 30 farms were examined with RT real-time PCR. Additionally, IAV and PRRSV infection statuses of PRV1-positive samples were examined. The results showed that the virus is highly prevalent (76.7% farms positive) and different patterns of PRV1 circulation in herds with mild-moderate respiratory disease were observed. Co-infections with IAV and PRRSV were infrequent and detected in 8 (23.5%) and 4 (11.8%) out of 34 PRV1-positive nasal swab pools from diseased pens, respectively. In one pen PRV1, IAV, and PRRSV were detected at the same time. Interestingly, PRV1 mean Ct value in samples with co-infections was significantly lower (29.8 ± 3.1) than in samples with a single PRV1 infection (32.5 ± 3.6) (p < 0.05), which suggested higher virus replication in these populations. On the other hand, the virus detection in pig populations exhibiting respiratory clinical signs, negative for PRRSV and IAV, suggests that PRV1 should be involved in differential diagnosis of respiratory problems.

In Situ Hybridization of Feline Leukemia Virus in a Case of Osteochondromatosis.

Osteochondromatosis, also known as multiple cartilaginous exostosis, polyostotic osteochondroma, and multiple osteochondromas, comprises one-fifth of all primary bone tumors in cats, with no breed or sex predisposition or hereditary pattern. Unlike in dogs, horses, and humans, it is predominantly seen in young cats (2-4 years old), after the maturation of the skeleton. Although the pathogenesis of osteochondromatosis is not fully understood, it is considered to be related to infection by feline leukemia virus (FeLV) or other retroviruses, such as the feline sarcoma virus. However, the presence of viral particles within tumor lesions has only been demonstrated by electron microscopy. The malignant transformation of osteochondromas, most typically to osteosarcoma or chondrosarcoma, has also been attributed to the viral infection. Here we report the case of osteochondromatosis in a 3.5-year-old male domestic European shorthair cat with concurrent FeLV infection confirmed by polymerase chain reaction. Viral RNA was visualized in representative tissues (spleen, mesenteric lymph node, liver, kidney, lung, brain) and in the osteochondromas with RNAscope in situ hybridization, which supports that FeLV infection may be involved in the pathogenesis of osteochondromatosis.

High Prevalence of Porcine Circovirus 3 in Hungarian Pig Herds: Results of a Systematic Sampling Protocol.

Porcine circovirus type 3 (PCV3) is an emerging pathogen that has been reported worldwide in all ages of healthy and clinically ill pigs. The presence of this virus in Hungary has been confirmed in a commercial farm experiencing reproductive failures, but there were no data on the circulation of PCV3 in the country. Here we report the prevalence and the genetic diversity of PCV3 in Hungarian herds. To estimate the prevalence, 1855 serum samples, 176 oral fluid and 97 processing fluid samples were collected in a systematic, cross-sectional method from 20 large scale swineherds and tested by real-time qPCR. PCV3 was present in at least one type of diagnostic matrix in 19 out of the 20 (95%) pig farms. The highest detection rates were observed in the processing fluid samples (61%), but 41% of the oral fluid and 23% of the serum samples were positive. The virus was found in all age groups, and slightly more adult animals were infected than growing pigs, but the viral burden was lower amongst them. Phylogenetic analysis of nine complete genomes, obtained from either the sampled herds or organ samples of PCV3-positive carcasses, showed high nucleotide identity between the detected sequences, which all belonged to the PCV3a genotype. Our results indicate that PCV3 is widespread in Hungary, but in most cases, the virus seems to circulate subclinically, infecting all age groups and production phases without the presence of apparent clinical disease.

Point-of-Care and Label-Free Detection of Porcine Reproductive and Respiratory Syndrome and Swine Influenza Viruses Using a Microfluidic Device with Photonic Integrated Circuits.

Swine viral diseases challenge the sector's sustainability by affecting productivity and the health and welfare of the animals. The lack of antiviral drugs and/or effective vaccines renders early and reliable diagnosis the basis of viral disease management, underlining the importance of point-of-care (POC) diagnostics. A novel POC diagnostic device utilizing photonic integrated circuits (PICs), microfluidics, and information and communication technologies for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A (SIV) was validated using spiked and clinical oral fluid samples. Metrics including sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device. For PRRSV, the device achieved a sensitivity of 83.5%, specificity of 77.8%, and DOR values of 17.66, whereas the values for SIV were 81.8%, 82.2%, and 20.81, respectively. The POC device and PICs can be used for the detection of PRRSV and SIV in the field, paving the way for the introduction of novel technologies in the field of animal POC diagnostics to further optimize livestock biosecurity.

First report of porcine parainfluenza virus 1 (species Porcine respirovirus 1) in Europe.

Porcine respirovirus 1, also known as Porcine parainfluenza virus 1 (PPIV-1) was first identified in Hong Kong in 2013, later in the USA and most recently in Chile. Here, we report the first detection of PPIV-1 outside these three regions. We screened 22 farms in Hungary by testing 15 nasal swab samples obtained from 3-week-old piglets (3 randomly chosen piglets from 5 litters in each farm). Only one farm was found to be positive. We subsequently sampled the positive farm by taking cross-sectional 20 nasal swab samples from 2-, 4-, 6- and 8-week-old piglets. Virus detection by qRT-PCR showed that although all investigated age groups were positive to PPIV-1, a higher number of infected animals and higher viral loads were found among 4-week-old animals. Based on the phylogenetic analyses of partial F and L genes, the 3 Hungarian strains are genetically closely related to the very first PPIV-1 strain identified in Hong Kong in 2013, whereas the overall genetic difference compared to the recently described North American isolates was around 10%.

In Situ Hybridization of PRRSV-1 Combined with Digital Image Analysis in Lung Tissues of Pigs Challenged with PRRSV-1.

Betaarterivirus suid 1 and 2 are the causative agents of porcine reproductive and respiratory syndrome (PRRS), which is one of the most significant diseases of the swine industry, causing significant economic losses in the main pig producing countries. Here, we report the development of a novel, RNA-based in situ hybridization technique (RNAscope) to detect PRRS virus (PRRSV) RNA in lung tissues of experimentally infected animals. The technique was applied to lung tissues of 20 piglets, which had been inoculated with a wild-type, highly pathogenic PRRSV-1 strain. To determine the RNAscope's applicability as a semi-quantitative method, we analysed the association between the proportion of the virus-infected cells measured with an image analysis software (QuPath) and the outcome of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed in parallel. The results of the quantitative approach of these two molecular biological methods show significant association (pseudo R2 = 0.3894, p = 0.004). This is the first time RNAscope assay has been implemented for the detection of PRRSV-1 in experimental animals.

In situ hybridization of feline leukemia virus in a primary neural B-cell lymphoma.

An 8-y-old castrated male, outdoor European shorthair cat was presented with a history of hindlimb weakness and paralysis. Disease progression was continuous from the onset; deep algesia disappeared at the final stage. Radiography of the vertebral column was unremarkable; along with patient history and physical examination results, magnetic resonance imaging suggested inflammatory lesions in the spinal cord, although neoplasia could not be ruled out. Feline leukemia virus (FeLV) positivity was confirmed by a serum ELISA prior to euthanasia. Upon postmortem examination, hemorrhages were present in the spinal cord at the level of vertebrae T7-8. Histologic and immunohistochemical analysis revealed primary diffuse large B-cell lymphoma of the spinal cord with multifocal myelomalacia and hemorrhages. To determine the presence of a pathogen within the lesion, we developed a novel in situ hybridization protocol for FeLV (RNAscope). The reaction revealed large amounts of FeLV viral RNA in the tumor cells.

Prevalence of feline immunodeficiency virus and feline leukaemia virus in domestic cats in Hungary.

OBJECTIVES: Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting cats worldwide. The objectives of the study were to estimate the prevalence of these retroviruses in domestic cats in Hungary and to characterise the phylogenetic relationships of FIV strains. METHODS: A total of 335 anticoagulated whole-blood samples obtained from both a healthy and ill cat population were examined for the presence of FIV and FeLV with two methods: ELISA and PCR. Statistical analysis was carried out to analyse the data obtained. Sequencing and phylogenetic analysis of partial polymerase (pol) gene sequences was performed to describe circulating FIV subtypes. RESULTS: Statistical analysis showed 11.8% and 9.9% true prevalence of FeLV and FIV, respectively, with ELISA. The apparent prevalence calculated from the PCR results were 17.3% for FeLV and 13.1% for FIV. Phylogenetic analysis of partial pol gene sequences obtained from 22 FIV strains showed that all observed Hungarian strains belonged to FIV subtype B. The strains were grouped into several monophyletic subgroups reflecting the geographic locations of the origin of the samples. The overall mean genetic similarity between the analysed strains was 98.2%. CONCLUSIONS AND RELEVANCE: We report the first thorough overview of the prevalence of FeLV and FIV in Hungary, which is relatively high, and give insight into the genetic diversity of Hungarian strains of FIV.

Detection and phylogenetic characterization of atypical porcine pestivirus strains in Hungary.

Atypical porcine pestivirus (APPV) is a recently identified RNA virus within the Flaviviridae family, causing congenital tremor (CT) in the piglets of infected sows. We have investigated 25 cases of CT from 2005, 2007, 2010 and 2016-2018, originating from six different farms. RT-PCR has been performed on these samples and all of the affected piglets were positive to APPV. Our phylogenetic analysis showed that Hungarian strains show a high degree of variability and are clustered into five distinct lineages. Four strains originating from one farm have shown exceptional similarity (99.9%) to an Austrian sequence, whereas another one from a different herd was grouped close to a Chinese strain (96.4% similarity). Our results suggest multiple events of introduction of the virus from various sources into Hungary. This is the first report of the presence and clinical relevance of APPV in the Hungarian pig population.

Genetic diversity of PRRSV 1 in Central Eastern Europe in 1994-2014: origin and evolution of the virus in the region.

More than 20 years after the first outbreaks, the phylogenetic picture of PRRSV is still incomplete and full of gaps, especially in regards of PRRSV 1. Due to the exceptional diversity observed at the eastern borders of Europe and the low number of available sequences from Central Eastern European countries, the authors collected and analyzed both recent as well as already submitted sequences comparing them to a large backbone set of available ORF5 sequences representing the full spectrum of PRRSV 1 Subtype 1 diversity to conduct a systematic phylogenetic analysis and reclassification elucidating the diversity of the virus in these countries. Moreover, further analyses of the EUROSTAT data regarding the live pig movement trends revealed their influence of virus diversity and evolution. The results indicate that besides the effect of local, isolated divergent evolution and the use of modified live vaccines, the most important factor influencing a given country's virus diversity is the transboundary movement of live, infected animals.