Porcine Circovirus Type 3 (PCV3) in Poland: Prevalence in Wild Boar Population in Connection with African Swine Fever (ASF).

Human health is dependent on food safety and, therefore, on the health of farm animals. One of the most significant threats in regard to swine diseases is African swine fever (ASF). Infections caused by porcine circoviruses (PCVs) represent another important swine disease. Due to the ubiquitous nature of PCV2, it is not surprising that this virus has been detected in ASFV-affected pigs. However, recent data indicate that coinfection of PCV3 and ASFV also occurs. It is still unclear whether PCV infection plays a role in ASFV infection, and that subject requires further analysis. The aim of this study was to assess whether PCV3 and PCV4 are present in the wild boar population in Poland (real-time PCR). The analysis was performed on wild boar samples collected for routine ASF surveillance in Poland, between 2018 and 2021. By extension, the obtained data were compared in regard to ASFV presence in these samples, thus investigating the odds of ASFV infection on the grounds of the PCV carrier state in free-ranging Suidae in Poland. In addition, sequencing of PCV3 and phylogenetic analysis were performed, based on a full genome and a capsid gene. In the current study, we demonstrated the high prevalence of PCV3 in the wild boar population in Poland; meanwhile, PCV4 was not detected. The odds of ASFV infection on the grounds of the PCV3 carrier state in free-ranging Suidae in Poland was more than twice as high. Ten full genome sequences of PCV3 were obtained, all of them belonging to clade 3a. The similarity between them was in the range of 98.78-99.80%.

SARS-CoV-2 infection impairs NK cell functions via activation of the LLT1-CD161 axis.

Introduction: Natural killer (NK) cells plays a pivotal role in the control of viral infections, and their function depend on the balance between their activating and inhibitory receptors. The immune dysregulation observed in COVID-19 patients was previously associated with downregulation of NK cell numbers and function, yet the mechanism of inhibition of NK cell functions and the interplay between infected cells and NK cells remain largely unknown. Methods: In this study we show that SARS-CoV-2 infection of airway epithelial cells can directly influence NK cell phenotype and functions in the infection microenvironment. NK cells were co-cultured with SARS-CoV-2 infected epithelial cells, in a direct contact with A549ACE2/TMPRSS2 cell line or in a microenvironment of the infection in a 3D ex vivo human airway epithelium (HAE) model and NK cell surface expression of a set of most important receptors (CD16, NKG2D, NKp46, DNAM-1, NKG2C, CD161, NKG2A, TIM-3, TIGIT, and PD-1) was analyzed. Results: We observed a selective, in both utilized experimental models, significant downregulation the proportion of CD161 (NKR-P1A or KLRB1) expressing NK cells, and its expression level, which was followed by a significant impairment of NK cells cytotoxicity level against K562 cells. What is more, we confirmed that SARS-CoV-2 infection upregulates the expression of the ligand for CD161 receptor, lectin-like transcript 1 (LLT1, CLEC2D or OCIL), on infected epithelial cells. LLT1 protein can be also detected not only in supernatants of SARS-CoV-2 infected A549ACE2/TMPRSS2 cells and HAE basolateral medium, but also in serum of COVID-19 patients. Finally, we proved that soluble LLT1 protein treatment of NK cells significantly reduces i) the proportion of CD161+ NK cells, ii) the ability of NK cells to control SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells and iii) the production of granzyme B by NK cells and their cytotoxicity capacity, yet not degranulation level. Conclusion: We propose a novel mechanism of SARS-CoV-2 inhibition of NK cell functions via activation of the LLT1-CD161 axis.

Aerogels based on cationically modified chitosan and poly(vinyl alcohol) for efficient capturing of viruses.

In this study, we developed a new filtering bioaerogel based on linear polyvinyl alcohol (PVA) and the cationic derivative of chitosan (N-[(2-hydroxy-3-trimethylamine) propyl] chitosan chloride, HTCC) with a potential antiviral application. A strong intermolecular network architecture was formed thanks to the introduction of linear PVA chains, which can efficiently interpenetrate the glutaraldehyde(GA)-crosslinked HTCC chains. The morphology of the obtained structures was examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The aerogels and modified polymers' elemental composition (including the chemical environment) was determined using X-ray photoelectron spectroscopy (XPS). New aerogels with more than twice as much developed micro- and mesopore space and BET-specific surface area were obtained concerning the starting sample chitosan aerogel crosslinked by glutaraldehyde (Chit/GA). The results obtained from the XPS analysis showed the presence of cationic 3-trimethylammonium groups on the surface of the aerogel, which can interact with viral capsid proteins. No cytotoxic effect of HTCC/GA/PVA aerogel was also observed on fibroblast cells of the NIH3T3 line. Furthermore, the HTCC/GA/PVA aerogel has been shown that efficiently traps mouse hepatitis virus (MHV) from suspension. The presented concept of aerogel filters for virus capture based on modified chitosan and polyvinyl alcohol has a high application potential.

Zoonotic spill-over of SARS-CoV-2: mink-adapted virus in humans.

OBJECTIVES: This work aimed to analyse possible zoonotic spill-over of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the spill-over of mink-adapted SARS-CoV-2 from farmed mink to humans after adaptation that lasted at least 3 months. METHODS: Next-generation sequencing and a bioinformatic approach were applied to analyse the data. RESULTS: In an isolate obtained from an asymptomatic patient testing positive for SARS-CoV-2, we found four distinguishing mutations in the S gene that gave rise to the mink-adapted variant (G75V, M177T, Y453F, and C1247F) and others. CONCLUSIONS: Zoonotic spill-over of SARS-CoV-2 can occur from mink to human.

Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8, Poland, July to August 2021.

Routine genomic surveillance on samples from COVID-19 patients collected in Poland during summer 2021 revealed the emergence of a SARS-CoV-2 Delta variant with a large 872 nt deletion. This change, confirmed by Sanger and deep sequencing, causes complete loss of ORF7a, ORF7b, and ORF8 genes. The index case carrying the deletion is unknown. The standard pipeline for sequencing may mask this deletion with a long stretch of N's. Effects of this deletion on phenotype or immune evasion needs further study.

Ozone Treatment Is Insufficient to Inactivate SARS-CoV-2 Surrogate under Field Conditions.

COVID-19 caused by SARS-CoV-2 caused a worldwide crisis, highlighting the importance of preventive measures in infectious diseases control. SARS-CoV-2 can remain infectious on surfaces for up to several weeks; therefore, proper disinfection is required to mitigate the risk of indirect virus spreading. Gaseous ozone treatment has received particular attention as an easily accessible disinfection tool. In this study, we evaluated the virucidal effectiveness of gaseous ozone treatment (>7.3 ppm, 2 h) on murine hepatitis virus (MHV)-contaminated stainless-steel surface and PBS-suspended virus under field conditions at ambient (21.8%) and high (49.8-54.2%) relative humidity. Surficial virus was soiled with 0.3 g/L of BSA. Parallelly, a half-hour vaporization with 7.3% hydrogen peroxide was performed on contaminated carriers. The obtained results showed that gaseous ozone, whilst quite effective against suspended virus, was insufficient in sanitizing coronavirus contaminated surfaces, especially under low RH. Increased humidity created more favorable conditions for MHV inactivation, resulting in 2.1 log titre reduction. Vaporization with 7.3% hydrogen peroxide presented much better virucidal performance than ozonation in a similar experimental setup, indicating that its application may be more advantageous regarding gaseous disinfection of surfaces contaminated with other coronaviruses, including SARS-CoV-2.

Effectiveness of Chemical Compounds Used against African Swine Fever Virus in Commercial Available Disinfectants.

African swine fever (ASF) causes huge economic losses and is one of most dangerous diseases of pigs. The disease is known for almost 100 years, an effective vaccine or treatment is still unavailable, only proper biosecurity measures, including disinfection, are being applied, in order to prevent disease outbreaks. Eight active substances, i.e., formaldehyde, sodium hypochlorite, caustic soda, glutaraldehyde, phenol, benzalkonium chloride, potassium peroxymonosulfate and acetic acid, were tested, in order to confirm their effectiveness against African swine fever virus (ASFV). This specific selection was done based on the World Organisation for Animal Health (OIE)'s recommendation and previous disinfectant studies on surfaces. The result of our study shows that most of them inactivate the virus, in recommended concentrations. In order to reduce the cytotoxicity of the four substances, Microspin S-400 HR columns were applied, therefore making it possible to demonstrate four logarithms virus titer reduction. Sodium hypochlorite, glutaraldehyde, caustic soda and potassium peroxymonosulfate showed the best ASFV inactivation rates, achieving titer reductions over 5 logs. Despite microfiltration, the virucidal activity of formaldehyde was not assessable, due to its high cytotoxicity. Our results showed that cleaning is particularly important, because removal of the soiling provides improved effectiveness of the tested chemical compounds.

The Spillover of African Swine Fever in Western Poland Revealed Its Estimated Origin on the Basis of O174L, K145R, MGF 505-5R and IGR I73R/I329L Genomic Sequences.

The African swine fever epidemic occurred in Poland at the beginning of 2014 and, up to date, the disease has been spreading mainly in the eastern part of the country. Unexpectedly, in November 2019 an infected wild boar case was confirmed in Lubuskie voivodship in western Poland. During the following weeks, several dozen African swine fever virus (ASFV)-positive animals were notified in the neighboring area, causing severe concern regarding further spread of the disease to the mostly pig-dense region in Poland, namely, Wielkopolskie voivodship. Moreover, almost a year after, several infected wild boar cases were confirmed for the first time in Germany, just beyond the Polish border, sending out a shock wave through the global pig market. The whole genome sequence of ASFV, isolated from the first case of ASF in western Poland, and three selected viruses from other affected areas, revealed the tandem repeat and single nucleotide polymorphism (SNP) variations in reference to the Georgia 2007/1 strain. These data, supported by the conventional sequencing of selected genomic regions from a total of 154 virus samples isolated between 2017 and 2020 in Poland, shed a new light on pathogen epidemiology. The sequence variations within the O174L gene detected in this study showed that cases identified in western Poland might be originating from the so-called southern Warsaw cluster. Moreover, the viruses originating from the northern Warsaw cluster do not possess single nucleotide polymorphism (SNP) mutations within the K145R and MGF 505-5R genes, which are specific to all of the other Polish ASFV strains. These results led to a conclusion of their distinct origin. Supporting these results, the nucleotide sequencing of I73R/I329L intergenic region revealed its new, previously undescribed variant, called IGR IV, with an additional three tandem repeats of 10 nucleotides in comparison to the reference sequence of the Georgia 2007/1 strain.

Attempts at the Development of a Recombinant African Swine Fever Virus Strain with Abrogated EP402R, 9GL, and A238L Gene Structure using the CRISPR/Cas9 System.

INTRODUCTION: African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus' mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain. MATERIAL AND METHODS: The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells. RESULTS: The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate. CONCLUSION: Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.

Analysis of the Clinical Course of Experimental Infection with Highly Pathogenic African Swine Fever Strain, Isolated from an Outbreak in Poland. Aspects Related to the Disease Suspicion at the Farm Level.

This paper was aimed to characterize clinical signs and pathomorphological lesions in twenty-two pigs, infected intranasally by different doses of African swine fever virus (Pol18_28298_O111), isolated during the outbreak in a pig farm that occurred in Eastern Poland throughout 2018. This article also attempts to indicate risk, related to virus load and shedding, and present possible difficulties with proper disease recognition at the farm level. The results revealed that even a very low dose (5 HAU) may initiate the infection. Various forms of the disease (acute, subacute, and chronic), mainly with prodromal clinical signs like fever, apathy, and reduced feed intake were observed. The most frequently observed lesions (82%) were: hyperemia and enlargement of lymph nodes and splenomegaly. The minimal incubation period was estimated at five days post-infection (dpi). Mortality ranged from 80-100%. Two pigs survived the infection. Some viremic animals presented delayed fever. In some cases, the fever was not detectable. Shortly after viremia, the virus was secreted ion the urine, feces, and saliva. The highest levels of virus were found in the internal organs and blood; however in the case of one pig (chronic form), viral DNA was not detected in the spleen, liver, bone marrow, and brain. Veterinary diagnosis may be difficult, and the final results should always be based on laboratory investigations.

Natural inactivation of African swine fever virus in tissues: Influence of temperature and environmental conditions on virus survival.

African swine fever virus can be transmitted through direct contact with infected animals and their excretions, or indirect contact with contaminated fomites. Risk assessment of the disease spreading requires quantitative knowledge about time and conditions needed for its inactivation in various material of pig origin. In this study we aimed to assess ASFV stability in naturally contaminated tissues during storage in selected environmental conditions. Virus half-life (T ½) and decimal reduction rate (D-value) were determined for temperatures relevant for freezing, chilling and ambient storage. A nonlinear regression model was developed to predict T ½ for temperatures between -20 °C and +23 °C. The half-life of the infectious ASFV in tissues ranged from 31.95 days at -20 °C to 0.38 days at +23 °C, with estimated D-values between 106.12-1.27 days, respectively. In order to assess the influence of environmental conditions on the rate of ASFV inactivation in decomposing tissue, viral half-life was evaluated at +4 °C and +23 °C in tissues stored within various matrices, mimicking possible natural conditions. Water, soil and leaf litter presence induced significantly faster ASFV inactivation. Straw, hay and grain provided stable conditions and prolonged virus viability for a considerable amount of time. In contrast to viable virus reduction over time, no change in ASFV DNA concentration was detected by real-time PCR. Based on estimated half-life values, the investigated tissues are predicted to remain infectious for 353-713 days at -20 °C, 35-136 days at +4 °C, and from 9 to 17 days at +23 °C. These data provide valuable information for the ASF preventive measures improvement.

African Swine Fever Virus - Persistence in Different Environmental Conditions and the Possibility of its Indirect Transmission.

Since 2007, African swine fever (ASF) has posed a serious threat to the European swine industry. In Poland, the numbers of reported outbreaks in pigs and affected areas grow every year. In 2018, the disease was noted in Western Europe, in Belgium specifically, where several hundred infected wild boars have been detected so far. In 2018, the virus unexpectedly emerged in pig holdings in eastern China, northern Mongolia, Vietnam, and Cambodia, causing worldwide concern about its further spread. Since there is still no vaccine available, the only approach to control the disease is biosecurity. Identification of potential sources of the virus is extremely important in light of its phenomenal survivability. The review summarises the current knowledge about ASFV survivability and resistance to environmental conditions, and discusses the role of indirect contact in spreading the disease.

The first complete genomic sequences of African swine fever virus isolated in Poland.

African swine fever (ASF) is a contagious, notifiable viral disease, which is considered a significant threat not only for European, but also for worldwide pork production, since recently the virus emerged within numerous Chinese pig herds. The disease was introduced in Poland in 2014 and up to the end of 2018,  213 outbreaks in pigs and 3347 cases in wild boars have been reported. The presented study describes the whole genome sequencing of seven Polish isolates, collected between 2016 and 2017, using next generation sequencing (NGS) technology. The complete, genomic sequences of these isolates were compared against five other closely related ASFV genomes, annotated in the NCBI database. The obtained sequences were from 189.393 to 189.405 bp long and encoded 187-190 open reading frames (ORFs). The isolates were grouped within genotype II and showed 99.941 to 99.956% nucleotide identity to the Georgia 2007/1 reference strain.