Antimetastatic effect of intratumoral Treg antagonists in mice with renal cancer.

The interplay of regulatory T cells (Tregs) within the tumour microenvironment presents a significant challenge in anticancer immunotherapy. This study investigates the potential of Treg blockade to enhance the efficiency of effector T cells. Two distinct treatment cocktails were examined: 3p-hpRNA (5' triphosphate hairpin RNA) combined with unmethylated CpG oligonucleotide (CpG); CpG in combination with OX40 receptor-specific monoclonal antibody (anti-OX40). Treatment efficacy was assessed using a murine model of kidney adenocarcinoma.Renca cells (renal cortical cells with adenocarcinoma) were subcutaneously engrafted in 30 BALB/c mice, then animals were allocated into three treatment groups: Group 1: CpG+anti-OX40, Group 2: CpG+3p-hpRNA, Group 3: untreated control. Treatment efficacy was evaluated based on tumour growth, the occurrence of metastases and overall survival.On day 28 post-implantation, experiments had to be terminated due to tumour progression. Although comparisons of survival times and primary tumour sizes thus became inconsequential, histological examinations provided valuable insights. We observed distinct variations in primary tumour characteristics among the different groups: Groups 1 and 2 displayed demarcations, while Group 3 exhibited diffuse tumours with necrosis. Lung metastases were evident in 70% of untreated mice, whereas none were observed in either of the treated groups.Our findings instil confidence in the potential efficacy of the treatments, thereby laying a solid foundation for future investigations.

A CTB-SARS-CoV-2-ACE-2 RBD Mucosal Vaccine Protects Against Coronavirus Infection.

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.

Asymptomatic and Mild SARS-CoV-2 Infections in a Hungarian Outpatient Cohort in the First Year of the COVID-19 Pandemic.

We aimed to estimate the proportion of the population infected with SARS-CoV-2 in the first year of the pandemic. The study population consisted of outpatient adults with mild or no COVID-19 symptoms and was divided into subpopulations with different levels of exposure. Among the subpopulation without known previous COVID-19 contacts, 4143 patients were investigated. Of the subpopulation with known COVID-19 contacts, 594 patients were investigated. IgG- and IgA-seroprevalence and RT-PCR positivity were determined in context with COVID-19 symptoms. Our results suggested no significant age-related differences between participants for IgG positivity but indicated that COVID-19 symptoms occurred most frequently in people aged between 20 and 29 years. Depending on the study population, 23.4-74.0% PCR-positive people (who were symptomless SARS-CoV-2 carriers at the time of the investigation) were identified. It was also observed that 72.7% of the patients remained seronegative for 30 days or more after their first PCR-positive results. This study hoped to contribute to the scientific understanding of the significance of asymptomatic and mild infections in the long persistence of the pandemic.

Metal-oxide inhalation induced fever - Immuntoxicological aspects of welding fumes.

Metal fume fever is a well-known occupational disease that arises from prolonged exposure to subtoxic levels of zinc oxide-containing fumes or dust. This review article aims to identify and examine the possible immunotoxicological effects of inhaled zinc oxide nanoparticles. The current most widely accepted pathomechanism for the development of the disease involves the formation of reactive oxygen species following the entry of zinc oxide particles into the alveolus resulting the release of pro-inflammatory cytokines by activation of the Nuclear Factor Kappa B transcriptional signal, thus evoking the symptoms. The role of metallothionein in inducing tolerance is believed to be a key factor in mitigating the development of metal fume fever. The other, poorly proven hypothetical route is that zinc-oxide particles bind to an undefined protein in the body as haptens to form an antigen and act as an allergen. After activation of the immune system, primary antibodies and immune complexes are developed and type 1. hypersensitivity reaction occurs, that can cause asthmatic dyspnoea, urticaria and angioedema. The development of tolerance is explained by the formation of secondary antibodies against primary antibodies. Oxidative stress and immunological processes cannot be completely separated from each other, as they can induce each other.

Effects of the combination of a monoclonal agonistic mouse anti-OX40 antibody and toll-like receptor agonists: Unmethylated CpG and LPS on an MB49 bladder cancer cell line in a mouse model.

PURPOSE: The basis of the antitumor immunotherapy, of which the purpose is the stimulation of the immune system. We have used two of the Pathogen Associated Molecular Patterns: unmethylated CpG oligonucleotide, a ligand of Toll-like receptor 9 (TLR9), and lipopolysaccharide (LPS) which is recognized by TLR4, combined with an agonistic OX40 receptor-specific monoclonal antibody (anti-OX40), which is expressed by activated regulatory T-cells (and by other activated T-cell populations as well). The objective of this study was to prove the effectiveness of the aforementioned compounds in an animal model, on a bladder cancer cell line. METHODS: We have instilled MB49 cells subcutaneously, to the left musculus biceps femoris. We have created three observation groups, each containing ten mice. After eleven days, all treated mice bearing the size of 5-8 mm (in diameter) tumor were administered CpG + anti-OX40 or LPS + anti-OX40 three times with a three-day lap between each treatment. Mice in the control group did not receive any treatment. RESULTS: All the specimens from the control and LPS + anti-OX40 groups have died by the sixtieth day of the observation period, however, five mice from the CpG + anti-OX40 group were still alive. The experiment lasted until the last surviving mouse died, which occurred on the 357th day after tumor implantation. DISCUSSION: The treatment with LPS did not make anti-OX40 more potent and did not increase the survival times. However, CpG + anti-OX40 has shown increased antitumor activity compared to the other two groups.

Analysis of the full-length VP2 protein of canine parvoviruses circulating in Hungary.

In recent years, the number of cases of disease caused by canine parvovirus 2 (CPV-2) in vaccinated dogs has increased. The aim of the present study was to identify CPV-2 strains present in Hungary. Forty-two out of 50 faecal specimens examined were positive, and 25 VP2 sequences were determined and analysed. Based on the current classification, the Hungarian viruses belong to New CPV-2a type, except two viruses that are recombinants of vaccine viruses and CPV-2a strains. The Tyr324Ile alteration was detected for the first time in Europe, and a "Hungarian-specific" substitution (Ala516Thr) was also identified in this study. The immunologically important parts of the currently spreading canine parvoviruses were examined and found to differ greatly from the vaccine strains that are widely used in Hungary.

Sporadic re-emergence of enzootic porcine transmissible gastroenteritis in Hungary.

Transmissible gastroenteritis (TGE) is a coronavirus-induced disease of pigs, characterised by diarrhoea and vomiting. The incidence of the disease had been decreasing since the late 1980s when deletion mutant variants (porcine respiratory coronavirus, PRCoV) of the virus emerged, repressing TGE gradually. Although disease manifestations are infrequent, the virus is still present in pig herds, causing sporadic outbreaks in a milder form. Identification and characterisation of the spike genes from TGEV and PRCoV, detected in such outbreaks, were performed in Hungary. Analysis of the amplified partial gene sequences showed that TGEV was present in herds with TGE clinical signs together with PRCoV. The sequences, apart from the deletions in PRCoV, were identical and at least two types of PRCoV spike proteins could be identified based on the length of the deleted sequence.

Emerging novel porcine parvoviruses in Europe: origin, evolution, phylodynamics and phylogeography.

To elucidate the spatiotemporal phylodynamics, dispersion and evolutionary processes underlying the emergence of novel porcine parvovirus 2 (PPV2), PPV3 and PPV4 species, we analysed all available complete capsid genes, together with ours, obtained in Europe. Bayesian phylogeography indicates that Romania (PPV2 and PPV4) and Croatia (PPV3) are the most likely ancestral areas from which PPVs have subsequently spread to other European countries and regions. The timescale of our reconstruction supported a relatively recent history of the currently circulating novel PPV species (1920s to 1980s) in the domestic or sylvatic host. While PPV2 strains exhibited a large genetic exchange characterized by significant recombination and gene flow between distinct regions and hosts, PPV3 and PPV4 showed a diversification reflected by the accumulation of geographically structured polymorphisms. The RNA-like evolutionary rates detected inter- and intrahost recombination and the positive selection sites provided evidence that the PPV2-4 capsid gene plays a prominent role in host adaptation.

Integrated circoviral rep-like sequences in the genome of cyprinid fish.

Recently a new group of circoviruses have been detected in tissues of Barbel fish and European catfish in Hungary. In our study circovirus genomes were screened in eight additional fish species for the detection and characterization of circoviruses. Two species of these bore circoviral sequences based on conventional PCR assay targeting the replication-associated protein coding gene fragments. Interestingly, the methods successfully used before failed to amplify other parts of the circular viral genome, suggesting the presence of partial, integrated genetic elements in the genome of the host. The successfully sequenced fragments of the Indian rohu (Labeo rohita) encoded mutations which may cause frameshifts or termination in the coding region described previously in other vertebrates. Phylogenetic analyses presumed that integration of the viral genetic elements might have progressed concurrently or following the diversification of cyprinid fish. Further studies on the nature of whole circovirus genomes and integrated elements may help to understand their potential role and evolution in different fish species.

Phylogeny and evolutionary genetics of porcine parvovirus in wild boars.

Porcine parvovirus (PPV) is widespread among swine and is responsible for reproductive failure of susceptible sows, characterized by embryonic and fetal death. Studies showed that PPV in domestic pig is genetically diverse and some strains differ from the ones used for vaccination. Organ samples from wild boars and domestic pigs were collected in Transylvania (Romania) and tested for the presence of PPV by polymerase chain reaction. Positive samples were grouped and 14 from the wild boar and 1 from the domestic pig PPVs were selected for VP1/VP2 sequence analysis and comparison with available GenBank data. The molecular clock analysis revealed that PPV has a relatively recent evolutionary history, originated approximately 120 years ago and the main divergence occurred in the last 20-60 years. Phylogenetic and residue substitution analysis showed that the viruses could be divided into 6 distinct clusters and that wild boar PPVs were partially different and independent from domestic pig PPVs. PPVs of wild boars proved to be more diverse than viruses of domestic pigs. The presence of the highly virulent 27a-like PPV strains in wild boars was also detected.

Novel circovirus in European catfish (Silurus glanis).

Circular single-stranded DNA viral genomes had been identified worldwide in different species and in environmental samples. Among them, viruses belonging to the genus Circovirus of the family Circoviridae are present in birds and pigs, and recently, they were detected in barbels. The present study reports the identification of a new circovirus in fish. PCR amplification and sequencing were used to identify the novel circular DNA virus in European catfish (Silurus glanis). Full genome characterization and phylogenetic analysis showed that the virus belonged to the family Circoviridae and that it was distantly related to the previously described barbel circovirus.

Detection, prevalence and analysis of emerging porcine parvovirus infections.

A number of newly identified porcine parvoviruses had been described during the last decade, but the presence and prevalence of these viruses are unknown in Hungary and only partly known for Europe. The present study was conducted to detect and measure the prevalence of these viruses, namely porcine parvovirus (PPV) 2, PPV3, PPV4, porcine bocavirus (PBoV) 1, PBoV2, PBo-likeV and the 6V and 7V parvoviruses. The prevalence of PPV1 and porcine circovirus type 2 (PCV2) was also investigated. Faecal samples, blood serum samples, organ tissues, foetuses and semen were collected from different swine herds in Hungary and tested by polymerase chain reaction methods specific for the different viruses. The results indicated that all of the examined parvoviruses were present in Hungary, hence in Europe. The prevalence was 18.1% for PCV2, 0.5 % for PPV1, 6.4% for PPV2, 9.7% for PPV3, 6.4% for PPV4, 1.5% for PBo-likeV, 4.8% for PBoV1 and PBoV2 and 1.8% for 6V and 7V. Based on the analysis of partial PPV4 and PBo-likeV sequences, these viruses showed a high degree of sequence conservation, whereas PPV3 and the majority of PPV2, PBoV1, PBoV2, 6V and 7V sequences showed higher variability. Possible sites of recombination were also identified between PBoV1 and PBoV2 genomes.

Detection of natural inter- and intra-genotype recombination events revealed by cap gene analysis and decreasing prevalence of PCV2 in wild boars.

Porcine circovirus type 2 (PCV2), the causative agent of a number of PCVAD (porcine circovirus associated diseases), is ubiquitous in domestic pig and wild boar populations. In the present study, using recombination detection program, phylogenetic analysis and base-by-base comparison of 28 PCV2 ORF2s (capsid protein coding gene) from wild boars and 8 from domestic pigs of Transylvania, recent natural intra- (PCV2b-1B/PCV2b-1C) and inter-genotype (PCV2a-2D/PCV2b-1C) recombination events were detected. Notably, one potential recombinant (F1-21) was detected in domestic pig with possible parental strains of wild boar origin. The estimated recombinant breakpoints comprised epitopes A, B and C of ORF2, without major changes in amino acid sequences. The prevalence of PCV2 in the wild boar population during the 5-year period following the first outbreaks of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs in Romania showed a decrease from 13.4% to 8.3%. To our knowledge, this is the first study to show the existence of ORF2-based intra- and inter-genotype recombination in wild boar populations and the possible recombination between PCV2 strains of wild boars and domestic pigs. Our results suggest a certain independence of PCV2 infection in wild boar populations and demonstrate the possibility of infection with multiple PCV2 genotypes under natural circumstances. On the other hand, PCV2 genotypes specific for wild boars could be detected in domestic pig at lower frequency suggesting the possible spread of wild boar PCV2 to domestic swine. The recombination events described here may contribute to the genetic diversity of PCV2 and may also be the source of emergence of new PCV2 strains.

Genetic diversity of pigeon circovirus in Hungary.

Pigeon circoviruses (PiCV) had been identified worldwide and are responsible for immune suppression and a variety of diseases collectively referred to as young pigeon disease syndrome. Samples from racing pigeons were collected throughout Hungary and analyzed for the presence of PiCV by polymerase chain reaction. The capsid protein coding gene was amplified from ten PiCVs of different origins, and compared with known PiCV sequences. The results indicated that PiCV was highly variable, the viruses formed five distinct genetic groups. Differences of the 3' end of the gene suggested the possibility of genetic recombination among these groups.

Distribution and genetic diversity of porcine hokovirus in wild boars.

Porcine hokovirus (PHoV), a newly discovered member of the family Parvoviridae and the proposed genus Hokovirus, is considered phylogenetically distinct from other parvoviruses. Here, we report a comprehensive spatio-temporal study of PHoV infection in Romanian wild boars. The prevalence of PHoV differed significantly in samples from 2006/2007 (22.76%) and 2010/2011 (50.54%), and also increased with age. Sequence analysis of PHoVs from 2006/2007 showed a close relationship to PHoVs from pigs from England and wild boars from Germany, while the PHoVs from 2010/2011 were mostly similar to isolates from Hong Kong. The most variable regions were detected in the NS1 gene and proved to be suitable for analysis of the genetic diversity of the virus. It was observed that PHoVs from older wild boar samples differed from those collected recently. These results suggested that porcine hokovirus could be a newly emerging virus of both domestic and wild pigs with yet unknown implications.

Genetic detection and analysis of porcine bocavirus type 1 (PoBoV1) in European wild boar (Sus scrofa).

Novel porcine parvoviruses showing the genetic characteristics of bocaviruses have recently been identified. The first such porcine bocavirus (PoBoV1), described as boca-like virus (PBo-likeV), was discovered in PMWS affected pigs in Sweden. Later, several other bocaviruses with divergent genomes were reported under various names in domestic pigs. This is the first report of the presence of bocaviruses in European wild boars. 842 wild boar samples originating from the Western region of Romania (Transylvania) were collected during the 2006/2007 and the 2010/2011 hunting seasons and tested for the presence of PoBoV1 by polymerase chain reaction and sequencing. The results showed 12.94% (109/842) overall positivity, with an increasing prevalence from the 2006/2007 (9.14%, 43/470) to the 2010/2011 (17.74%, 66/372) season (P < 0.01). Differences between the prevalence of the virus in 6-12-month-old-animal (77.06%, 84/109) and 12-36-month-old-animal (22.94%, 25/109) (P < 0.01) indicated that the infection occurred mainly in younger pigs. Comparative sequence analysis of partial VP1/2 genes from wild boars and those available in the GenBank showed only minor differences, indicating that PoBoV1 circulating within the wild boar populations and domestic pigs from different geographic regions were highly similar.

First detection and analysis of a fish circovirus.

Circoviruses are present worldwide in birds and pigs but their occurrence in fish has not yet been reported. Recently, increased mortality was observed in barbel fry (Barbus barbus) in Hungary. This paper reports the detection of previously unknown circular viral DNA genomes in barbels by the use of a circovirus-specific wide-range nested PCR. The analysis of two complete genomes (Barbel circovirus, BaCV1 and BaCV2) indicated that they belonged into a new genetic group within the family Circoviridae, distinct from known circoviruses and circovirus-like genomes. Their genome size was 1957 bases and contained two major ORFs similar to the capsid and replication-associated protein genes of circoviruses. A connection between the presence of the virus and clinical manifestations of the infection could not be proved.

Prevalence of porcine circoviruses in Transylvanian wild boars, detected by real-time PCR--short communication.

Porcine circoviruses (PCV) are widespread in domestic pigs worldwide and there is growing information about the presence of PCV in other suid species. Based on serological studies with sera of wild boars, it was established that PCV1 was present in these animals and antibodies specific to PCV2 were also detected in wild boars living in captivity or in sylvatic areas, both with or without clinical signs of PMWS. Studies including PCV2 genome or antigen detection confirmed the previous findings. This is the first report about the presence of PCV in Transylvanian wild boar populations. Four hundred and sixty-nine samples were collected and grouped according to geographic origin, tested for the presence of PCV DNA using a real-time quantitative polymerase chain reaction assay, and 13.52% of the animals proved to be positive for one or in three cases both of the PCV genotypes. PCV2 was detected in all of the PCV-positive samples.

Detection of porcine circovirus in rodents - short communication.

Porcine circoviruses (PCV) are present worldwide, infecting domestic pigs and wild boars alike. Studies under laboratory conditions indicated that PCV can be taken up by mice and the virus can replicate in these animals. The possible role of rodents in maintaining and transmitting PCV2 infection in the field has not been investigated yet. The present study reports the detection of PCV2, the pathogenic form of the virus, in mice and rats. A number of rodents, such as mice, rats and voles, were collected at PCV2-infected farms and also outside pig herds and tested for the presence of the virus by polymerase chain reaction (PCR). The results indicated that PCV2 can be present both in mice and rats (65.0% and 23.8% positivity, respectively) on the infected premises, but those rodents that were collected outside pig farms remained negative for PCV2.