The surface modification of the silica-coated magnetic nanoparticles and their application in molecular diagnostics of virus infection.

The study presents a series of examples of magnetic nanoparticle systems designed for the diagnosis of viral diseases. In this interdisciplinary work, we describe one of the most comprehensive synthetic approaches for the preparation and functionalization of smart nanoparticle systems for rapid and effective RT-PCR diagnostics and isolation of viral RNA. Twelve different organic ligands and inorganic porous silica were used for surface functionalization of the Fe3O4 magnetic core to increase the number of active centres for efficient RNA binding from human swab samples. Different nanoparticle systems with common beads were characterized by HRTEM, SEM, FT-IR, XRD, XPS and magnetic measurements. We demonstrate the application of the fundamental models modified to fit the experimental zero-field cooling magnetization data. We discuss the influence of the nanoparticle shell parameters (morphology, thickness, ligands) on the overall magnetic performance of the systems. The prepared nanoparticles were tested for the isolation of viral RNA from tissue samples infected with hepatitis E virus-HEV and from biofluid samples of SARS-CoV-2 positive patients. The efficiency of RNA isolation was quantified by RT-qPCR method.

SARS-CoV-2: Zoonotic origin of pandemic coronavirus.

A novel disease, of unknown origin, causing a deadly pneumonia of human patients was reported in December 2019 in Wuhan, Hubei province of China. Later called coronavirus disease (COVID-19), it rapidly spread across China and worldwide. Intensive research revealed that the etiological agent of the global COVID-19 pandemic was a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome contains typical coronavirus genes but the receptor binding domain (RBD) in the S protein is highly specific. The site for furin-like protease cleavage of the S protein into S1 and S2 subunits is also unique. Further analyses suggested that SARS-CoV-2 is of zoonotic origin. The analysis of the SARS-CoV-2 genome, especially the S gene, shows that natural evolutionary process between a bat-CoV and a pangolin-CoV or other animal coronavirus could have been important in creating SARS-CoV-2, with transmission of novel virus to the human population. On the other hand, new analyses indicate that SARS-CoV-2 is not a recombinant virus. Keywords: COVID-19; SARS-CoV-2; genome; bat coronavirus; pangolin coronavirus.

Genetic variability of pig and human rotavirus group A isolates from Slovakia.

The aim of this work was the genetic typing of RVA isolates originating from pigs and human patients in Slovakia. Seventy-eight rectal swabs from domestic pigs and 30 stool samples from humans were collected. The whole VP7 (G genotypes), VP6 (I genotypes) and partial VP4 (P genotypes) ORFs were amplified by RT-PCR. Genetic variability was higher amongst porcine sequences, where four G genotypes (G3, G4, G5, G11), two P genotypes (P[6], P[13]) and one I5 genotype were detected. Human RVA strains were represented by two G genotypes (G1, G3), two I genotypes (I1, I2), and one P genotype (P[8]). Genetic analysis did not show a relationship between Slovakian porcine and human RVA strains, but phylogenetic grouping of some Slovakian porcine sequences with Hungarian human sequences in both G and P genotypes was observed.

Systemic immune response of gnotobiotic mice infected with porcine circovirus type 2 after administration of Lactobacillus reuteri L26 Biocenol™.

In our previous study we confirmed an antiviral activity of probiotic Lactobacillus reuteri L26 which was mediated by stimulation of local intestinal immunity. The aim of this paper was to evaluate the influence of L. reuteri L26 on the systemic immune response in gnotobiotic mice infected with porcine circovirus type 2 (PCV2). A total of 30 germ-free mice were divided into 3 groups and animals in noninfected and infected control groups (NC and IC; n=10) received sterile de Man-Rogosa-Sharpe broth for 7 days and animals in experimental group L+PCV (n=10) were inoculated with L. reuteri L26. Subsequently, mice in L+PCV and IC groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). The results showed an increase of percentage of cytotoxic cells (CD8+ and CD49b+CD8-) and oxidative burst of phagocytes, up-regulation of the gene expression of RANTES, granulocyte-macrophage colony-stimulating factor, interferon-γ and immunoglobulin A in blood above all in the later phase of infection (14 dpi) in L+PCV group accompanied by higher load of PCV2 in the serum. These findings indicate that L. reuteri L26 has a potential to induce systemic immune reaction, but in gnotobiotic mice immune stimulation can increase virus replication.

Influence of Lactobacillus reuteri L26 Biocenol™ on immune response against porcine circovirus type 2 infection in germ-free mice.

Probiotic bacteria are frequently used for prevention of bacterial infections of the gastrointestinal tract, but there are only limited studies on their efficacy against viral gut infections in animals. The aim of this study was to investigate the effect of probiotic Lactobacillus reuteri L26 BiocenolTM on the innate and adaptive immune responses in germ-free Balb/c mice, experimentally infected by porcine circovirus type 2 (PCV2), which confers immunosuppressive effect. A total of 30 six-week-old female mice were divided into 3 groups and animals in experimental group LPCV (n=10) were inoculated with L. reuteri L26, animals in the control group (C; n=10) and experimental group PCV (n=10) received sterile De Man-Rogosa-Sharpe broth for 7 days. Subsequently, mice from both experimental groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). Virus load in faeces, ileum and mesenteric lymph nodes (MLN); as well as gene expression of selected cytokines, immunoglobulin A (IgA) and polymeric Ig receptor (PIgR) in the ileum, and percentage of CD8+, CD19+ and CD49b+CD8- cells in the MLN were evaluated. Our results showed that L. reuteri significantly decreased the amount of PCV2 in faeces and in the ileum, and up-regulated the gene expression of chemokines, interferon (IFN)-γ, IgA and PIgR in the ileum. Increased IFN-γ mRNA level was accompanied by higher proportion of natural killer cells and up-regulated IgA and PIgR gene expressions were in accordance with significantly higher percentage of CD19+ lymphocytes in the MLN. These findings indicate that probiotic L. reuteri has an antiviral effect on PCV2 in the intestine which is mediated by stimulation of local gut immune response.

Genetic variability of the S1 subunit of enteric and respiratory bovine coronavirus isolates.

Bovine coronavirus (BCoV) is considered an important pathogen in cattle worldwide. It is a causative agent of enteric and respiratory diseases of cattle. The S1 subunit of the viral S glycoprotein is responsible for virus binding to host-cell receptors, induction of neutralizing antibody and hemagglutinin activity. The entire S1 genomic region (2304 bp) of two enteric bovine coronavirus isolates from Austria, one respiratory and one enteric isolate from Slovakia were analyzed at the genetic level. The comparative analysis of those four isolates revealed 97.1-98.6% similarity at the nucleotide and 95.6-98.6% at the amino acid level. No differences between enteric and respiratory isolates were observed at the genetic level. The isolates were clustered in the phylogenetic tree with European isolates independently of their enteric or respiratory origin.

Genetic typing of porcine reproductive and respiratory syndrome virus isolates from central European countries.

All thirty-three but one porcine reproductive and respiratory syndrome virus (PRRSV) isolate originating from pigs in Austria, Czech Republic and Slovakia were typed on the basis of partial ORF5 sequence as PRRSV-1, subtype EU-1. The single isolate of PRRSV-2 originated from Slovakia.

Genetic characterization of a border disease virus isolate originating from Slovakia.

In this study, a major part of genome of the pestivirus isolate 297 from Slovakia, comprising the 7195 nt-long 5΄-UTR-NS3 region was sequenced and analyzed. Conserved cleavage sites between individual viral proteins of this region were determined and the number of amino acids of respective proteins was estimated as follows: 168 for Npro, 100 for C, 227 for Erns, 195 for E1, 373 for E2, 70 for p7, 453 for NS2, and 683 for NS3. Based on sequence and phylogenetic analysis of 5΄-UTR, Npro, and E2 the isolate 297 was characterized as a border disease virus of genotype 3. It was found to be distinct from other BDV-3 strains analyzed so far, consequently forming a distinct branch within the phylogenetic clade. All these data expand a relatively limited knowledge of genetic properties of individual BDV genotypes and strains circulating in the Central Europe.

Genetic typing of porcine circovirus type 2 (PCV-2) isolates from Slovakia.

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n=10) into two known genotypes, PCV-2a (n=1) and PCV-2b (n=9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe.

Genetic analysis of a bovine viral diarrhea virus 2 isolate from Slovakia.

The identification and genetic characterization of bovine viral diarrhea virus (BVDV) isolate 17237 detected in western Slovakia is described. The analysis of 5'-untranslated region (5'-UTR), autoprotease (Npro) gene, and structural genes (C, Erns, E1, E2) was carried out. The percentage of nucleotide and deduced amino acid identity in analyzed genes implied that the isolate was closely related to the bovine viral diarrhea virus 2 (BVDV-2). Furthermore, the phylogenetic analysis revealed that this isolate fall into BVDV-2b subtype that is sporadic in Europe. The cleavage sites between viral proteins were similar to the ones of a reference strain of BVDV-2.

Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries).

The wide spread prevalence of bovine viral diarrhea virus type 1 (BVDV-1) in cattle and recent identification of BVDV-2 in goats in India warranted pestivirus surveillance in sheep. Nested reverse transcription-polymerase chain reaction (RT-PCR) was used to detect BVDV-2 in one of 1561 blood samples collected randomly from 78 sheep flocks in 11 states of India. Antigenic characterization of the isolated pestivirus using polyclonal and monoclonal antibodies typed the isolate as BVDV-2. When analyzed at genetic level in N(pro) (N-terminal autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein and envelope proteins E(rns) (ribonuclease secreted), E1 and E2 genomic organization was the same for all pestiviruses, the nucleic acid and amino acid sequences showed highest similarity with those of BVDV-2. When compared with BVDV-2 isolate 890, the sequence homology was 83.7% for C, 84.6% for E(rns) and E1, and 81.5% for E2. The cleavage site C/E(rns) was found totally conserved while N(pro)/C was conserved only from C-terminus of N(pro), E(rns)/E1 site was conserved only from C-terminus of E(rns) and E1/E2 site was conserved only from C-terminus of E1. Phylogenetic analysis of nucleotide sequences in 5' untranslated regions (UTR), N(pro), E2, NS3 and NS5B regions placed the sheep isolate in a separate clade within BVDV-2 subtype b. This was supported by the presence of unique mutations in the structural protein coding regions beside NS3 and NS5B. To our knowledge this is the first report on the sequence analysis of the entire structural gene coding region of a BVDV-2b isolate. This is also the first occurrence of BVDV-2 subtype b in sheep, providing the evidence that this subtype can also occur in species other than cattle.

Identification of bovine viral diarrhea virus type 1 in yaks (Bos poephagus grunniens) in the Himalayan region.

Since cattle are widely infected by bovine viral diarrhea virus (BVDV) in India, we searched for pestivirus infection in yaks. Of 71 pure and crossbred yaks from Himalayan region, pestivirus antigen was detected by Ag-ELISA in three animals. Pestivirus in leukocyte and cell culture isolated virus samples originating from positive yaks was also confirmed by RT-PCR using panpestivirus specific primers selected from 5'-untranslated region (5' UTR). The 5' UTR, N(pro) and E2 regions were sequenced and used for genetic typing. Phylogenetic analysis revealed that pestiviruses detected in three Himalayan yaks were similar genetically, belonging to BVDV-1. Antigenic characterisation of yak pestivirus also confirmed the typing as BVDV-1. This is the first report on the identification of BVDV type 1 in yaks.

Influence of communal alpine pasturing on the spread of pestiviruses among sheep and goats in Austria: first identification of border disease virus in Austria.

The purpose of this investigation was to determine the influence of communal Alpine pasturing on the spread of pestivirus infections among sheep and goats. The study included 481 sheep from 23 farms and 131 goats from 26 farms pastured on separated Alpine meadows in the western part of Austria. At the starting of pasturing on the sheep meadow, 325 (67.6%) animals were seropositive, on the goat meadows in 16 (12.2%) samples antibodies to pestiviruses were detected. At the end of pasturing, 74 seronegative sheep and two seronegative goats had seroconverted. Between the beginning and the end of pasturing the seroprevalence in sheep increased significantly from 67.6% to 83% (P<0.05). Moreover, in the peripheral blood mononuclear cells of four sheep, pestivirus-specific RNA was detected before as well as after pasturing; these animals remained serologically negative throughout the investigation. They were, therefore, identified as persistently infected. Sequence analysis in the N(pro) region revealed that the detected pestiviruses were the same at genetic level and they were grouped into the Border disease virus (BDV)-3 genotype. No pestivirus RNA was found in goat samples. The results of this survey indicate that communal Alpine pasturing does play a key role in the spread of BDV. Moreover, BDV has been identified and characterized for the first time in sheep in Austria, which until then had been regarded as being free from BD.

Genetic variability of bovine viral diarrhoea virus subtypes at 3'-nontranslated region.

Ten isolates belonging to different BVDV-1 subtypes and three BVDV-2 isolates were studied in 3'-nontranslated region (3'-NTR) by sequencing of PCR products and comparative computer-assisted nucleotide sequence analysis. The alignment of nucleotide sequences revealed that all BVDV-1 isolates except BVDV-1a isolates represented by NADL strain were characterised with a deletion of 38 nucleotides in the variable region located after the stop codon. For all BVDV-1 subtypes, a constant region at the end of viral genome was highly conserved. The variable and constant regions with no significant insertions or deletions were also identified in BVDV-2 isolates. The poly AT reach region was situated at different locations in both pestiviruses.

Pestiviruses in wild animals.

Pestiviruses are not strictly host-species specific and can infect not only domestic but also wild animals. The most important pestivirus, CSFV, infects domestic pigs and wild boars, which may cause a major problem for successful CSFV eradication programmes. Mainly BVDV specific antibodies have been reported in captive and free-living animals. Virus has been isolated from some of these animal species, but since BVDV can contaminate cell cultures and foetal calf serum, early reports of BVDV isolation have to be considered with caution. Genetic typing of early pestivirus isolates from wild species revealed that the majority were BVDV-1. Of the pestiviruses identified so far three species (CSFV, BVDV-1, giraffe pestivirus) and three genotypes (BDV-2, BDV-4, pronghorn) appear to circulate in wildlife animal populations. The potential for pestiviruses to spread between farm animals and free-living animals is discussed as are epidemiological and technical problems, and the future direction of research.

Genetic analysis of indian bovine viral diarrhea virus 1 isolates in N(pro) and entire gene region coding structural proteins.

Three Indian Bovine viral diarrhea virus 1 (BVDV-1) isolates were analyzed at genetic level in N(pro) (viral autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein, E(rns), and envelope proteins E1 and E2. All these isolates were found to be of b subtype based on the entire 504 nt region of N(pro) and 1119 nt region of E2. However, in comparison with other isolates of this subtype, they were allocated inside the BVDV-1 subtype b cluster to a separate clade with a longer distance. Of six cysteine residues in N(pro) only three were totally conserved in all three isolates. The isolates showed 94.9-99.3% and 92.2-99.0% identities for the entire C-E2 gene region at nucleotide and amino acid levels, respectively. The lowest identity values (88.5-91.7%) were observed for E2 amino acid sequences. The identity of the isolates with Osloss, a reference BVDV-1 subtype b strain, was in the range of 82.1-89.9% for nucleotide and 78.6-89.2% for amino acid sequences in the C-E2 region. The N(pro)/C and E(rns)/E1 cleavage sites were highly conserved. The C/E(rns) and E1/E2 cleavage sites were more conserved from the N-end of E(rns) and the C-end of El, respectively. These findings suggest that some unique mutations have occurred in the described Indian BVDV-1 isolates, though they all belong to the BVDV-1 subtype b.

Detection of bovine torovirus in neonatal calf diarrhoea in Lower Austria and Styria (Austria).

Faeces of 230 calves with and without diarrhoea collected during the winter period 2004/2005 in 100 Austrian farms (Styria and Lower Austria) were examined for viral, bacterial and parasitic enteropathogens. Torovirus-specific nucleic acid confirmed by reverse transcriptase-polymerase chain reaction was found in 12 of 230 calves (5.2%). Ten of these calves were clinically ill, several of them showing signs of dehydration and abnormal faecal consistency at the time of sampling. Computer assisted analysis of two nucleotide sequences obtained from Austrian bovine samples revealed 93% similarity to Breda strain, but only 71% or 52% similarity to Equine Berne or Porcine Markelo torovirus strains respectively. Phylogenetic analysis grouped Austrian torovirus samples into the Bovine torovirus cluster indicating the first detection of Bovine torovirus in Austria. In addition, the following agents were detected in bovine faecal samples: Bovine coronavirus, 25.7%; Escherichia coli, 17%; Cryptosporidium spp., 11.7%; Eimeria spp., 10.4%; Rotavirus, 9.1%; Clostridium perfringens, 9.1% and Giardia spp., 6.1%. Salmonella spp. was not detected.

Genetic diversity of BVDV: consequences for classification and molecular epidemiology.

Genetic typing of bovine viral diarrhoea virus (BVDV) is important for the precise classification of viruses as well as for the development of molecular epidemiology. BVDV isolates were usually typed based on comparison of genomic sequences from the 5'-untranslated region (5'-UTR), N(pro) and E2 region. Recently we have identified 11 genetic groups (subgenotypes) of BVDV-1. Our further experiments confirmed a new subgenotype, BVDV-1k, isolated from cattle in Switzerland. BVDV isolates from India were typed as BVDV-1b whereas BVDV-1c is a predominant subgenotype in Australia. The results of genetic typing of BVDV indicate that distribution of subgenotypes has no relationship to the geographic origin of viral isolates.