Anelloviridae taxonomy update 2023.

The family Anelloviridae comprises negative single-stranded circular DNA viruses. Within this family, there are 30 established genera. Anelloviruses in the genus Gyrovirus have been identified infecting various avian species, whereas those in the remaining 29 genera have been found primarily infecting various mammal species. We renamed the 146 anellovirus species with binomial species names, as required by the International Committee on Taxonomy of Viruses (ICTV) using a "genus + freeform epithet" format.

Examination of Immunogenic Properties of Recombinant Antigens Based on p22 Protein from African Swine Fever Virus.

Introduction: The single member of the Asfarviridae family is African swine fever virus (ASFV). This double-stranded DNA virus infects wild and farmed swine and loses the pig industry large sums of money. An inner envelope, capsid, and outer envelope are parts of the ASFV particle containing structural proteins playing different roles in the process of infection or host immune defence evasion. When expressed by the baculovirus system, the p22 protein from the inner envelope was found to induce partial protection against a virulent virus strain. This study aimed to express a part of this protein in a different system and evaluate its immunogenicity. Material and Methods: We designed two proteins, the extracellular (C terminal) part of the p22 protein (p22Ct) and its fusion with the heat-labile enterotoxin B subunit from Escherichia coli (LTB-p22Ct), which is supposed to be a potent enhancer of the immune response. Both proteins were produced in the E. coli expression system and subsequently used for mice immunisation to analyse their safety and immunogenicity. Results: The protein fused with LTB did not show the expected adjuvant properties and did not prove safe, because abscess formation was observed after immunisation. In contrast, immunisation with the p22Ct protein alone induced a higher antibody titre but caused no adverse symptoms. Conclusion: These results show the high potential of the p22Ct region as an immunogenic protein for ASFV serological detection purposes.

Taxonomic updates for the genus Gyrovirus (family Anelloviridae): recognition of several new members and establishment of species demarcation criteria.

The genus Gyrovirus was assigned to the family Anelloviridae in 2017 with only one recognized species, Chicken anemia virus. Over the last decade, many diverse viruses related to chicken anemia virus have been identified but not classified. Here, we provide a framework for the classification of new species in the genus Gyrovirus and communicate the establishment of nine new species. We adopted the 'Genus + freeform epithet' binomial system for the naming of these species.

Taxonomic update for mammalian anelloviruses (family Anelloviridae).

Anelloviruses are small negative-sense single-stranded DNA viruses with genomes ranging in size from 1.6 to 3.9 kb. The family Anelloviridae comprised 14 genera before the present changes. However, in the last five years, a large number of diverse anelloviruses have been identified in various organisms. Here, we undertake a global analysis of mammalian anelloviruses whose full genome sequences have been determined and have an intact open reading frame 1 (ORF1). We established new criteria for the classification of anelloviruses, and, based on our analyses, we establish new genera and species to accommodate the unclassified anelloviruses. We also note that based on the updated species demarcation criteria, some previously assigned species (n = 10) merge with other species. Given the rate at which virus sequence data are accumulating, and with the identification of diverse anelloviruses, we acknowledge that the taxonomy will have to be dynamic and continuously evolve to accommodate new members.

The Main DNA Viruses Significantly Affecting Pig Livestock.

Swine DNA viruses have developed unique mechanisms for evasion of the host immune system, infection and DNA replication, and finally, construction and release of new viral particles. This article reviews four classes of DNA viruses affecting swine: porcine circoviruses, African swine fever virus, porcine parvoviruses, and pseudorabies virus. Porcine circoviruses belonging to the Circoviridae family are small single-stranded DNA viruses causing different diseases in swine including poly-weaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, and porcine respiratory disease complex. African swine fever virus, the only member of the Asfivirus genus in the Asfarviridae family, is a large double-stranded DNA virus and for its propensity to cause high mortality, it is currently considered the most dangerous virus in the pig industry. Porcine parvoviruses are small single-stranded DNA viruses belonging to the Parvoviridae family that cause reproductive failure in pregnant gilts. Pseudorabies virus, or suid herpesvirus 1, is a large double-stranded DNA virus belonging to the Herpesviridae family and Alphaherpesvirinae subfamily. Recent findings including general as well as genetic classification, virus structure, clinical syndromes and the host immune system responses and vaccine protection are described for all four swine DNA virus classes.

Assessment of Antimicrobic, Antivirotic and Cytotoxic Potential of Alginate Beads Cross-Linked by Bivalent Ions for Vaginal Administration.

Antimicrobial agent abuse poses a serious threat for future pharmacotherapy, including vaginal administration. The solution can be found in simple polymeric systems with inherent antimicrobial properties without the need to incorporate drugs, for instance alginate beads cross-linked by bivalent ions. The main goal of the presented study was to provide improvement on the well-documented cytotoxicity of Cu2+ cross-linked alginate. Alginate beads were prepared by external ionotropic gelation by cross-linking with Cu2+, Ca2+ and Zn2+ ions, separately and in mixtures. Morphological properties, swelling capacity, ion release and efficacy against the most common vaginal pathogens (C. albicans, E. coli, E. faecalis and virus strain-human herpesvirus type 1) were evaluated. The prepared particles (particle size 1455.68 ± 18.71-1756.31 ± 16.58 µm) had very good sphericity (0.86 ± 0.04-0.97 ± 0.06). In mixture samples, Cu2+ hampered second ion loading, and was also released incompletely (18.75-44.8%) compared to the single ion Cu2+ sample (71.4%). Efficacy against the selected pathogens was confirmed in almost all samples. Although anticipating otherwise, ion mixture samples did not show betterment over a Cu2+ cross-linked sample in cytotoxicity-pathogen efficacy relation. However, the desired improvement was found in a single ion Zn2+ sample whose minimal inhibition concentrations against the pathogens (0.6-6.12 mM) were close to, or in the same mathematical order as, its toxic concentration of 50 (1.891 mM). In summary, these findings combined with alginate's biocompatibility and biodegradability give the combination solid potential in antimicrobial use.

Dynamics and Differences in Systemic and Local Immune Responses After Vaccination With Inactivated and Live Commercial Vaccines and Subsequent Subclinical Infection With PRRS Virus.

The goals of our study were to compare the immune response to different killed and modified live vaccines against PRRS virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. In the experiment, we immunized four groups of piglets with two commercial inactivated (A1-Progressis, A2-Suivac) and two modified live vaccines (B3-Amervac, B4-Porcilis). Twenty-one days after the final vaccination, all piglets, including the control non-immunized group (C5), were i.n., infected with the Lelystad strain of PRRS virus. The serum antibody response (IgM and IgG) was the strongest in group A1 followed by two MLV (B3 and B4) groups. Locally, we demonstrated the highest level of IgG antibodies in bronchoalveolar lavages (BALF), and saliva in group A1, whereas low IgA antibody responses in BALF and feces were detected in all groups. We have found virus neutralization antibody at DPV 21 (days post vaccination) and higher levels in all groups including the control at DPI 21 (days post infection). Positive antigen specific cell-mediated response in lymphocyte transformation test (LTT) was observed in groups B3 and B4 at DPV 7 and in group B4 at DPV 21 and in all intervals after infection. The IFN-γ producing lymphocytes after antigen stimulation were found in CD4-CD8+ and CD4+CD8+ subsets of all immunized groups 7 days after infection. After infection, there were obvious differences in virus excretion. The virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at DPI 3. Significantly lower virus load was found in groups A1 and B3 at DPI 21. Negative samples appeared at DPI 21 in B3 group in saliva. It can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. Immunization with inactivated vaccine A1-Progressis induces high levels of antibodies produced both systemically and locally. Immunization with MLV-vaccines (Amervac and Porcilis) produces sufficient antibody levels and also cell-mediated immunity. After infection virus secretion gradually decreases in group B3, indicating tendency to induce sterile immunity.

PARV4 found in wild chimpanzee faeces: an alternate route of transmission?

Human parvovirus 4 (PARV4, family Parvoviridae, genus Tetraparvovirus) displays puzzling features, such as uncertain clinical importance/significance, unclear routes of transmission, and discontinuous geographical distribution. The origin, or the general reservoir, of human PARV4 infection is unknown. We aimed to detect and characterize PARV4 virus in faecal samples collected from two wild chimpanzee populations and 19 species of captive non-human primates. We aimed to investigate these species as a potential reservoir and alternate route of transmission on the African continent. From almost 500 samples screened, a single wild Pan troglodytes schweinfurthii sample tested positive. Full genome analysis, as well as single ORF phylogenies, confirmed species-specific PARV4 infection.

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.

Adenovirus infection in savanna chimpanzees (Pan troglodytes schweinfurthii) in the Issa Valley, Tanzania.

Adenoviruses are a widespread cause of diverse human infections with recently confirmed zoonotic roots in African great apes. We focused on savanna-dwelling chimpanzees in the Issa Valley (Tanzania), which differ from those from forested sites in many aspects of behavior and ecology. PCR targeting the DNA polymerase gene detected AdV in 36.7% (69/188) of fecal samples. We detected five groups of strains belonging to the species Human mastadenovirus E and two distinct groups within the species Human mastadenovirus C based on partial hexon sequence. All detected AdVs from the Issa Valley are related to those from nearby Mahale and Gombe National Parks, suggesting chimpanzee movements and pathogen transmission.

New adenoviruses from new primate hosts - growing diversity reveals taxonomic weak points.

The knowledge of the closest human relatives of human adenoviruses (AdVs) such as adenoviruses found in nonhuman primates is still limited, despite the growing importance of adenoviruses in vaccine development, gene and cancer therapy. We examined 153 stool samples of 17 non-human primate species and detected adenoviral DNA sequences of DNA polymerase (DPOL) gene in 54 samples (35%), originating from 12 out of 17 primate species. We further sequenced 15 hexon gene fragments and based on the phylogenetic analysis we propose two new provisional species SAdV-H and SAdV-I. Our study shows extensive diversity of adenoviral strains forming separate clades often from closely related host species from old world monkeys suggesting the existence of new species of AdVs and shows the necessity for clear ICTV guidelines for final establishment of so far provisional AdV species.

New species of Torque Teno miniviruses infecting gorillas and chimpanzees.

Anelloviridae family is comprised of small, non-enveloped viruses of various genome lengths, high sequence diversity, sharing the same genome organization. Infections and co-infections by different genotypes in humans are ubiquitous. Related viruses were described in number of mammalian hosts, but very limited data are available from the closest human relatives - great apes and non-human primates. Here we report the 100% prevalence determined by semi-nested PCR from fecal samples of 16 captive primate species. Only the Mandrillus sphinx, showed the prevalence only 8%. We describe three new species of gorillas׳ and four new species of chimpanzees׳ Betatorqueviruses and their co-infections in one individual. This study is also first report and analysis of nearly full length TTMV genomes infecting gorillas. Our attempts to sequence the complete genomes of anelloviruses from host feces invariably failed. Broader usage of blood /tissue material is necessary to understand the diversity and interspecies transmission of anelloviruses.

Expression and serological reactivity of hemorrhagic enteritis virus hexon protein.

The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus.

Genetic and phylogenetic characterization of novel bocaparvovirus infecting chimpanzee.

Primate bocaparvoviruses were first described in 2005, since then further human and gorilla bocaparvoviruses have been identified. To uncover diversity of non-human primates' bocaparvoviruses, their phylogenetic relationship and potential to cross the host species barrier, we tested 153 fecal samples from 17 captive primate species. The only one captive female of central chimpanzee (coded CPZh2) has been identified as bocaparvovirus positive. Based on the full genome phylogenetic analyses, CPZh2 strain shows close relationship to HBoV3 and GBoV. Further recombination analysis confirmed expected mosaic origin of CPZh2 strain. According the phylogenetic position, following the ICTV recommendations, we propose a novel genotype within the Primate bocaparvovirus 1 species infecting chimpanzee.

In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife.

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.

Antimicrobial Properties of Microparticles Based on Carmellose Cross-Linked by Cu(2+) Ions.

Carmellose (CMC) is frequently used due to its high biocompatibility, biodegradability, and low immunogenicity for development of site-specific or controlled release drug delivery systems. In this experimental work, CMC dispersions in two different concentrations (1% and 2%) cross-linked by copper (II) ions (0.5, 1, 1.5, or 2.0 M CuCl2) were used to prepare microspheres with antimicrobial activity against Escherichia coli and Candida albicans, both frequently occurring pathogens which cause vaginal infections. The microparticles were prepared by an ionotropic gelation technique which offers the unique possibility to entrap divalent copper ions in a CMC structure and thus ensure their antibacterial activity. Prepared CMC microspheres exhibited sufficient sphericity. Both equivalent diameter and copper content were influenced by CMC concentration, and the molarity of copper (II) solution affected only the copper content results. Selected samples exhibited stable but pH-responsive behaviour in environments which corresponded with natural (pH 4.5) and inflamed (pH 6.0) vaginal conditions. All the tested samples exhibited proven substantial antimicrobial activity against both Gram-negative bacteria Escherichia coli and yeast Candida albicans. Unexpectedly, a crucial parameter for microsphere antimicrobial activity was not found in the copper content but in the swelling capacity of the microparticles and in the degree of CMC surface shrinking.

Whole genome sequencing and phylogenetic analysis of feline anelloviruses.

Torque teno felis virus (FcTTV) was detected in the cat population in the Czech Republic. A total of 110 serum samples were tested by a nested PCR technique using specific primers, situated in the highly conserved untranslated region of the virus genome. The frequency of feline TT virus in the Czech Republic was found to be 33.63%. Sequencing of PCR product from several virus strains showed that all of them are closely related and belong to the same virus species. Whole genome sequencing of three strains was performed to compare overall genetic heterogeneity of feline TT viruses. One of these three strains showed more that 10% difference at the nucleotide level. Furthermore we didn't find any correlation between FcTTV infection and sex or health status of examined animals.

Preliminary epitope mapping of Torque teno sus virus 1 and 2 putative capsid protein and serological detection of infection in pigs.

The aim of this work is to identify antigenic regions within the ORF1 protein of Torque teno sus virus 1 (TTSuV1) and Torque teno virus sus 2 (TTSuV2) that could be used as antigens to detect virus-specific antibodies following infection in pigs. Protein sequences of TTSuV ORF1 genes were analysed to predict linear antigenic epitopes. Synthesized peptides were analysed for serological reactivity with swine sera. Such an antigenic region was identified at the C terminus of the ORF1 protein of both viruses and showed serological reactivity with 78 % (TTSuV1) and 88 % (TTSuV2) of swine sera. An ELISA with an immunodominant peptide as antigen was used to examine the sera of piglets, aged 4-20 weeks, and adults. Results indicated that TTSuV1- and TTSuV2-specific antibodies were detectable at 4 weeks. Antibody titres increased from week 10 and peaked at week 20. A relatively high antibody titre persisted to adulthood.

Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells.

Expression and purification of whole and nuclear localization signal (NLS) deleted ORF2 capsid protein of porcine circovirus 2 (PCV2) is demonstrated in the present study. Gene coding for both protein forms were cloned into pDest17 vector and expressed in BL21 (DE3)AI cells and in BL21-CodonPlus (DE3)-RIPL E. coli cells. The later cells were used to overcome difficulties with the heterologous expression of viral proteins in prokaryotic systems. Whole 30 kDa recombinant ORF2 protein was successfully expressed in BL21-CodonPlus (DE3)-RIPL cells only, 3 mg of pure protein was consistently obtained per liter of bacterial culture. NLS deleted ORF2 protein was expressed in both cell types. Resulting proteins reacted with PCV2 positive swine serum in immunofluorescent test and immunoblot.

Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus.

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.