Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) genetic diversity and occurrence of wild type and vaccine-like strains in the United States swine industry

Porcine reproductive and respiratory syndrome virus genotype 2 (PRRSV-2) genetic diversity in the U.S. was assessed using a database comprising 10 years’ worth of sequence data obtained from swine production systems routine monitoring and outbreak investigations. A total of 26,831 ORF5 PRRSV-2 sequences from 34 production systems were included in this analysis. Within group mean genetic distance (i.e. mean proportion of nucleotide differences within ORF5) per year according to herd type was calculated for all PRRSV-2 sequences. The percent nucleotide difference between each sequence and the ORF5 sequences from four commercially available PRRSV-2 vaccines (Ingelvac PRRS MLV, Ingelvac PRRS ATP, Fostera PRRS, and Prevacent PRRS) within the same lineage over time was used to classify sequences in wild-type or vaccine-like. The mean ORF5 genetic distance fluctuated from 0.09 to 0.13, being generally smaller in years in which there was a relative higher frequency of dominant lineage. Vaccine-like sequences comprised about one fourth of sequences obtained through routine monitoring of PRRS. We found that lineage 5 sequences were mostly Ingelvac PRRS MLV-like. Lineage 8 sequences up to 2011 were 62.9% Ingelvac PRRS ATP-like while the remaining were wild-type viruses. From 2012 onwards, 51.9% of lineage 8 sequences were Ingelvac PRRS ATP-like, 45.0% were Fostera PRRS-like, and only 3.2% were wild-type. For lineage 1 sequences, 0.1% and 1.7% of the sequences were Prevacent PRRS-like in 2009–2018 and 2019, respectively. These results suggest that repeated introductions of vaccine-like viruses through use of modified live vaccines might decrease within-lineage viral diversity as vaccine-like strains become more prevalent. Overall, this compilation of private data from routine monitoring provides valuable information on PRRSV viral diversity.

DEPENDENCE OF THE PHENOTYPIC COMPOSITION OF T-LYMPHOCYTES IN PATIENTS WITH CHRONIC VIRAL HEPATITIS C ON THE VIRUS GENOTYPE (IN DYNAMICS OF TREATMENT OF MEDICINES OF DIRECT ANTIVIRAL ACTION)

The aim of the study was to investigate the phenotype of effector T lymphocytes in patients with chronic viral hepatitis C (CVHC) in the dynamics of treatment with direct antiviral drugs depending on the genotype of the virus. 50 patients with CVHC were examined. The diagnosis was made on the basis of epidemiological and clinical laboratory data when specific serological markers of CHCV and RNA of hepatitis C virus (HCV) were detected. The determination of HCV RNA was carried out by the method of quantitative polymerase chain reaction in real time. The degree of liver fibrosis in patients with CVHC was assessed using ultrasound elastography. Patients were treated for 3 months with direct antiviral drugs according to the recommendations of the European Association for the Study of the Liver. The control group included 46 healthy donors with negative serological and molecular studies for the presence of viral hepatitis markers. The study of the subpopulation composition of helper and cytotoxic T-lymphocytes was carried out by direct immunofluorescence of whole peripheral blood. It was found that in CVHC patients were found characteristic features in the phenotypic composition of effector T-lymphocytes before and after treatment with direct antiviral drugs in depending on the genotype of HCV. The patients with HCV genotypes 1 and 3 had an increase in the content of terminal differentiated effector (TEMRA) T-helpers and effector memory (EM). Only patients with HCV genotype 2 had a decrease in the level of EM T-helper cells in the blood. A decrease in the relative number of T-helpers of central memory (СM) was independent of the HCV genotype. The level of effector subpopulations of cytotoxic T-lymphocytes in patients with CVHC was consistent with or exceeded control levels in depending on the genotype of HCV. The level of all investigated subpopulations of effector cytotoxic T-lymphocytes in patients with HCV genotype 1 is equal to the control values. The number of naive cytotoxic T cells and CM in peripheral blood in patients with HCV genotype 2 was increased. The content of naive cytotoxic T-lymphocytes, CM and TEMRA in patients with genotype 3 HCV in the blood was increased. The highest viral load was detected in patients with CVHC with genotype 1 HCV. Liver fibrosis was most pronounced in patients with CVHC infection with HCV genotypes 2 and 3. After 3 months of treatment with direct antiviral drugs the patients with CVHC had a reduced content of CM T-helpers regardless of the HCV genotype. In addition, patients with HCV genotypes 1 and 3 had a decrease in the number of naive T-helpers and patients with HCV genotypes 2 and 3 had a normalization of the content of naive cytotoxic T lymphocytes.

A new Hendra virus genotype found in Australian flying foxes

Background Hendra virus (HeV) has caused lethal disease outbreaks in humans and horses in Australia. Flying foxes are the wildlife reservoir from which the virus was first isolated in 1996. Following a heat stress mortality event in Australian flying foxes in 2013, a novel HeV variant was discovered. This study describes the subsequent surveillance of Australian flying foxes for this novel virus over a nine year period using qRT-PCR testing of tissues from flying foxes submitted primarily for Australian bat lyssavirus diagnosis. Genome sequencing and characterisation of the novel HeV variant was also undertaken. Methods Spleen and kidney samples harvested from flying fox carcasses were initially screened with two real-time qRT-PCR assays specific for the prototype HeV. Two additional qRT-PCR assays were developed specific for the HeV variant first detected in samples from a flying fox in 2013. Next-generation sequencing and virus isolation was attempted from selected samples to further characterise the new virus. Results Since 2013, 98 flying foxes were tested and 11 were positive for the new HeV variant. No samples were positive for the original HeV. Ten of the positive samples were from grey-headed flying foxes (GHFF, Pteropus poliocephalus), however this species was over-represented in the opportunistic sampling (83% of bats tested were GHFF). The positive GHFF samples were collected from Victoria and South Australia and one positive Little red flying fox (LRFF, Pteropus scapulatus) was collected from Western Australia. Immunohistochemistry confirmed the presence of henipavirus antigen, associated with an inflammatory lesion in cardiac blood vessels of one GHFF. Positive samples were sequenced and the complete genome was obtained from three samples. When compared to published HeV genomes, there was 84% sequence identity at the nucleotide level. Based on phylogenetic analyses, the newly detected HeV belongs to the HeV species but occupies a distinct lineage. We have therefore designated this virus HeV genotype 2 (HeV-g2). Attempts to isolate virus from PCR positive samples have not been successful. Conclusions A novel HeV genotype (HeV-g2) has been identified in two flying fox species submitted from three states in Australia, indicating that the level of genetic diversity for HeV is broader than first recognised. Given its high genetic relatedness to HeV, HeV-g2 is a zoonotic pathogen.

Is Human Bocavirus Infection Associated with Gastroenteritis in Children? An Updated Systematic Review and Meta-analysis

Background: Human bocavirus (HBoV) figures as an increased risk factor of respiratory and gastrointestinal tract infections among children. A great deal of data is available to support the pathogenic role of HBoV in acute respiratory diseases. However, the association between HBoV infection and gastroenteritis remains controversial due to the ambiguous results. The present work aims to clarify the role of HBoV as a cause of gastroenteritis in children. Methodology/Principal findings: A systematic search of the literature was carried out from 1 January 2016 to 29 August 2021 in Embase, PubMed, Scopus, Web of Science, and the Chinese bibliographic database of biomedicine (CBM). Data from included studies were analyzed by use of a random-effects model. The pooled estimates of HBoV prevalence among all cases of gastroenteritis were generated and stratified by potential confounders. The pooled odds ratio (OR) and 95% confidence interval (CI) were computed for HBoV infection in relation to the risk of gastroenteritis. The overall prevalence of HBoV in children with gastroenteritis (9.1%, 95% CI: 6.7-11.8%) was considerably higher than that detected in children without gastroenteritis (4.0%, 95% CI: 1.1-8.5%). HBoV prevalence tended to be higher in cases of gastroenteritis under five years of age (12.1%, 95% CI: 6.8-18.7%). The highest frequency of HBoV was found in Egypt (57.8%, 95% CI: 47.7-67.6%). The predominant genotypes of HBoV circulating in children with gastroenteritis were genotype 1 (HBoV1, 3.8%, 95% CI: 2.7-5.2%) and genotype 2 (HBoV2, 2.4%, 95% CI: 1.3-3.7%). HBoV infection was significantly associated with an increased risk of gastroenteritis in children (OR 1.620, 95% CI: 1.023-2.566). Conclusion: The HBoV prevalence in pediatric cases of gastroenteritis is higher than that in children without gastroenteritis, demonstrating an increasing global burden of gastroenteritis in children caused by HBoV infection. Targeted intervention to reduce the HBoV burden should be established.

Examination of the Usage of a New Beak-Abrasive Material in Different Laying Hen Genotypes (Preliminary Results)

The aim of the experiment was to investigate the use and effect of a new beak-abrasive material not yet examined on mortality of non-beak trimmed laying hens of different genotypes housed in an alternative pen. The study was performed on 636 females belonging to three genotypes of Bábolna TETRA Ltd. (a1 = commercial brown layer hybrid (C); a2 = purebred male line offspring group (maternal); a3 = purebfigure ed female line offspring group (paternal)). A total of 318 hens, i.e., 106 hens/genotype distributed in six pens (53 hens/pen), were evaluated. Cylindrical beak-abrasive blocks of 5.3–5.6 kg were suspended (0.1–0.4 mm diameter gravel, limestone grit, lime hydrate, and cement mixture) in six alternative pens. In six control pens without abrasive material, 318 hens, i.e., 106 hens/genotype (2 pens control group/genotype, i.e., C1 = commercial brown layer hybrid, C2 = purebred male line offspring group, C3 = purebred female line offspring group; 53 hens/pen;) were placed where there were no beak-abrasive materials. The rate of change in the weight of the beak-abrasive materials and the mortality rate were recorded daily. In the six pens equipped with beak-abrasive materials, infrared cameras were installed, and 24 h recordings were made. The number of individuals pecking the beak-abrasive material, the time and duration of dealing with the material were recorded. Data coming from one observation day are given. During the 13 experimental weeks of observation, the weight loss of beak-abrasives differed significantly in the different genotypes (a1 = 27.4%; a2 = 29.6%; a3 = 56.6%). During the only day analyzed, the hens from all the genotypes mostly stayed between 17:00 and 21:00 h in the littered scratching area where the beak-abrasive material was placed (a1 = 48.4%; a2 = 49.2%; a3 = 54.4%). In the case of each genotype, the rate of the hens dealing with beak-abrasives in the first two periods of the day was relatively low (0.2%–0.7%). Peaks of the activity were between 17:00 and 21:00 (a1 = 0.8%; a2 = 1.3%; a3 = 1.8%). The a3 dealt with the beak-abrasive materials to a significantly greater extent in the period from 13:00 to 17:00 (0.8%) and from 17:00 to 21:00 (1.8%) than the a1 (0.2% and 0.8%, respectively). Due to the use of the beak-abrasive materials, the mortality rate decreased the most in the genotypes that used them (a1 with beak-abrasive material 0.0% vs. C1 9.4%; a2 with beak-abrasive material 2.9% vs. C2 12.4%; a3 with beak-abrasive material) 15.4% vs. C3 5.7%). It can be concluded that the insertion of beak-abrasive materials increased the behavioral repertoire of hens, which is particularly beneficial from an animal welfare point of view. Further and longer-term research is needed to determine whether the insertion of the beak-abrasive material has a beneficial effect on the mortality data of the experimental groups through enrichment, either through physical abrasion of the beak or both.

Pilot Sub-Study of the Effect of Hepatitis C Cure by Glecaprevir/Pibrentasvir on the Gut Microbiome of Patients with Chronic Hepatitis C Genotypes 1 to 6 in the Mythen Study

In this small pilot sub-study, longitudinal gut microbiota composition changes, after successful treatment of hepatitis C virus (HCV) with the co-formulated glecaprevir/pibrentasvir (GLE/PIB), were analyzed before treatment (baseline) and 12 weeks post-treatment. Participating patients provided a fresh stool sample the week before their study visit, from which microbial DNA was extracted and sequenced for the 16S rRNA region in an Illumina MiSeq2 platform. Microbial and statistical analyses were conducted to determine the alpha-diversity (number of different taxa within a sample) and beta-diversity (number of overlapping taxa between samples). Stool samples from 58 patients were eligible for analysis. There were 27 patients with HCV genotype 1, 10 with genotype 2, 16 with genotype 3, and 5 with genotype 4. No statistically significant differences in gut microbiota diversity, species richness, or microbial community pattern were found at baseline and at post-treatment Week 12. Lack of statistically significant differences remained consistent in further analysis by demographic and baseline disease characteristics. Surprisingly, no statistically significant changes in alpha- and beta-diversity were seen in the microbiota after GLE/PIB treatment, though there was a trend toward less richness over time. Further investigation is needed into this unexpected outcome to better understand the role of HCV treatment and the gut microbiota.

Development of a nanobody-based competitive ELISA for efficiently and specifically detecting antibodies against genotype 2 porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes considerable economic loss to the global pig industry. Efficient detection assay is very important for the prevention of the virus infection. Nanobodies are the advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, to develop a nanobody-based competitive ELISA (cELISA) for specifically detecting antibodies against PRRSV, three nanobodies against PRRSV-N protein were screened by Camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-horseradish peroxidase (HRP) fusion protein against PRRSV-N protein was successfully constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting antibodies against PRRSV in pig sera, exhibiting a cut-off value of 23.19% and good sensitivity, specificity, and reproducibility. Importantly, the cELISA specifically detect anti-genotype 2 PRRSV antibodies. The cELISA showed more sensitive than the commercial IDEXX ELISA kit by detecting the sequential sera from the challenged pigs. The compliance rate of cELISA with the commercial IDEXX ELISA kit was 96.4%. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera. Collectively, the developed nanobody-based cELISA showed advantages of simply operation and low production cost and can be as an assay for epidemiological investigation of genotype 2 PRRSV infection in pigs and evaluation after vaccination.

Amplicon sequencing of Primate Erythroparvovirus 1 B19V from clinical samples. v1

This protocol describes amplicon based sequencing of Primate Erythroparvovirus 1 (B19V ) from clinical samples using the MinION (Oxford Nanopore Technologies). The primers for amplification were designed using PrimalScheme (https://primalscheme.com/) with B19V genotype 3 virus as the template, however these would allow partial recovery of B19V genotype 2 genomes. The protocol was modified from the Zika and SARS-CoV-2 sequencing protocols (https://dx.doi.org/10.17504/protocols.io.bbmuik6w).

Morphological and mechanical characterization of bone phenotypes in the Amish G610C murine model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a hereditary bone disease where gene mutations affect Type I collagen formation resulting in osteopenia and increased fracture risk. There are several established mouse models of OI, but some are severe and result in spontaneous fractures or early animal death. The Amish Col1a2G610C/+ (G610C) mouse model is a newer, moderate OI model that is currently being used in a variety of intervention studies, with differing background strains, sexes, ages, and bone endpoints. This study is a comprehensive mechanical and architectural characterization of bone in G610C mice bred on a C57BL/6 inbred strain and will provide a baseline for future treatment studies. Male and female wild-type (WT) and G610C mice were euthanized at 10 and 16 weeks (n = 13–16). Harvested tibiae, femora, and L4 vertebrae were scanned via micro-computed tomography and analyzed for cortical and trabecular architectural properties. Femora and tibiae were then mechanically tested to failure. G610C mice had less bone but more highly mineralized cortical and trabecular tissue than their sex- and age-matched WT counterparts, with cortical cross-sectional area, thickness, and mineral density, and trabecular bone volume, mineral density, spacing, and number all differing significantly as a function of genotype (2 Way ANOVA with main effects of sex and genotype at each age). In addition, mechanical yield force, ultimate force, displacement, strain, and toughness were all significantly lower in G610C vs. WT, highlighting a brittle phenotype. This characterization demonstrates that despite being a moderate OI model, the Amish G610C mouse model maintains a distinctly brittle phenotype and is well-suited for use in future intervention studies.