Sequence analysis of human pegivirus from 3 patients indicates that the central nervous system constitutes a separate viral compartment from serum. Human pegivirus (HPgV), previously called hepatitis G virus or GB virus C, is a lymphotropic virus with undefined pathology. Because many viruses from the family Flaviviridae, to which HPgV belongs, are neurotropic, we studied whether HPgV could infect the central nervous system. We tested serum and cerebrospinal fluid samples from 96 patients with a diagnosis of encephalitis for a variety of pathogens by molecular methods and serology; we also tested for autoantibodies against neuronal antigens. We found HPgV in serum and cerebrospinal fluid from 3 patients who had encephalitis of unclear origin; that is, all the markers that had been tested were negative. Single-strand confirmation polymorphism and next-generation sequencing analysis revealed differences between the serum and cerebrospinal fluid–derived viral sequences, which is compatible with the presence of a separate HPgV compartment in the central nervous system. It is unclear whether HPgV was directly responsible for encephalitis in these patients. Pegivirus) within the family Flaviviridae (Human pegivirus (HPgV) was originally described as a hepatitis virus by 2 independent groups of researchers and called GB virus C and hepatitis G virus  such as West Nile virus (WNV) and tick-borne encephalitis virus (TBEV), are neurotropic and a prominent cause of encephalitis in Europe and North America , demonstrating that the virus can be present in the brain under certain circumstances . We obtained written informed consent from all patients or from close relatives of patients unable to give consent due to their condition. The Internal Review Board of the Medical University of Warsaw approved the study.We prospectively enrolled patients with encephalitis at the Warsaw Hospital for Infectious Diseases  from June 2012 through July 2015. The details of this study were published previously (We collected CSF and serum samples from patients at admission (5–7 days after symptom onset) and kept them frozen at −80°C until analysis. We tested the samples from all 96 patients for the presence of 5′ untranslated region (UTR) HPgV RNA. We performed real-time quantitative PCR (qPCR) or real-time quantitative reverse transcription PCR (qRT-PCR) to detect human herpesvirus (HHV) 1 and 2, varicella zoster virus (VZV), cytomegalovirus (CMV), HHV-6, enteroviruses , TBEV, WNV, and human adenovirus (HAdV) in CSF samples. We used commercial serologic tests to test CSF and paired serum samples for HHV-1, HHV-2, VZV, TBEV, and WNV as described  from 400 µL of CSF or serum and suspended RNA in 20 µL of water, 5 µL of which was subsequently used for each amplification reaction. We amplified the HPgV 5′ UTR by nested reverse transcription PCR (RT-PCR) as described previously . Each primer contained the following: sequences complementary to the adapters on a flow cell; an 8-nt index sequence; sequences corresponding to the Illumina sequencing primers; and sequence-specific nested primers for the 5′ UTR and E2 region. Amplification of the 5′ UTR region included initial denaturation at 94°C for 5 min, 20 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, and final elongation at 72°C for 10 min. HPgV E2 amplification included denaturation at 94°C for 5 min followed by 20 cycles of 94°C for 18 s, 55°C for 20 s, 72°C for 90 s, and 1 cycle at 72°C for 10 min. We trimmed the libraries by using the LabChip XT apparatus  with the DNA 300 Assay Kit (PerkinElmer); the range of fraction collection was 370–430 bp for 5′ UTR and 460–530 bp for E2.We assessed the quality and average length of next-generation sequencing libraries by using Bioanalyzer . We equimolarily pooled the indexed samples and sequenced them on Illumina MiSeq with 301 bp-end reads according to the manufacturer’s protocol.https://github.com/marcelm/cutadapt/); (http://hannonlab.cshl.edu/fastx_toolkit/index.html) for additional processing. We removed all Phred quality score reads <20 using FASTQ/A Artifacts Filter and preprocessed the remaining reads  using R scripts   (http://www.cbs.dtu.dk/services/BepiPred/) , and free energies of the hypothetical secondary structures were only occasionally and mildly affected  Figure (We detected HPgV sequences in CSF from 3/96 patients with encephalitis. We classified all 3 cases as encephalitis of unknown origin because they were negative for serologic and molecular markers of common CNS pathogens. Furthermore, we demonstrated that these viral sequences differed from those circulating in serum. The presence of viral RNA in CSF could be due to a compromised blood–brain barrier, which was possible in these patients with encephalitis. However, the presence of differences in circulating and CSF-derived sequences is more compatible with the existence of separate viral compartments and thus independent replication. Similar compartmentalization in which distinct blood and CNS viral populations indicate separately evolving populations has been described for other viruses, most prominently for HIV (It is unclear how HPgV could access the CNS. Although initially HPgV was thought to be a hepatotropic virus, the viral negative strand, which is the putative replicative intermediate of the virus, was not detected in liver tissue (Whether HPgV was the causative factor of encephalitis in the patients we describe is not clear. Viral pathogens are typically present only transiently in CSF and this window could be easily missed, particularly when the spinal tap is done too late in the course of illness (The identified 5′ UTR and E2 region sequence differences between CSF and serum compartments could have biological meaning. The 5′ UTR contains an internal ribosomal entry site that allows cap-independent viral translation (On the amino acid level, we saw 2 unique E2 region changes in CSF variants compared with serum: in patient 2, serine was changed to phenylalanine at aa position 508, and in patient 3, proline was changed to leucine at position 572. Both were within regions predicted to contain B-cell epitopes, thus suggesting that they were the effect of immune pressure. Furthermore, the change in patient 3 was located in the region of E2 that was experimentally shown to contain a strong antigenic site and likely to be involved in cell binding or fusion (RNA viruses in particular are characterized by a high degree of genetic heterogeneity; probably because the lack of proofreading 3′–5′ exonuclease activity in viral RNA polymerases causes low fidelity. As a result, viruses circulate in the infected host as a population of closely related but nonidentical genomes, referred to as quasispecies (In summary, we detected HPgV sequences in the CSF of 3 patients with encephalitis of unclear origin, and these sequences from CSF differed from those circulating in serum. These findings are compatible with the presence of a separate viral compartment in the CNS. Determining if the pegivirus was responsible for encephalitis or if it was present along with another cause of encephalitis will require further research, including histopathological analysis.Additional information about human pegivirus in patients with encephalitis, Poland.


NB-LRR defense genes in monocots, some of which arose in the Triticeae species era. These miRNAs regulate different types of NB-LRR genes, most of them with coil-coiled domains, and trigger the generation of secondary small interfering RNAs (siRNA) as a phased pattern in the target site regions. In addition to acting in response to biotic stresses, they are also responsive to abiotic stresses such as heat, drought, salt, and light stress. Their copy number and expression variation in Triticeae suggest a rapid birth and death frequency. Altogether, non-conserved miRNAs as conserved transcriptional regulators in gymnosperms and angiosperms regulating the disease resistance genes displayed quick plasticity including the variations of sequences, gene copy number, functions, and expression level, which accompanied with NB-LRR genes may be tune-regulated to plants in natural environments with various biotic and abiotic stresses.Disease resistance genes encoding proteins with nucleotide binding sites and Leucine-Rich Repeat (NB-LRR) domains include many members involved in the effector-triggered immunity pathway in plants. The transcript levels of these defense genes are negatively regulated by diverse microRNAs (miRNAs) in angiosperms and gymnosperms. In wheat, using small RNA expression datasets and degradome datasets, we identified five miRNA families targeting  NB-LRRs with a large gene family are an important class of disease resistance genes. Of the total coding genes, 1.19–3.48% were defined as NB-LRR genes in plants [NB-LRR genes have been demonstrated as ancient and conserved genes in plants [CC-NB-LRR) or TNL (TIR-NB-LRR) domains [Triticum, the defense genes Sr31 and Sr50 with CNL [Secale cereale) can confer the resistance to stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt). Sr35 gene with CNL from Triticum monococcum confers the resistance to Ug99 Stem Rust Race Group [The competition between plants and pathogens has never disappeared, and sometimes, it becomes very intense. During evolution, plants evolved multiple adaption mechanisms for biotic stresses. The important defense mechanisms include PAMP-Triggered Immunity (PTI) triggered by Pathogen-Associated Molecular Patterns (PAMP) as a preliminary defense  and Effen plants . TIR andn plants . Althougn plants , comparan plants . Approxice Group . NB-LRR class genes will do damage to the growth and development of plants [Most of the genes involved in the disease defense pathways always have very rapid evolutionary rates in the coevolution process with pathogens . The higf plants . Thus, pf plants .Arabidopsis can be elicited by Flagelin 22 (flg22). Repressing auxin signaling through miR393 overexpression increases bacterial resistance to virulent Pto DC3000 by negatively targeting F-box auxin receptors transcripts Transport Inhibitor Response 1 (TIR1) [Nuclear transcription Factor Y, Alpha (NFYA) module in Arabidopsis influences Ralstonia solanacearum pathogenicity [Magnaporthe oryzae [Arabidopsis miR396/GRF module mediates innate immunity against P. cucumerina infection without growth costs [OsMADS directly monitors RNA-dependent RNA polymerase 1 (OsRDR1) transcription involved in the rice antiviral pathway [TCP21 can facilitate rice ragged stunt virus infection via the jasmonic acid pathway [OsSCL6) [METHYLTRANSFERASE 2 (MET2) in Arabidopsis and elevates the resistance to infection by fungal pathogens [MicroRNAs (miRNAs), as a class of small noncoding RNAs, function in posttranscriptional gene regulation. Small miRNAs play important roles in a variety of biological processes, such as development, hormone responses, and stress adaptation ,15,16,171 (TIR1) . The miRgenicity  and in re oryzae . The Ara pathway . Increas pathway . Overexp[OsSCL6) . Interfeathogens . In addition to basal defense, miRNAs can also directly regulate disease resistance genes in the ETI pathway. These disease resistance genes have proteins that include NB, LRR, TIR, and CC domains, most of which are mediated by the generation of 21-nt phased siRNAs (phasiRNAs) . The supTIR-NB-LRR and CC-NB-LRR gene classes by miRNAs has mostly been characterized in dicots. In reported posttranscriptional regulation networks, miRNAs can trigger 21-nt phasiRNAs generation from NB-LRR transcripts, which are further processed by RNA-Dependent RNA polymerase 6 (RDR6) and DICER-LIKE 4 (DCL4) [Brassica miR1885 was validated to be induced by a Turnip mosaic virus (TuMV) infection, which induced the cleavage of TIR-NB-LRR transcripts [TIR-NB-LRR immune receptors in Tobacco, both nta-miR6019 and nta-miR6020 contributed to the resistance to Tobacco mosaic virus (TMV) [Arabidopsis, miR472 influenced disease resistance genes mediated by the RDR6 silencing pathway [Medicago, miR2109, miR482/miR2118, and miR1507 were found to regulate the NB-LRR gene family [NB-LRR gene class with TIR or CC domains in legumes, which were proposed to function in the modulation of defense response during rhizobium colonization [NB-LRR genes in Norway Spruce [NB-LRR genes such as the absence of TIR domain, and the neofunctionalization of disease resistance-associated miRNAs such as miR2118 in monocots. Until now, only one miRNA family was validated as the regulator of NB-LRR genes. MiR2009 was first predicted to target Mla alleles in wheat [Mla-mediated immune signaling, which is fine-tuned at a later stage of MLA activity to avoid overloading of immune responses in barley cells [The regulation of the 4 (DCL4) . For exanscripts . By cleaus (TMV) ,33. Sl-mus (TMV) . In Arab pathway . In Medie family . MiR1507nization ,36. Addiy Spruce . Howeverin wheat . In barley cells .Triticeae. These periodic nascent miRNAs by regulating the NB-LRR genes may maintain the potential fitness or growth costs in Triticeae species at the posttranscriptional level. Increasing numbers of small RNAs from high-throughput sequencing are available in the public database, including various developmental and stress samples. Multiple efforts have been made to identify the differential expression of miRNAs in response to biotic and abiotic stresses ,35,36,37Comparing the miRNAs from the miRbase , PNRD 4, and PmiNB-LRR genes by triggering the biogenesis of 21-nt phased siRNAs [mildew resistance locus (Mla1). Here, we designated tae-miR2009 as tae-miR9863a in the next section.In barley, the hvu-miR9863 had been characterized to regulate d siRNAs . In whead siRNAs , is homohttp://unafold.rna.albany.edu/) [TAE-MIR3117A displayed three loci on chromosomes 1DL, 2DL, and 7BL; TAE-MIR3084A had one locus on chromosome 3al; TAE-MIR5071A has one locus on chromosome 7BL; and TAE-MIR7757A had two loci on chromosomes 3AL and 3DL  . After f and 3DL . The lenTriticum turgidum), diploid , and nascent allohexaploid , tae-miR9863a had higher expression levels in the young seedling, spike, and seed developmental stage; tae-miR7757a had moderate expression levels; and tae-miR3804a had relatively low expression levels. For example, tae-miR9863a had a TPM value higher than 27,000 in Aegilops tauschii seedling. Tae-miR7757a had a TPM value ranging from 100 to 1700, and tae-miR3084a had a TPM value ranging from 3 to 40 , and Fusarium infection and abiotic stresses such as thermal stress (high and low temperatures), salt stress, drought stress, light, and UV stresses ) of gene expression level between the mock/ck and the stressed samples greater than 1 or less than −1 as upregulated or downregulated, respectively. Among biotic stresses, 24 h after Fusarium infection, tae-miR3084a, miR5071a, miR7757a, and miR9863a were downregulated while tae-miR3117a-b showed no changes in the expression level. For a powdery mildew infection, only tae-miR7757a was downregulated while other miRNAs did not show any changes in their expression levels. However, for the BYSMV infection, the disease resistance associated miRNAs were upregulated for tae-miR3084a, tae-miR5071a, and tae-miR7757a , Mla16-1 (Accession number: ACZ65488.1), RPP13 (Accession number: EMS57774.1) Sr35 (Accession number: AGP75918.1), and Yr10 (Accession number: AAG42168.1). In this study, we identified 163 NB-LRR genes targeted by miRNAs in total. To understand the response of these targets in the infection of pathogens, we used the transcriptome datasets to investigate the variation of gene expression level with the fold change more than 1.5 times . Targets of Tae-miR3117/tae-miR5071/tae-miR7757/tae-miR9863 can respond to the diseases of both stripe rust and Powdery mildew pathogens , Ehrhartoideae (rice), Pooideae (Brachypodium), and Triticeae . Then, based on the conserved and species-specific miRNAs among grasses and Triticeae, we deduced the evolution history of the five miRNA families. According to the divergence time among species, we created the phylogenetic trees in grasses [Triticum species [Brachypodium, barley, and wheat; miR9863a was conserved in Brachypodium, barley, and wheat; miR5071a was conserved in barley and wheat; and miR3117a and miR3084a were conserved in only Triticum. Thus, we can assume that tae-miR7757a emerged before the diversity of grasses approximately 45-60 million years ago (mya). MiR3117b appeared before the diversity of Ehrhartoideae and Pooideae approximately 40–54 mya, and miR9863a developed before the diversity of Brachypodium and Triticeae approximately 32–39 mya. MiR5071a was present before the diversity of barley and Triticum species approximately 10–15 mya. In contrast, miR3117a and miR3084a only appeared in Triticum species approximately 4 mya.To investigate the evolution of disease resistance-associated miRNAs in grasses, we mapped the mature miRNA sequences to grass genomes, including  grasses ,66,67 an species . As showTriticum species, we mapped the mature miRNA sequences to their genome, including AA (Triticum urartu), AmAm (Triticum monococcum), SS , DD (Aegilops tauschii), AABB (Triticum durum), and AABBDD (Triticum aestivum). As shown in In Flowering plants are derived from a common ancestor approximately 135 to 250 mya. During the evolution process, plants passed through countless encounters with pathogens. The pathogens evolved various tactics to adapt to the surrounding environment via changeable genomes. Thus, plants had to constantly develop strategies to defend against pathogenic virulence. The most important mechanism was to increase the copy number and diversity of disease resistance genes at the dosage and structure levels. The increase in gene expression level could be regulated when no pathogenic infection was present for cost benefit. Thus, plants developed another mechanism, i.e., transcript regulatory miRNAs, to control gene expression. To affect multiple disease resistance gene variations, miRNAs had to be fickle for survival with rapid plasticity including gene copy number variation, expression level variation, and function variation, etc.NB-LRR transcripts in plants. Here, using the small RNA transcriptome datasets, we also detected that some miRNAs could respond to the biotic stresses. Tae-miR3084, tae-miR5071, tae-miR7757, and tae-miR9863 in wheat were downregulated after 24 h of a Fusarium infection, and miR7757 was also downregulated after a powdery mildew infection  [http://structuralbiology.cau.edu.cn/PNRD) [http://pmirexat.nabi.res.in) [http://unafold.rna.albany.edu/) to confirm their precursor sequences.To identify the disease resistance-associated miRNAs in wheat, we first downloaded the miRNA sequences from the miRbase (ase.org) , PNRD (hcn/PNRD) , and Pmi.res.in)  websites.res.in) . TargetsTriticum species, we first downloaded the genome sequences of various grass species including Oryza sativa (v7), Brachypodium distachyon (v3.1), Zea mays (PH207 v1.1), and Sorghum (v3.1.1) from the pytozome website (http://www.phytozome.net); Hordeum vulgare (IBSC_v2) from the ensemble plant website; and Triticum species including Triticum aestivum (TGACv1), Triticum durum (v1), Aegilops tauschii (v1), Triticum urartu (v1), Triticum monococcum (v1), Aegilops sharonensis (v1), and Aegilops speltoides (v1) from the URGI website (https://wheat-urgi.versailles.inra.fr/Seq-Repository/Assemblies). Next, we aligned these miRNAs sequences to perfect matches in the genomes of grasses and Triticum. According to the presence and absence of these miRNAs, we developed a birth and death phylogenic tree with the evolutionary time scale of these species.To detect the presence/absence of disease resistance-associated miRNAs in grasses and virus, and Fusarium stress from GSM675614-GSM675615 [The small RNA datasets used in this analysis were downloaded from the NCBI GEO database . The smaSM675615 , GSM1508SM675615 , and GSMSM675615  and GSM6SM675615 ; and colSM675615 . The degSM675615 , GSM9119SM675615  were dowhttp://mev.tm4.org/, version_4.9.0, GitHub, San Francisco, CA, USA).MiRNA read normalization as transcripts per million (TPM) was performed for each sample in the small RNA datasets downloaded from the GEO database. The expression levels were shown with the MeV software , USA) [To identify the domain categories of these targets, we used the Hmmscan program against the Pfam database with an  domains . We also domains . The regS), USA) .http://meme-suite.org/tools/meme, version 5.0.4, National Institutes of Health, USA) to identify the motifs [http://www.mbio.ncsu.edu/bioedit/bioedit.html, version 7.2.5, Tom Hall, Ibis Therapeutics, A division of Isis Pharmaceuticals, Carlsbad, CA, USA) to align the sequences, removed the high variation sequences manually, and then used the MEGA program (version 10.0.4) with the neighbor-joining method to draw the phylogenetic tree. The parameters such as poisson model, uniform rate among sites, homogeneous pattern, and pairwise deletion for the missing data were selected [To show the target consensus regions, we used the MEME program  value more than five and the upregulated fold change more than 2 times after infection by pathogens were identified as upregulated.To investigate the response of target genes to disease, we downloaded the expression data from the website of According to the small RNA distribution on the target transcripts of disease resistance-associated miRNAs, we identified the phased clusters using the Short Stack package with a phased score of more than 15  and showMicroRNAs, as an ancient posttranscriptional regulator, have a conserved regulation mechanism in eukaryotic organisms. MiRNAs have evolved independently in the lineages since a lack of universally conservation among plants and animals . In plan


Orthobunyavirus and are associated with human febrile disease in tropical and subtropical areas of South and Central America. While numerous GRCVs have been isolated from mosquitoes, animals, and humans, genetic analysis of these viruses is limited. In this study, we characterized 65 GRCV isolates from febrile patients identified through clinic-based surveillance in the northern and southern Peruvian Amazon. A 500 base pair region of the S segment and 750 base pair regions of the M and L segments were sequenced. Pairwise sequence analysis of the clinical isolates showed nucleotide identities ranging from 68% to 100% and deduced amino acid sequence identities ranging from 72% to 100%. Sequences were compared with reference strains of the following GRCVs: Caraparu virus (CARV), Murutucu virus (MURV), Oriboca virus (ORIV), Marituba virus (MTBV), Itaqui virus (ITQV), Apeu virus (APEUV), and Madrid virus (MADV). Sequence comparison of clinical isolates with the prototype strains based on the S and L segments identified two clades; clade I included isolates with high genetic association with CARV-MADV, and clade II included isolates with high genetic association with MURV, ORIV, APEUV, and MTBV. Genetic relationships based on the M segment were at time inconsistent with those based on the S and L segments. However, clade groupings based on the M segment were highly consistent with relationships based on microneutralization assays. These results advance our understanding of the genetic and serologic relationships of GRCVs circulating in the Peruvian Amazon.Group C orthobunyaviruses (GRCVs) are a complex of viruses in the genus  Servico Especial de Saude Publica of Brazil and the Rockefeller Foundation conducted studies to isolate arboviruses in the Brazilian Amazon region and isolated a novel group of vector-borne viruses, designated as group C (GRCV). These isolates were grouped serologically as 6 viruses named Marituba, Oriboca, Apeu, Murutucu, Caraparu and Itaqui [During 1954–1959, the d Itaqui . Humans d Itaqui ,5,8.GRCV classification was initially based on antigenic relationships determined using complement fixation, neutralization, and hemagglutination inhibition tests. The earliest studies on the serological relationships showed broad cross reactions between prototypes, depending on the assays: hemagglutination inhibition and virus neutralization tests indicated similarities between Oriboca virus and Itaqui virus, Apeu virus and Caraparu virus, Murutrucu virus and Marituba virus, while complement fixation tests indicated similarities between Itaqui virus and Caraparu virus, Apeu virus and Marituba virus, Murutucu virus and Oriboca virus .Peribunyaviridae, genus Orthobunyavirus and represented in the International Committee on Taxonomy of Viruses (ICTV) database  by the following species: Caraparu orthobunyavirus (CARV), Oriboca orthobunyavirus (ORIV), Madrid orthobunyavirus (MADV) and Marituba orthobunyavirus (MTBV).Currently GRCVs are grouped in the family Orthobunyavirus, the virions are spherical, enveloped in lipid, and possess an outer fringe of glycoprotein. The genome is composed of three segments of negative-sense, single-stranded RNA: large (L) ~ 7000 nucleotides (nt), medium (M) ~ 4500 nt, and small (S) ~ 1000 nt. The L segment encodes an internal large protein RNA-dependent RNA polymerase (RdRp). The M segment encodes the two external viral glycoproteins Gn and Gc, which are inserted in the viral membrane, and also additionally encodes a non-structural protein NSm. The S segment encodes two proteins in overlapping reading frames, the internal nucleocapsid (N) protein and a non-structural protein (NSs) [Like the other members of the genus in (NSs) –11.In Peru, the first isolations of GRCV occurred in 1970 and 1971 from animal samples collected in the Amazon region . More reSerum samples were collected from subjects participating in a passive clinic-based surveillance study of febrile disease between 1995 and 2013 , coveredSerum samples from febrile patients were inoculated onto Vero cell cultures and identified as GRCVs using an immunofluorescent assay as previously described . Six GRCMore than 500 base pair region of the S segment  and more than 750 base pair regions of the M  and L  segments were amplified using one step reverse transcription and PCR (RT-PCR). Primers  were desRT-PCR was performed in a total volume of 50 μl containing 1 μM of each primer  after a single band was apparent in the gel. The amplicons from the partial M and L segments were recovered from excised DNA bands from the agarose gel and purified using Wizard SV Gel and PCR Clean-Up System . Sequencing was performed using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the same primers as used for RT-PCR. Sequencing reactions were purified using centri-sep columns  or an ethanol precipitation method. Nucleotide sequences were determined by running purified sequencing reactions on the ABI 3130 XL Genetic Analyzer (Applied Biosystems).Nucleotide sequences were assembled using Sequencher 4.8 software . In addition to the sequences that were generated for this study (65 isolates and 6 reference strains), the following sequences from GenBank were included in the genetic analysis: Madrid Virus—BT4075   and IQT9ClustalW multiple alignment, pairwise comparisons, multiple sequence analyses and phylogenetic tree construction were performed using BioEdit 7.2  and MEGA50, where one TCID50 is the dilution of the original stock which results in half the wells being infected and half the wells remaining uninfected, calculated using the method of Reed and Muench [5 / ml in cell culture media with 4% Fetal Bovine serum (FBS). The plates were incubated at 37°C in 5% CO2. After 3 days, supernatant media were removed and the cells were fixed and stained with a solution of naphthol blue black and acetic acid [Hyperimmune mouse ascitic fluid (HMAF) against APEUV BeAn848, CARV BeAn3994, ITQV , MTBV Bd Muench . After itic acid . The neuIn total, 65 GRCV isolates were identified between 1995 and 2013 from acute phase serum samples from febrile patients . GRCVs wBased on partial S segment sequence, GRCV isolates grouped into two main clades . PairwisM segment sequences segregated into three clades . Clade IClustering of isolates based on L segment analysis mirrored that of the S segment. Based on partial L segment analysis, L segment sequences separated into two clades , with 71Clinical isolates fell into four antigenically distinct groups  which trThe genetic characteristics of reference strains CARV BeAn3994 and MADV BT4075 tracked together for S, M, and L in clade I, and MURV BeAn974 and MTBV BeAn15 tracked together for S, M, and L in clade II. ITQV PE00036 tracked with CARV BeAn3994 and MADV BT4075 for S and L as part of clade I, but with ORIV BeAn17 for M, as part of clade III. APEUV BeAn848 and ORIV BeAn17 tracked with MURV BeAn974 and MTBV BeAn15 for S and L segments as part of clade II, but APEUV BeAn848 tracked with CARV BeAn3994 for the M segment as part of the clade I, and ORIV BeAn17 tracked with ITQV PE00036 for M, as part of clade III. These results are based on neighbor-joining method; similar topologies were identified using maximum likelihood and maximum parsimony approaches –S6 Figs.Based on genetic and antigenic characteristics, clinical isolates could be grouped with prototype strains. Specifically, 41 isolates were most closely related to CARV , 17 isolates were most closely related to MURV-MTBV serocomplex , and 2 with ITQV . No ORIV isolates were detected. Additionally, six reassortants relative to CARV reference strains were identified (90% - 91% M segment amino acid identity). These isolates grouped with Itaya virus, a novel reassortant orthobunyavirus isolated from febrile patients in the cities of Iquitos and Yurimaguas in Peru ,17. ItayIn this study, we identified 41 isolates of CARV, 17 isolates of MURV, 6 isolates of Itaya virus, and 2 isolates of ITQV associated with human febrile illness in the northern and southern Amazon basin of Peru. The ITQV isolates represent the first association of ITQV with human illness in Peru. Overall, while the Peruvian isolates were most closely related genetically to the indicated reference viruses, they still displayed greater than 10% divergence at the nucleotide level in comparison to the reference viruses, consistent with the fact that the reference viruses were collected in neighboring Brazil several decades earlier.Our data confirm that the M segment gene product is the key determinant of virus neutralization specificity. Similarly, early studies on GRCVs ,3,4 descFor all isolates described here, S and L segments segregated together, both grouping into two major clades. For S and L, clade I was divided into two lineages, with lineages most closely aligned with geographic distribution rather than year of isolation. Lineages 1 and 3 of clade I contained only isolates from the northern Amazon region of Peru, while lineage 2 mainly contained isolates from the southern Amazon region of Peru. These data suggest that there has been little flow of orthobunyaviruses within Peru over the past 20 years.While the majority of isolates grouped consistently across all three segments, there were clear inconsistencies between S and L versus M segment for a subset of viruses, likely representing reassortants. In this virus set, six isolates of Itaya virus were identified, including four new cases in addition to the originally identified isolate  and the These data demonstrate the diversity of GRCVs that infect and cause febrile illness in humans in the Peruvian Amazon. Our data also suggest that reassortment among GRCVs is a relatively common phenomenon, as described for other bunyaviruses . FurtherS1 Table(DOCX)Click here for additional data file.S1 FigThe phylogenetic tree was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model. Bootstrap values were inferred from 500 replicates on the bases of partial coding sequence of N and NSs protein. Members of the Simbu, California and Bunyamwera serogroups were used as outgroups to root the tree.(TIF)Click here for additional data file.S2 FigThe MP tree was constructed using the Maximum Parsimony method. Bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed on the bases of partial coding sequence of N and NSs protein. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm. Members of the Simbu, California and Bunyamwera serogroups were used as outgroups to root the tree.(TIF)Click here for additional data file.S3 FigThe phylogenetic tree was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model. Bootstrap values were inferred from 500 replicates on the bases of partial coding sequence of the polyprotein. Members of the Simbu, California and Bunyamwera serogroups were used as outgroups to root the tree.(TIF)Click here for additional data file.S4 FigThe MP tree was constructed using the Maximum Parsimony method. Bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed on the bases of partial coding sequence of the polyprotein. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm. Members of the Simbu, California and Bunyamwera serogroups were used as outgroups to root the tree.(TIF)Click here for additional data file.S5 FigThe phylogenetic tree was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model. Bootstrap values were inferred from 500 replicates on the bases of partial coding sequence of RdRp. Members of the Simbu, California and Bunyamwera serogroups were used as outgroups to root the tree.(TIF)Click here for additional data file.S6 FigThe MP tree was constructed using the Maximum Parsimony method. Bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed on the bases of partial coding sequence of RdRp. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm. Members of the Simbu, California and Bunyamwera serogroups were used as outgroups to root the tree.(TIF)Click here for additional data file.


Culicoides-transmitted orbivirus that infects domestic and wild ruminants in many parts of the world. Of the eight proposed serotypes, only EHDV-1, 2 and 6 have been reported to be present in the Americas. Following the identification of a virulent EHD-6 reasssortant virus in the USA in 2007 (EHDV-6 Indiana), with outer coat protein segments derived from an Australian strain of EHDV and all remaining segments derived from a locally circulating EHDV-2 strain, questions have remained about the origin of the Australian parent strain and how it may have arrived in the USA. When EHDV-6 was identified in asymptomatic cattle imported into the Caribbean island of Trinidad in 2013, full genome sequencing was carried out to further characterise the virus. The EHDV-6 Trinidad was a reassortant virus, with 8 of its 10 segments, being derived from the same exotic Australian EHDV-6 strain as the VP2 and VP5 present in the EHDV-6 Indiana strain from the USA. Analyses of the two remaining segments revealed that segment 8 showed the highest nucleotide identity (90.4%) with a USA New Jersey strain of EHDV-1, whereas segment 4 had the highest nucleotide identity (96.5%) with an Australian EHDV-2 strain. This data strongly suggests that the Trinidad EHDV-6 has an Australian origin, receiving its segment 4 from a reassortment event with an EHDV-2 also from Australia. This reassortant virus likely came to the Americas, where it received its segment 8 from a locally-circulating (as yet unknown) EHDV strain. This virus then may have gained entry into the USA, where it further reassorted with a known locally-circulating EHDV-2, the resulting strain being EHDV-6 Indiana. This study therefore identifies, for the first time, the likely minor parent virus of the EHDV-6 currently circulating in the USA.Epizootic hemorrhagic disease virus (EHDV) is a  •A unique reassortant EHD-6 was circulating asymptomatically in Trinidad in 2013•Nine of the ten segments making up the EHD-6 Trinidad virus had a likely Australian origin•The EHDV-6 Trinidad is likely to be the minor parent virus of the EHDV-6 Indiana strain currently circulating in the USA. In contrast, mule deer, black-tailed deer and pronghorn antelope are less affected by the virus, having higher survival rates (Epizootic hemorrhagic disease virus (EHDV) is a al rates . EHDV haal rates . More real rates .Reoviridae and is closely related to Bluetongue virus (BTV). The genomes of both viruses comprise ten segments of linear double-stranded (ds) RNA, which encode for seven structural (VP1-VP7) and non-structural proteins (NS1-NS4 and possibly NS5) (EHDV belongs to the genus Orbivirus within the family bly NS5) . Structubly NS5) . Virus sbly NS5) . Based obly NS5) . EHDV sebly NS5) , with noSimilar to other Orbiviruses, EHDV evolution is largely driven by two main forces; random mutation and segment reassortment. The former occurs during natural transmission cycles and plays an important role in the diversification of EHDV strains and their pathogenicity. A small number of nucleotide substitutions can have an impact on overall pathogenicity as has been shown for BTV serotype 8 (BTV-8) . SegmentIn 2006, a novel reassortant strain of EHDV-6 (Indiana) was detected in the USA, in which segments 2 and 6 originated from an Australian virus  and the remaining 8 segments originated from a North American EHDV-2 strain from Alberta . FollowiIn 2013, a group of EHDV-naive cattle imported from the USA onto the island of Trinidad, seroconverted for EHDV antibodies within six months of their arrival on the island . The detThe objective of this study was to perform full genome sequencing on the Trinidadian EHDV-6 isolate, in order to identify the degree of reassortment within the virus. Phylogenetic sequence comparison of each segment would then enable conclusions to be made about the likely provenance of each segment of the virus, giving clues to how the virus may have evolved, and how it may be related to the EHDV-6 strains currently circulating and causing severe disease in the USA.22.1n = 60) were confirmed as EHDV RNA negative using the EHDV real-time PCR assay  from a blood sample, collected from a Jersey cow, two months after its arrival into Trinidad. This isolate, named as TAT2013/02 [KC2], was deposited in The Pirbright Institute, Orbivirus Reference Collection and is available through the European Virus Archive goes global catalogue . TAT2013/02 isolate was repassaged two more times in KC cells as previously described  and ssRNA was removed by precipitation in 2 M lithium chloride  overnight as described . The dsR2.4A pre-alignment quality check was performed using the FASTQC programme and the Trim Galore programme was used for adapter trimming and quality trimming of reads at the Phred quality threshold of 30 and removal of short reads (<50 bp). Subsequently, reads were aligned to the reference genome (EHD6/AUS1981/07 virus) for segments 1,2,3,4,5,6,7,9, and 10, and for segment 8 to the EHD1/USA/1995/01 virus sequence , using the BWA-MEM tool and a combination of samtools (mpileup) and bcftools to derive the consensus sequence (GenBank accession numbers: MK919254-MK919263).2.5n = 51 on 1/12/2018) are publicly available through GenBank. In order to save computation time, 30 full-genome reference sequences were selected to represent all EHDV serotypes distributed worldwide and were retrieved from GenBank . Amino acid sequences for each coding region  were aligned individually using the Muscle algorithm. The evolutionary distances between a pair of nucleotide sequences were calculated separately for each coding region using the Maximum Composite Likelihood Model (n = 30) and the EHD6/Trinidad/2013 virus, then full coding region were aligned on the amino acid level. The detection of potential recombination events was performed with the following methods: RDP, GENECONV, Maximum Chi Square, CHIMAERA, BOOTSCANing, Sister Scanning (SISCAN) and 3SEQ. Sequences were considered as reassortants if they were identified by at least by 5 recombinant detection methods. For graphical representation of the relationship between the possible reassortant virus (EHD6/Trinidad/2013) and its potential parental sequences, the distance plot was constructed using a window size of 200, a step size of 20 and the similarity model in the RDP4.A number of full genome EHDV sequences  and 11,470 . These positions correspond to nucleotides 9574–11,508 encoding for the minor structural protein VP4. The beginning and end breakpoint positions of the second recombinant event were estimated at a location of 15,844  and 17,090 , which corresponds to the NS2 coding region. The genomic exchange of the NS2 and VP4 regions between EHD6/Trinidad/2013 virus and the parental sequences was confirmed by six and five different detection methods, respectively . The maj3.1Based on the phylogenetic analysis of segment 2 (VP2) and segment 6 (VP5), EHD6/Trinidad/2013 virus isolate clustered, with ≥95% ultrafast bootstrap support, into the serological group B which contains closely-related EHDV strains such as EHD6/AUS/1981/07 and the EHDV-6 reassortants from the USA: 2006 CC304–06, 2012 12–3437-8 and 2016 OV208 a&2c. Phy4http://btv.glue.cvr.ac.uk/#/home). Despite the recent EHDV-6 outbreaks in beef and dairy cattle in Israel . Eight out of the 10 segments showed a high degree of identity (97.2–97.9%) between these two viruses. This Australian strain was therefore identified as the major parental strain of the Trinidad virus. Other strains with equally high levels of VP2 homology with this Australian major parental strain have previously been reported in the Caribbean region, in Martinique and Guadeloupe in 2010 , howeverThe EHD6/Trinidad/2013 virus contained two reassorted segments, segments 4 encoding the VP4 protein and segment 8 encoding the NS2 protein. For segment 4, the highest percent identity (96.5%) was observed with the segment 4 of an Australian EHDV-2 virus (EHD2/AUS/1979/05), although the nucleotide identity between EHD6/Trinidad/2013 and EHD6/AUS/1981/07 was also quite high at 92.4%. These two Australian strains (EHDV-2 and EHDV-6) were identified in 1979 and 1981, so it is possible that they were co-circulating. It is therefore likely that the reassortment of segment 4 seen in the Trinidad virus occurred in Australia, at the time that these two viruses were co-circulating. Although EHDV-2 has been reported to be present on the island of Guadeloupe in 2011, the VP2 of this virus clustered with EHDV-2 strains from the USA, rather than from Australia . AlthougCulicoides vectors infected with EHDV could have transported the virus by boat or by wind between the two countries. Unfortunately, the full genomes of EHDV-1 and EHDV-6 from French Guiana were not available, so comparison of the segment 8 sequences between the Trinidad and French Guiana viruses was not possible.The reassortment of segment 8 in EHD6/Trinidad/2013 virus was highly supported by six independent methods  when the 31 full genome sequences of EHDV were analysed using the RDP. The RDP analysis did not however identify a minor parent for segment 8 of the EHDV/Trinidad/2013 virus from the 31 full genome sequences of EHDV that were analysed. Due to lack of sequencing data enabling the identification of the minor parent, the closest relative was selected as the plausible minor parent for segment 8 of the EHD6/Trinidad/2013 virus as an EHDV-1 strain from the USA (EHD1/USA/1955/01). Due to a lack of active epidemiological surveillance and the often subclinical nature of EHD, many EHDV serotypes/reassortants may remain undetected in the Caribbean, South America and elsewhere in the world. It is therefore possible that the minor parent strain for NS2 could be circulating, yet unidentified, in Trinidad. Although EHDV-1 was not identified to be circulating at the single location where the cattle were sampled in this study, it is possible that this serotype is present elsewhere on the island. A more extensive sampling exercise across the whole of the island would be necessary to see if this was the case. Alternatively, the Trinidad EHDV-6 could have received its NS2 by reassorting with a locally circulating virus in a neighbouring South American country, with the reasssorted virus then being transported to Trinidad. Co-circulation of EHDV-1 and EHDV-6 was reported in young cattle in French Guiana in the summer of 2011 . The proThis data strongly suggests that the EHD6/Trinidad/2013 virus had an Australian origin, receiving 9 of its 10 segments from two viruses circulating in Australia. This reassortant virus then likely came to the Americas, where it received its segment 8 from a locally circulating, as yet unknown, EHDV strain. This virus then may have gained entry into the USA, where it further reassorted with a known locally circulating EHDV-2 strain, giving rise to the EHDV-6 Indiana strain, with segments 2 and 6 originating from the EHDV-6 Trinidad strain and its remaining 8 segments originating from the locally circulating EHDV-2 strain. This study therefore identifies, for the first time, the likely minor parent virus (EHD6/Trinidad/2013) of the EHDV-6 Indiana strain currently circulating in the USA.Culicoides vectors. Critically however, when trying to understand more about the mechanisms behind viral virulence, it is vital to carry out full genome sequencing of all 10 segments of the virus, in order to know whether the virus has undergone reasssortment and to identify the parent strains of the reassorted segments. Knowing which segments have reassorted, as well as the virulence of the parent viruses, could provide evidence as to which of the segments are playing a role in virulence.Similar to BTV, the reasons behind the wide range of virulence observed between different EHDV strains within the same serotype is not fully understood. These differences in virulence could in part be attributed to different susceptibilities of hosts and/or differences in the competence of the local The EHD6/Trinidad/2013 double-reassortant did not cause clinical signs of disease in the group of naïve Holstein and Jersey cattle in this study. This was also the case in cattle infected with its major parental virus from Australia, EHD6/AUS/1981/07 . InteresTo better understand the origin of emerging EHDV strains, their transmission and their potential for generating reassortants, full genome sequencing is required. This study shows how full genome sequencing of Orbiviruses can enhance the ability to support epidemiological investigations on tracing viral incursions from distinct geographical locations. In addition, we also identified ‘missing links’ in the epidemiology of EHDV as the donor virus of segment-8 in EHDV-6 from Trinidad still reminds unknown. Improved pipelines for full genome sequence analysis, along with cheaper and more accessible sequencing platforms, should lead to an increase in published EHDV sequences, making the tracing of incursions more accurate and more representative to different regions of the world. It will also enable the direct comparison of reassortment frequencies between EHDV and BTV. Although a reverse genetics system has been developed for EHDV , the inf5In this study, a double-reassortant EHDV-6 strain, with an EHD6/AUS/1981/07 backbone (8 segments) and minor parental strains from EHD2/AUS/1979/05 (segment 4) and an unknown EHDV strain (segment 8), was identified to be circulating asymptomatically in cattle in Trinidad, West Indies. These data provide evidence that the minor parent of EHDV-6 Indiana is circulating in the Caribbean region and likely spread northwards from the Caribbean, or South America, to North America, before reassorting with a locally circulating EHDV-2 strain in the USA. This addresses the long-unanswered question related to the origin of the Australian parent strain of EHDV Indiana and how it may have arrived in the USA.Full-genome EHDV reference sequences.Table 1The following are the supplementary data related to this article.


Regarding viral infection of intestinal mucosa, there have been only a few studies on limited diseases, targeting a few herpes family viruses. In this study, we analyzed 12 kinds of DNA viruses including 8 species of herpes family viruses in the gastrointestinal mucosa of patients with hematologic malignancies, inflammatory bowel diseases, collagen diseases, or other miscellaneous forms of gastroenteritis using the multiplex virus PCR assay, which we recently developed. The virus PCR assay yielded positive results in 63 of 102 patients; Epstein-Barr virus (EBV) was the most frequently detected, followed by cytomegalovirus (CMV), human herpes virus 6 (HHV-6), HHV-7, parvovirus B19, and herpes simplex virus type 1. The frequencies of viral detection in the 4 diseases were similar involving these 6 viruses. Regarding CMV colitis, the multiplex virus PCR assay was superior to the immunohistopathologic method in detecting CMV. All viruses were more efficiently detected in the mucosa than in the blood in individual patients. These results suggest that CMV, EBV, and HHV-6 were commonly detected in the gastrointestinal mucosa of patients with these 4 diseases, and our multiplex virus PCR assay was useful for the early diagnosis of gastrointestinal virus infection, especially CMV colitis. Viral infections include meningitis/encephalitis caused by herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), human herpes virus type 6 (HHV-6), varicella-zoster virus (VZV), and JC virus (JCV), pneumonia by cytomegalovirus (CMV), herpes zoster/varicella by VZV, herpes simplex by HSV-1 or HSV-2, viral cystitis by adenovirus (ADV) or BK virus (BKV), and colitis by CMV. To make an exact diagnosis of these viral infections, viral genome analysis by polymerase chain reaction (PCR) in individual specimens has been performed. The specimens include cerebrospinal fluid (CSF) for meningitis/encephalitis by HSV-1 In recent years, we developed a multiplex virus PCR. The multiplex PCR assay is designed to qualitatively and simultaneously measure the genomic DNA of 12 viruses including CMV and EBV. When a specific PCR signal is obtained, the viral load is determined by a quantitative real-time PCR. It takes only 3 hours to complete both the qualitative multiplex and quantitative real-time PCR procedures 2.2.1.All patients were referred to the Laboratory of Cell Therapy in Shinko Hospital by their attending physicians for multiplex virus PCR analysis because of suspected gastrointestinal viral infection  from August 2011 to June 2016. Patients examined in the present study included those who received HSCT, those with hematologic malignancies under chemotherapy, collagen disease under immunosuppressive treatment, inflammatory bowel disease, and miscellaneous gastroenteritis associated with or without melena. The present retrospective analysis was a single institutional clinical study designated the “Multiple virus-analytic study by multiplex virus PCR in immunocompromised patients”, which had been approved by the Ethical Committee of Shinko Hospital.2.2.Mucosal biopsy specimens were obtained during endoscopic examination for both histopathologic and viral analyses from individual patients who provided written informed consent for the multiplex PCR examination. Two healthy persons who underwent gastroendoscopy also provided written informed consent for the PCR assay. The size of the mucosal biopsy specimen for the viral PCR was the same as that for histopathologic examination. Colon mucosal viral PCR assay of healthy persons was not performed because the risk of colonal mucosa biopsy in them was controversial among physicians in the Department of Gastroenterology and Hepatology of Shinko Hospital.2.3.3 copies for CMV, 2.5 × 103 for EBV, 2.5 × 103 for HHV-6, 5 × 103 for HHV-7, 5 × 103 for HHV-8, 1 × 103 for VZV, 1 × 103 for BKV, 5 × 103 for JCV, 1 × 103 for ParvoB19, 2.5 × 103 for HSV-1, 1 × 103 for HSV-2, and 5 × 103 copies/mL plasma for HBV The mucosal specimen was incubated in a mixed solution with 180 μL of Buffer ATL  and 20 μL of Protease K (Qiagen) at 56 °C until complete lysis of the specimen. Genomic DNA was extracted from the solution using the QIAamp DNA Mini kit (Qiagen), and then subjected to multiplex qualitative PCR and subsequently to quantitative real-time PCR. In each assay, GAPDH was quantified as an internal control, and the viral load is indicated in copy/μg DNA. When the specimen was the peripheral blood, EDTA-2Na-chelated whole blood (200 μL) was obtained from individual patients, and then the plasma was subjected to the PCR assay. The method for both qualitative multiplex PCR and quantitative real-time PCR was previously described in detail 2.4.Mucosal CMV infection was determined by the presence of CMV-caused inclusion bodies in H-E staining and positive CMV immunostaining with a mouse monoclonal antibody 2.5.To evaluate differences between rates of patients with positive virus detection, Pearson's Chi-squared test with Yates' continuity correction, Fisher's Exact Test, or the Bonferroni method (for multiple comparison) was employed. To compare mean values, Student's t-test was used.3.3.1.A total of 102 patients were examined for possible gastrointestinal viral infection, of whom 81 patients had colon disorders and 21 had upper gastrointestinal disorders. In the 102 patients, the largest number of patients (30 patients) included was those with inflammatory bowel diseases, followed by miscellaneous gastroenteritis with or without melena (26 patients), hematologic malignancies (26 patients) under chemotherapy (15 patients) and post-HSCT (11 patients), and collagen diseases (20 patients).3.2.1 to 4.2 × 106 copies/μg DNA. In this study, we could detect each virus at levels as low as 1.0 × 101 copies/μg DNA when compared with those in the plasma, although the standard unit was different.As shown in 3.3.p = 0.041; EBV + CMV versus CMV + HHV-6: p = 0.017).A total of 81 patients were examined for multiplex virus PCR because of an ulcer or a mucosal defect. 3.4.In 3 copies/μg DNA. Two years later, similar results were obtained. This person is quite healthy but develops a reddish skin rash each time after febrile common cold-like upper respiratory symptoms, confirming the latency of these 2 viruses.It was interesting that both HHV-6 and HHV-7 were detected in the gastric mucosa in one of 2 healthy persons, and the HHV-6 load was above 1 × 103.5.3 copies/μg DNA  and EBV (1.2 × 101 copies) plus CMV (9.3 × 103 copies) were detected in colon mucosa and gastric mucosa, respectively. Another patient with Behcet's disease showed negative virus detection in both lower and upper gastrointestinal tracts. These 2 patients showed a similar pattern of detection in terms of positive results in the former and negative results in the latter in both the upper and lower tracts. While in a patient with Crohn's disease, EBV (3.4 × 101 copies) and CMV (3.3 × 102 copies) were only detected in the colon mucosa.Three patients were examined with multiplex virus PCR for both the upper gastrointestinal tract and colon with a close interval. No specific treatment was performed during the interval in these patients. In one patient with Behcet's disease, EBV  were also not significantly different between the 2 groups. Immunohistopathologic diagnosis of CMV colitis was made in 6 of 13 patients (46.2%) in whom more than 1 × 103 CMV copies were detected, while the diagnosis was made in only 3 of 16 patients (18.8%) with a load of less than 1 × 103 CMV, without a significant difference (data not shown).CMV was detected in the colon mucosa in 29 patients, as shown in 3 CMV copies, also recovered from abdominal distress with supportive treatments suggesting that more or less 1 × 103 CMV copies could not be a turning point between CMV disease and latency.Thirteen of 29 patients in whom CMV was detected by multiplex PCR assay were treated with ganciclovir based on histopathologic diagnosis or positive PCR result with improved abdominal distress. However, remaining 16 patients including 5 with more than 1 × 103.8.In some patients, virus PCR tests were simultaneously performed with intestinal mucosa and blood specimens. Therefore, we compared the incidence of virus detection between the intestine and blood. EBV was not detected in the blood in 4 of 10 patients in whom EBV was detected in the colon mucosa. CMV was not detected in the blood in 2 of 10 patients with positive mucosal CMV. As for HHV-6, this virus was not detected in the blood in 8 patients with detectable mucosal HHV-6. Conversely, only a small number showed blood viral detection among mucosal virus-negative patients (data not shown), indicating the higher detection of these viruses in the colon mucosa than in the blood.3.9.3 copies/μg DNA. In Patient C, both HHV-6 and EBV were detected. These patients, however, did not have a skin rash or atypical lymphocytes in the peripheral blood, suggesting local viral infection, and all 4 patients recovered with fluid therapy. Patient D with an excised MALT lymphoma did not have abdominal distress, but underwent colonofiberscopy and mucosal biopsy for evaluation after the lymphoma excision. Although the biopsied mucosa looked normal, HHV-7 was detected with a viral load of 3.2 × 103 copies/μg DNA, suggesting the mucosal latency of this virus.CMV colitis is an established clinical entity; however, whether gastroenteritis is caused by other herpes-family viruses is unknown. As shown in 4.In the present study, we examined gastrointestinal mucosa for possible target for viral infection. Thus, we have detected viral DNA with using the multiplex virus PCR assay in patients with an immunodeficient disease or status. There have been a few studies regarding gastrointestinal EBV, CMV, or HHV-6 infection using a quantitative PCR method that can assay a single virus. In the present study, we simultaneously assayed 12 kinds of viruses including these 3 viruses in the gastrointestinal mucosa involving a larger number of diseases or statuses than examined previously. Therefore, this is the first report of a study on many kinds of gastrointestinal virus infections in patients with various abnormal immune statuses. The detected viruses in the present study in both lower and upper gastrointestinal tracts were EBV, CMV, HHV-6, HHV-7, HSV-1, and ParvoB19, suggesting that these 6 but not the remaining 6 kinds of virus are involved in immunodeficiency-related gastroenteritis.In the large intestine, the most frequently detected virus was EBV, followed by CMV, HHV-6, HHV-7, HSV-1, and ParvoB19 . The detIt should be noted that the incidence of virus detection was high in patients with miscellaneous colitis (58.8%), showing a similar pattern of detection to other diseases/statuses in terms of the virus species detected and frequent combination of EBV and CMV. The majority of these patients did not have underlying disease and their abdominal distress including melena was transient, being improved with conservative treatment. Thus, in a future study, follow-up of virus PCR examination will be needed to determine the role of these viruses in this type of colitis.As for exceptional virus detection, HSV-1 could chronically affect the GI tract in immunocompromised hosts without severe symptoms, as observed in patients in the present study We simultaneously performed the multiplex virus PCR assay in both upper and lower gastrointestinal tracts. The virus detection showed roughly similar patterns at both sites. Therefore, it is of marked interest that an upper GI tract virus examination could be employed as a substitute for colonofiberscopy because a high CMV detection rate in upper GI tract biopsies from heart transplant patients was reported In the diagnosis of CMV, there was a discrepancy between the results of our PCR assay and immunohistopathologic diagnosis. Generally, multiple antibodies are necessary to perform a precise immunohistopathologic procedure We analyzed the possibility of colitis caused by a herpes family virus other than CMV. In these patients with miscellaneous colitis , an ulceThe limitations of this study include: retrospective analysis, too small patient population to draw a definite conclusion, unclear indication of gastrointestinal virus PCR, vague indication for ganciclovir treatment in patients with positive colonal CMV, and unclear criteria for virus infection or latency. In the future, the significance of viral detection in the gastrointestinal mucosa and appropriate therapeutic indications for antiviral treatment should be determined with a prospective study.


There is speculation that beef bull semen quality is inferior to that of dairy bulls although few scientific studies are available in the literature. The aim of this study was to evaluate sperm quality in beef bull semen and to determine which parameters could be indicative of fertility after insemination. Sperm quality, assessed by computer assisted sperm motility analysis and flow cytometric evaluation of membrane integrity, levels of reactive oxygen species, mitochondrial membrane potential, acrosome status and DNA fragmentation index, was evaluated in beef and dairy bull semen.For beef bulls, normal morphology  and WOBBLE  were significantly correlated with 56-day non-return rate, whereas sperm quality was not significantly correlated with the fertility index score for dairy bulls. Membrane integrity , normal morphology , and high respiratory activity  were higher for dairy bulls than for beef bulls. The DNA fragmentation index was lower for dairy bull spermatozoa than beef , whereas some sperm kinematics were higher. Multivariate analysis indicated that type of bull (beef versus dairy) had an impact on sperm quality.Different assays of sperm quality may be needed for appropriate analysis of beef and dairy bull semen. These finding could be important for cattle breeding stations when evaluating semen quality. Artificial insemination (AI) is one of the most effective tools available to dairy cattle producers to improve herd productivity and profitability . WhereasAlthough the market for beef bull semen for inseminating beef cows is generally stable, it is increasing in countries such as Brazil, where sales of beef semen have risen from approximately 3 million to nearly 12 million in less than 20 years . TherefoCommercial straws of frozen semen from 14 beef bulls  frozen in Triladyl extender  were available from the Estonian Animal Breeders Association, Rapla, Estonia. Semen from 19 dairy bulls (10 Swedish Red and 9 Holstein) and 4 beef bulls  frozen in Andromed (Minitüb) was supplied by VikingGenetics, Skara, Sweden. The straws were thawed in a water bath at 37 °C for 12 s.A Nucleocounter SP-100 (Chemometec) was used to measure sperm concentration. Aliquots (50 µL) of frozen-thawed semen were diluted with 5 mL S100 reagent to permeabilise the sperm membranes and a cassette containing propidium iodide (PI) was loaded with the mixture. The cassette was inserted into the fluorescence reader, which displayed the sperm concentration after approximately 30 s.Wet smears were air dried and stained with carbolfuchsin-eosin,  for evalSperm kinematics were assessed objectively using a CASA system, consisting of a phase-contrast Olympus BX 51 microscope  connected to the SpermVision™ . An aliquot (5 µL) of semen was deposited on a warmed microscope slide at 38 °C and covered with a coverslip (18 × 18 mm). Sperm images in 8 fields were digitized for analysis of the kinematic patterns using the SpermVision™ software. The mean values were calculated for each of the following parameters based on approximately 1000 spermatozoa : total m6 spermatozoa/mL for staining with the Live-Dead Sperm Viability KIT L-7011 (Invitrogen). An aliquot (300 µL) of the sperm suspension was stained with 0.6 µL SYBR14  and 3 µL PI , and incubated for 10 min at 38 °C. The stained samples were evaluated using a BD LSR flow cytometer , the excitation being induced by an argon-ion laser (488 nm). Green fluorescence was detected with a FL 1 band-pass filter (530/30 nm) while red fluorescence was measured using a FL 3 long-pass filter (> 670 nm). A total of 50,000 sperm-specific events was evaluated and classified as membrane intact , membrane damaged .Membrane integrity was analysed by flow cytometry after SYBR14-PI staining . Aliquot2O2) were determined using HE and DCFDA, respectively, while HO was added to enable live and dead cells to be differentiated.Reactive oxygen species were determined using a modification of the protocol described by Guthrie and Welch , in whic6 spermatozoa/mL, the spermatozoa were stained as follows: two aliquots (300 µL) of each sample were stained with 9 µL of HO (40 µM), 9 µL HE (40 µM) and 9 µL DCFDA, (2 mM). In addition, 3 µL Menadione  were added to one of the aliquots to stimulate ROS production. After mixing gently, the samples were incubated at 38 °C for 30 min.After adjusting the sperm concentration to approximately 2 × 102O2 negative; Live, H2O2 positive; Dead, H2O2 negative; Dead, H2O2 positive (%).The analysis was carried out using the BD LSR flow cytometer . Excitation was achieved with an argon-ion laser (488 nm) and a HeCd laser (325 nm); green fluorescence was detected with a FL1 band pass filter (530/30 nm), red fluorescence with a FL3 long pass filter (> 670 nm) and blue fluorescence with a FL4 band pass filter (510/20 nm). In total, 30,000 sperm-specific events were evaluated and classified as the proportions of: Live, Superoxide negative; Live, Superoxide positive; Dead superoxide positive; Live, H6/mL, were mixed with 1.2 µL 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide (JC-1) (stock 3 mM) and incubated for 40 min at 38 °C as described by Garner and Thomas [Aliquots of semen (300 µL) at a sperm concentration of approximately 2.5 × 10d Thomas . The JC-Chromatin integrity was evaluated using the metachromatic dye acridine orange (AO). The DNA fragmentation index (%DFI) was expressed as the proportion of cells with denatured, single stranded DNA (red fluorescence) out of the total population . An aliquot (20 µL) of thawed semen was mixed 1:1 (v/v) with TNE buffer, snap-frozen in liquid nitrogen and stored at − 80 °C. For analysis, samples were thawed on ice, and an aliquot (10 µL) was mixed with 90 µL of TNE followed by 200 µL of acid-detergent solution. After 30 s, AO (600 µL) was added and the sample was analyzed within 3–5 min using a FACStar Plus Flow cytometer with settings and software as described previously by Morrell et al. .6/mL, was mixed with 3 µL of FITC-PNA  and 3 µL of PI, and was incubated at 38 °C for 10 min [Sperm acrosome status and cell viability were assessed using fluorescein isothiocyanate-PNA (FITC-PNA) labeling and PI, respectively. An aliquot (300 µL) of semen, previously diluted to a sperm concentration of 2 × 10r 10 min , 15. TheThe 56-day non-return rate after first insemination in 130 to > 1000 cows was available for the beef bulls. For the 19 dairy bulls, a fertility index score was provided for inseminations in > 1000 cows, which had been calculated by adjusting the non-return rate to account for factors such as the age and parity of the female, farm location, inseminator etc.The statistical analyses were performed using the SAS software . To evaluate the difference between the two groups of bulls, analysis of variance (PROC GLM) was applied, using a statistical model including the effect of breed. The relationships between sperm quality and fertility (either the 56-day non-return rate or the fertility index score) were analysed within bull type (beef or dairy) using Spearman rank correlation. Only bulls with more than 130 cows were included in the correlation analysis. In all cases, P < 0.05 was considered to be significant.http://onlinelibrary.wiley.com/doi/10.1002/cem.1006/full.To investigate the interaction of extender and type of semen i.e. beef or dairy bull semen, multivariate analysis was performed with Partial Least Squares Regression (PLS) using Simca software , as described on the website 6/mL and 55 ± 19 × 106/mL for beef and dairy bull semen, respectively (P < 0.001).The mean concentration (± SD) was 88 ± 20 × 10Normal morphology for beef and dairy bulls was 76 ± 8% and 87 ± 6%, respectively (P < 0.05).Except for total motility, progressive motility and BCF, the sperm kinematics were significantly different for the two types of semen Table . Most kiBeef bull semen contained a lower proportion of membrane intact spermatozoa Table  than dai2O2 negative sperm cells between the two types of bulls, although no significant differences were observed for the other categories of ROS. In samples stimulated with menadione, trends towards significance were seen for live H2O2 positive spermatozoa (P < 0.055) and dead, H2O2 negative spermatozoa (P < 0.066) between the two types of bulls.In both control samples and samples stimulated with menadione Table , there wThe FSC-SSC pattern obtained and the regions of analysis used in the assay for acrosome status are shown in Fig. Figure The %DFI was significantly higher (P < 0.01) for beef bulls (6.1 ± 2.9) than for dairy bulls (3.8 ± 1.1).For beef bulls, the kinematic WOBBLE Fig. a was sigThe PLS scatter plot showing the distribution of the individual bulls according to type (beef or dairy) and extender is shown in Fig. In this study, some interesting relationships between sperm quality and pregnancy rates were observed, not the least the fact that sperm motility  was not significantly related to pregnancy rate for these bulls. Significant differences in sperm quality between the two types of bulls were seen, as were correlations between sperm quality and pregnancy rate for beef bulls , whereas there were no links between sperm quality parameters and fertility for the dairy bulls. There were significant differences between sperm motility patterns in semen from beef and dairy bulls; however, the semen was frozen in different extenders on the two commercial cattle stations, which could have affected the assessment of kinematics. Multivariate analysis showed that the sperm quality of beef bull semen frozen in Andromed was more similar to that of beef bull semen frozen in Triladyl than to sperm quality of dairy bull semen frozen in Andromed. Therefore, we deduce that type of bull semen (beef versus dairy) has more effect on sperm quality than the extender itself , and therefore it is justifiable to combine the data for all of the beef bulls in this study, regardless of extender.The sperm concentration in the straws was different for beef and dairy semen. However, this should not have affected the motility analysis since the sperm concentration was not too high to be analysed by the CASA instrument. Sperm concentration was adjusted to the same levels in all samples for staining in the flow cytometric analyses. However, sperm concentration could be important depending on the type of abnormality that is present. So-called compensable abnormalities are ones that impede the ability of the spermatozoa to reach the oviducts or to interact with the oocyte ; increasAccording to a study in fresh bull semen , high spReactive oxygen species are believed to cause decreased motility, decreased viability, and a number of morphological defects, particularly in the mid-piece. Although viability was superior in the dairy bulls in the present study, no significant differences were observed in total and progressive motility between breed types. These results are in contrast to those obtained by Fiaz et al. , who obs®, Bioxcell® and Triladyl® extender for cryopreservation of bull semen [A comparative study on AndroMedll semen  showed tll semen . AccordiThe effect of bull type on other sperm quality parameters such as sperm morphology, may be explained by breed differences in adaptability to environmental conditions  and scroInterestingly, multivariate analysis indicated that the type of bull (beef or dairy) had more of an effect on sperm quality than the extender. Since different extenders were used on the two commercial semen stations, the variable “extender” could also reflect differences in sperm handling procedures or freezing protocols. However, regardless of such potential sources of variation, beef bulls clustered together rather than clustering according to extender.The observed differences in sperm quality between beef and dairy breeds may be due in part to intensive selection among dairy sires both in terms of production traits in their offspring and in sperm quality. The relationships between sperm quality and pregnancy rate for the beef bulls are interesting and warrant further study with a larger number of bulls. It would also be interesting to study possible breed differences among beef breeds, especially since a breed effect has been observed in dairy bulls . The lacSignificant relationships were seen between normal morphology and the 56-day non-return rate for beef bulls, and also for WOBBLE and the 56-day non-return rate, although no correlations were found between sperm quality and the fertility index score for dairy bulls. There were some differences in sperm quality between dairy and beef bulls. Multivariate analysis indicated that type of bull (beef versus dairy) had more effect on sperm quality than the extender used. Thus, different parameters of sperm quality may be needed as indicators of fertility for the two types of bull. These finding could be important for cattle breeding stations when evaluating semen quality.


We show that label-free, selective, sensitive detection of influenza viruses by home appliances is possible in principle.We describe rapid, label-free detection of Influenza A viruses using the first radial mode of oscillations of lead zirconate titanate (PZT) piezoelectric discs with a 2 mm radius and 100 µm thickness fabricated from a piezoelectric membrane. The discs are modified with a synthetic sialylglycopolymer receptor layer, and the coated discs are inserted in a flowing virus suspension. Label-free detection of the virus is achieved by monitoring the disc radial mode resonance frequency shift. Piezo transducers with sialylglycopolymer sensor layers exhibited a long lifetime, a high sensitivity and the possibility of regeneration. We demonstrate positive, label-free detection of Influenza A viruses at concentrations below 10 Early detection of influenza is the most effective way to protect against the flu . Many inMost similar reports use antibodies as receptors ,9; theseSeveral different virus families, including Orthomyxoviridae, Paramyxoviridae and Parvoviridae, use sialoglycoconjugates for attachment . Some viMoreover, several types of sensors for detecting various types of avian influenza viruses were developed with different detection principles and types of readout systems, namely, fluorescent , electroThe most promising systems for the home appliance detection of influenza virus are microelectromechanical systems (MEMS). The main advantages of MEMS sensors are the small size and simple readout system based on electronic components.Currently, numerous publications describe the potential of using MEMS for virus detection by surface acoustic waves resonators (SAW) ,24, a caIn our previous work , we desc2.2.1.−1 [5, 106 and 107 virions ml−1 were prepared by diluting the initial virus solution (approx. 1 × 108 virions ml−1) with virus-free allantoic liquid from non-infected embryos.Influenza A virus A/Duck/Moscow/4182/2008  was used as our investigation target in all experiments. It contained an H5N3 surface glycoprotein composition. The stock of viruses was prepared in the allantoic cavity of 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs. Allantoic liquid is a biological medium with many different proteins. This complicated mixture provides a good background for determining sensor selectivity due to total protein concentration of approximately 2 mg ml−1 . Sample 2.2.PZT discs were modified with synthetic sialylglycoconjugates based on a polymer matrix (3′SL-PAA) which biospecifically bind haemagglutinin proteins on the influenza viruses. Polyacrylamide conjugates bearing Neu5Acα2-3Galβ1-4Glcβ (3SL) with a high molecular weight were synthesized as previously described –35. Neu52.3.−3 M in ethanol, 16 h). We used a ground carbide-grade milling cutter as a tool to produce 4 mm discs from prepared gold-coated PZT plate. The 4 mm discs were incubated in a 10−3 M 3′SL-PAA aqueous solution overnight for better adsorption of 3′SL-PAA on the aminated gold surface. Some binding sites were deactivated after adsorption; furthermore, numerous active receptors in the polymer structure do not interact with the surface. The PZT discs were washed with Milli-Q water after 3′SL-PAA immobilization.The fabrication process of PZT disc sensors from the PZT plates of 100 µm thickness with silver electrodes is presented in 2.4.rF is the ‘centre-of-mass’ resonance frequency, iF and iA are the frequency and amplitude of the ith point of the curve. Data were provided from a FemtoScan controller for scanning probe microscopy . Data processing were performed with FemtoScan Online software (Advanced Technologies Center).We designed a special board to measure the resonance frequency shift of PZT discs . To measa). The holder was placed in an Eppendorf tube, where it could be incubated in water or various solutions during experiments. This type of holding allows performing measurements at the radial mode with the shape of oscillation shown in the computer simulation model presented in In all experiments, we used PZT discs with a 2 mm radius and 100 µm thickness fabricated from a piezoelectric membrane using a common drilling machine. Each disc was clamped in the centre using a special holder with two conductive springs a. The hob. It was found that the Q factor changed little when air is replaced with water.Using the radial mode of oscillation can exclude much of the influence of liquid on the resonance properties of the sensor. Resonance peaks of the piezo disc in water and air are presented in figure 3pN = frD, where pN is a material specific constant, rf is the eigenfrequency of first radial mode, and D is the disc diameter. The eigenfrequency of the first radial mode is approximately 500 kHz for the PZT disc with 4 mm diameter, which corresponds to the experimental results in figure 3b.The first radial oscillation mode of a disc  can be c−1 was used for all virus detection experiments , a two-way six-port valve  and the liquid chamber. A peristaltic pump flow rate of 1 ml mineriments .Figure 2.5.−1 were used (working frequency in liquid was approx. 10 kHz). The line scan rate was typically 2.1 Hz, with 512 × 512 pixels per image. AFM images were processed using FemtoScan Online software  [Samples for AFM investigations of a sensor layer were prepared on freshly cleaved mica using the same procedure described earlier . AFM experiments were performed in tapping mode with a Nanoscope 3a multimode atomic force microscope  equipped with a tapping-mode fluid cell for operation in aqueous environments. NP-S1 silicon nitride cantilevers  with a spring constant of approximately 0.5 N m Russia) .3.3.1.et al. [f/f is independent of the length and width of the piezoelectric cantilevers and is only proportional to the surface stress s resulting from virus binding and inversely proportional to the thickness h of the resonator: Δf/f ∝ s/h, where f is the initial resonance frequency and Δf is the resonance frequency shift defined as the difference in the resonance frequency of the cantilever in solution before and after exposure to virus particles . We developed a special holder with central clamping  to contaa,b demonstrates the successive AFM images of the same area of the model biosensor surface (3′SL-PAA covered 4-aminothiophenol modified Au-Cr sputtered mica surface) after exposure to 30 µl of Influenza A virus solution (6 × 106 vp ml−1). The first image (a) demonstrates the presence of particles (globular features) 20–50 nm in height, which we identify as influenza virions bound specifically to the sensor surface. In the second image (b), we observe the disappearance of some particles (e.g. inside the marked areas), which can be explained by shifts due to the AFM tip during scanning. The appearance of holes (with apparent depth ≈ 10 nm and width over 100 nm) at the positions of the viral particles after their removal  indicate that bound virions are partially embedded in the polymer matrix.Direct evidence of virus binding to the sensor surface was visualized using AFM in an aqueous environment. Figure 7st image a demonstnd image b, we obs3.2.5, 106 and 107 virions ml−1. Steady-state resonance shifts for different concentrations and the experimental dependence of the frequency shift on the concentration are presented in 5 virions ml−1 and a signal-to-noise ratio of approximately 4. Sensitivity can be easily increased several fold by using a thinner PZT substrate because the sensitivity is inversely proportional to the thickness of the resonator. Applying Pb(Mg1/3Nb2/3)O3–PbTiO3 (PMN-PT) or other materials with better electromechanical constants could also improve the sensor quality. Using piezo discs with a smaller diameter should increase the sensitivity because the density of viruses in the receptor layer would be higher. The periodic noise in all graphs could be caused by the peristaltic pump. This could be avoided by optimizing the flow system.Interactions between the virions and receptor layer lead to additional lateral forces  or addit3.3.5 vp ml−1) in There are three independent measurements for different (PZT) piezoelectric discs modified with a synthetic sialylglycopolymer receptor layer for the same concentration  exhibited good results for the regeneration receptor layer. We showed a regeneration possibility of urea for this receptor layer in our previous work . The expLess than 3% of the sensitivity was lost after the first regeneration. The equilibrium value of the resonance shift after the second regeneration was over 80% of the measurements with initial receptor layer. The fluctuation amplitude in the plateaus was equal for the experiments before and after regeneration.6 vp ml−1) to block receptors. The blockage of 3′SL-PAA was controlled by measuring the resonance frequency shift (blue curve in 6 vp ml−1) was injected into the system again and the resonance frequency shift was measured. The red curve in Sensitivity of the sensor depends on the number of free 3′SL-PAA receptors present on the polymer layer according to the Gibbs equation relating adsorption to dependence of the surface stress . The fol3.5.f/f is only proportional to the surface stress s resulting from virus binding and inversely proportional to the thickness h of the resonator: Δf/f ∝ s/h. In our case, the AFM data confirmed that the thickness of the sensor layer did not change before and after virus particle interactions. In our previous paper, we measured the surface stress generation in synthetic sialylglycopolymer receptor layer [f/f ∝ s presented in f/f at 0 virus particle concentration due to small non-specific interactions of proteins from the allantoic fluid.We assumed that the relative resonance frequency shift Δor layer  by virus3.6.Methods for detecting influenza viruses can be divided into several groups: genetic analysis, immunoassays, electrochemical, MEMS. Laboratory and field methods can also be distinguished. Most of them are still oriented toward clinical diagnostics, but some can be used for environmental monitoring. The ‘gold standard’ for diagnostics are conventional PCR and ELISA, which are suitable for clinical use. Many novel methods based on improvement of traditional approaches or completely new technologies help to overcome limitations of traditional methods by increasing sensitivity, decreasing analysis time and extending the applicability. It is very complicated to compare the sensitivities of different methods because different units are used by researchers to define virus concentrations. These could be virus particles, plaque-forming units (PFUs), haemagglutination units (HAUs), molecular or weight concentrations of viruses or viral proteins. For example, for Influenza A virus particle-to-PFU ratio could be greater than 10 : 1 . Table 14.5 virions ml−1 and demonstrated meaningful differences between the frequency shifts for concentrations of 0, 105, 106 and 107 virions ml−1. Sensitivity can be easily increased several-fold using a thinner PZT substrate, because it is inversely proportional to the thickness of the resonator. The suggested sensor satisfied the detection time and reproducibility requirements for home appliance use. We demonstrated that PZT discs with a PAA-3′SL receptor layer can be successfully regenerated. Two consecutive flow regenerations of the sensor in a 10% urea water solution led to sensitivity loss of less than 20%. We demonstrated the principle of label-free, selective, sensitive detection of Influenza A virus particles that can potentially be used to detect pathogens in home appliances.We demonstrated the possible use of PZT piezo disc resonators with a polymer receptor layer for detecting influenza virions. The interaction between virus particles and the receptor layer on the surface of PZT discs leads to an increased frequency of the radial oscillation mode. This increase may be due to intermolecular forces in the receptor layer, which can add additional strain to the resonator system. The use of the radial oscillation mode allows excluding much of the influence of media on the resonance properties of the sensor. We detected viruses at concentrations of 10